mRNA gene expression was measured in intact female Sprague-Dawley rats at 6 (young), 26 (adult) and 52 (older) weeks of age at the time of fracture. Samples were collected at 0, 0.4, 1, 2, 4, and 6 weeks after fracture. RNA from two rats were pooled for each Affymetrix Rat U34A array.  Mid-shaft, simple, transverse left femoral fractures were induced after retrograde intramedullary rod fixation with a Bonnarens and Einhorn device.  Samples were collected from one third of the femoral length, centered on the fracture site, including the external callus, cortical bone, and marrow elements.
Keywords = rat

Bladders examined at 1, 4 and 24 hours after lipopolysaccharide, substance P or ovalbumin challenge.
Keywords: other

Our study seeks to identify genes differentially expressed between uterine leiomyomas and normal myometrial tissue. This series consists of samples derived from normal myometrium and uterine leiomyomas obtained from fibroid afflicted patients.Total RNA was extracted from samples, converted to biotin-labeled cRNA, hybridized to oligonucleotide arrays, and followed by model based expression analysis.

In order to select differentially expressed genes of interest, dChip model-based expression analysis was employed. Significant genes were identified, utilizing the dChip software, as having an average fold change of > +1.5 or < -1.5 between leiomyoma and normal myometrium and p < 0.05. Under these conditions the 226 genes in the following list were identified.

Hindlimb muscle, comparison of wild type and mdx mice, 7 to 112 Day (Porter lab)

Determination of gene expression changes in hindlimb muscle (gastrocnemius/soleus) of mdx (dystrophin-deficient) mice at postnatal ages 7, 14, 23, 28, 56, and 112.
Keywords: time-course

Diaphram, comparison of wild type and mdx mice, 7 to 112 Days (Porter lab)

Determination of gene expression changes in extraocular muscle of mdx (dystrophin-deficient) mice at postnatal ages 7, 14, 23, 28, 56, and 112 days.  3 independent replicates/age/strain. Data form part of publication: Human Molecular Genetics 13:257-269, 2004.
Keywords = microarray

multiple myeloma;osteolytic lesions;MRI;osteoblast;mesenchymal stem cell;Wnt signaling;DKK1;other

This series represents samples of multiple myeloma patients with and without bone lytic lesion by MRI.
Keywords = multiple myeloma

Systemic lupus erythematosous (SLE) is an autoimmune disease with an important clinical and biological heterogeneity. B lymphocytes appear central to the development of SLE which is characterized by the production of a large variety of autoantibodies and hypergammaglobulinemia. In mice, immature B cells from spontaneous lupus prone animals are able to produce autoantibodies when transferred into immunodeficient mice, strongly suggesting the existence of intrinsic B cell defects during lupus. In order to approach these defects in humans, we compared the peripheral B cell transcriptomes of quiescent lupus patients to normal B cell transcriptomes.

Genome-wide gene expression differences between Crohn’s and ulcerative colitis from endoscopic pinch biopsies:

Ulcerative colitis (UC) and Crohn’s disease (CD) are inflammatory bowel diseases (IBD) with variable, overlapping clinical features and complex pathophysiologies. To identify pathogenic processes underlying these disease subtypes, using single endoscopic pinch biopsies to estabolish 36 expression profiles, we elucidated gene expression patterns of active and inactive areas of UC and CD, and compared these to infectious colitis and healthy controls.
Keywords: RNA

Gene expression profiles of interstitial lung disease (ILD) patients

The mechanisms and molecular pathways underlying interstitial lung diseases (ILDs) are poorly understood. Systems biology approaches were used to identify perturbed networks in these disease states to gain a better understanding of the underlying mechanisms of disease. Through profiling genes and miRNAs, we found subsets of genes and miRNAs that distinguish different disease stages, ILDs from controls, and idiopathic pulmonary fibrosis (IPF) from non-specific interstitial pneumonitis (NSIP).  Traditional pathway analysis revealed several disease-associated modules involving genes from the TGF-beta, Wnt, focal adhesion and smooth muscle actin pathways that may be involved in advancing fibrosis.
A comprehensively integrative approach was used to construct a global gene regulatory network based on the perturbation of key regulatory elements, transcriptional factors and miRNAs.  The data also demonstrated that several subnetworks were significantly associated with key molecules involved in the diseases.  We present a broad overview of the disease at a molecular level and discuss several possibly key regulatory molecular circuits that could play central roles in facilitating the progression of ILDs.

Comparison of gene expression changes in CD4+CD8+ thymocytes following engagement of TCR with anti-Valpha or Vbeta antibodies
Keywords: parallel sample

Gene expression profiling in Treg cells deficient or mutant in candidate FoxP3 cofactors

The transcription factor FoxP3 partakes dominantly in the specification and function of FoxP3+ CD4+ T regulatory cells (Tregs), but is neither strictly necessary nor sufficient to determine the characteristic Treg transcriptional signature. Computational network inference and experimental testing assessed the contribution of several other transcription factors (TFs). Enforced expression of Helios or Xbp1 elicited specific signatures, but Eos, Irf4, Satb1, Lef1 and Gata1 elicited exactly the same outcome, synergizing with FoxP3 to activate most of the Treg signature, including key TFs, and enhancing FoxP3 occupancy at its genomic targets. Conversely, the Treg signature was robust to inactivation of any single cofactor. A redundant genetic switch thus locks-in the Treg phenotype, a model which accounts for several aspects of Treg physiology, differentiation and stability.

mRNA expression in the spinal cords of the G93A-SOD1 familial ALS transgenic mouse model was compared to that in nontransgenic (Normal mouse) and transgenic mice expressing wild-type (WT)SOD1.  Gene Ontology (GO)analysis was used to characterize differences in expression between G93A-SOD1 mouse and nontransgenic mouse spinal cord. Changes in multiple GO categories were found.  Many of these were associated with subsystems involving cell-cell communication and intracellular signal transduction.  Expression profiles of mice expressing WT-SOD1 did not differ from nontransgenic mice.  In contrast, protein profiling using proteomics technology indicated changes in mitochondrial protein expression in the G93A-SOD1 mouse spinal cord that were not found in the mRNA expression analysis.
Keywords: Disease state analysis, time course, transgenic mice

mTORC1 controls the systemically-induced adaptive regulation of stem cell quiescence into GAlert

A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function, the extent to which stem cells can regulate quiescence is unknown. Here, we show that the stem cell quiescent state is composed of two distinct functional phases: G0 and an “alert” phase we term GAlert, and that stem cells actively and reversibly transition between these phases in response to injury-induced, systemic signals. Using genetic models specific to muscle stem cells (or satellite cells (SCs)), we show that mTORC1 activity is necessary and sufficient for the transition of SCs from G0 into GAlert and that signaling through the HGF receptor, cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism, a novel adaptive response that positions stem cells to respond rapidly under conditions of injury and stress without requiring cell cycle entry or a cell fate commitment.
We performed microarray analysis on muscle stem cells, harvested immediately after isolation, to investigate the transcriptional response these cells have to injury and the role of mTORC1 and cMet have in this response

The Effect of Hepatitis C Virus Infection on Host Gene Expression

Hepatitis C Virus is a leading cause of chronic liver disease. The identification and characterisation of key host cellular factors that play a role in the HCV replication cycle is important for the understanding of disease pathogenesis and the identification of novel anti-viral therapeutic targets. Gene expression profiling of HCV infected Huh7 cells by microarray analysis was performed to identify host cellular genes that are transcriptionally regulated by infection. The expression of host genes involved in cellular defence mechanisms (apoptosis, proliferation and anti-oxidant responses), cellular metabolism (lipid and protein metabolism) and intracellular transport (vesicle trafficking and cytoskeleton regulation) was significantly altered by HCV infection. The gene expression patterns identified provide insight into the potential mechanisms that contribute to HCV associated pathogenesis. These include an increase in pro-inflammatory and pro-apoptotic signalling and a decrease in the anti-oxidant response pathways of the infected cell.

Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1α. We could then efficiently knockdown PGC-1β expression by shRNA expression. Loss of PGC-1α did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1α or PGC-1β coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1α and PGC-1β were deficient. These data show that PGC-1α is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis. Affymetrix microarray analysis of total RNA from wt, PGC-1α KO and PGC-1α KO
cells expressing an RNAi specific for PGC-1β knockdown was performed. Of the 461
mitochondrial genes analyzed, 181 were found to be at least 20% different between wt
and defective PGC-1α and β adipocytes (p < 0.05). More than 85% of these genes were downregulated in cells deficient for PGC-1alpha and PGC-1beta.

Expression of the brown fat thermogenic gene program requires PGC-1alpha

To investigate the specific role of PGC-1 coactivators in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severly reduced the induction of thermogenic genes. In order to assess the specific requirement for PGC-1α in the global transcriptional response to cAMP, we used Affymetrix arrays to compare the sets of genes induced in response to a 4 hr dbcAMP treatment in differentiated wt and KO cells. This analysis revealed that 88 genes were induced more than 3-fold in the wt cells; of these, 54 (61% of total) were similarly increased in both wt and KO. However, 28 genes (32% of total) were decreased by at least 50% in the KO cells compared to wt cells. These data were confirmed by quantitative PCR for a subset of genes. These data indicate that PGC-1α is required for proper expression of approximately one third of the genes induced in response to cAMP in brown fat cells, but this set of sensitive genes is enriched in those involved in adaptative thermogenesis.

Mesenchymal Stem/Progenitors and Other Endometrial Cell Types from Women with Polycystic Ovary Syndrome (PCOS) Display Inflammatory and Oncogenic Potential

Context: Endometrium in polycystic ovary syndrome (PCOS) presents altered gene expression indicating progesterone resistance and predisposing to reduced endometrial receptivity and endometrial cancer.  Objective: We hypothesized that an altered endocrine/metabolic environment in PCOS may result in an endometrial “disease phenotype” affecting the gene expression of different endometrial cell populations, including stem cells and their differentiated progeny.   Design and setting: A prospective study conducted at an academic medical center.  Patients and Main Outcome Measures: Proliferative phase endometrium was obtained from 6 overweight/obese PCOS (NIH criteria) and 6 overweight/obese controls. Microarray analysis was performed on fluorescence-activated cell sorting (FACS)-isolated endometrial epithelial cells (eEP), endothelial cells (eEN), stromal fibroblasts (eSF) and mesenchymal stem cells (eMSC). Gene expression data were validated using microfluidic Q-RT-PCR and immunohistochemistry (IHC).   Results: The comparison between eEPPCOS and eEPCtrl showed dysregulation of inflammatory genes and genes with oncogenic potential (CCL2, IL-6, ORM1, TNAIFP6, SFRP4, SPARC). eSFPCOS and eSFCtrl showed upregulation of inflammatory genes (C4A/B, CCL2, ICAM1, TNFAIP3). Similarly, in eMSCPCOS vs. eMSCCtrl the most upregulated genes were related to inflammation and cancer (IL-8, ICAM1, SPRR3, LCN2). IHC scoring showed increased expression of CCL2 in eEPPCOS and eSFPCOS compared to eEPCtrl and eSFCtrl and IL-6 in eEPPCOS compared to eEPCtrl.    Conclusions: Isolated endometrial cell populations in women with PCOS showed altered gene expression revealing inflammation and pro-oncogenic changes, independent of BMI, especially in eEPPCOS and eMSCPCOS, compared to controls. The study reveals an endometrial  “disease phenotype” in women with PCOS with potential negative effects on endometrial function and long-term health.

Celecoxib induced transcriptional alterations in gastric cancer cells

Celecoxib is recognized to have chemopreventive and anti-cancer property beyond its anti-inflammatory function. Celelcoxib treatment in AGS cells down regulates Wnt/β-catenin, STAT3, RXR and ERK/MAPK signaling pathways. Transcriptional profiling after celecoxib shows differential regulation of oncogenic pathway regulated genes.

Expression data from wildtype, MIST1-null, and induced MIST1 Mus musculus pancreata

Although early developmental processes involve cell fate decisions that define the body axes and establish progenitor cell pools, development does not cease once cells are specified.  Instead, most cells undergo specific maturation events where changes in the cell transcriptome ensure that the proper gene products are expressed to carry out unique physiological functions. Pancreatic acinar cells mature post-natally to handle an extensive protein synthetic load, establsih organized apical-basal polarity for zymogen granule trafficking, and assemble gap-junctions to perimt efficient cell-cell communication. Despite significant progress in defining transcriptional networks that control initial acinar cell specification and differentiation decisions, little is know regarding the role of transcription factors in the specification and maintenance of maturation events. One candidate maturation effector is MIST1, a secretory cell-restricted transcription factor that has been implicated in controlling regulated exocytosis events in a number of cell types. Embryonic knock-out of MIST1 generates acinar cells that fail to establish an apical-basal organization, fail to properly localize zymogen granule and fail to communicate intra-cellularly, making the exocrine organ highly suceptible to pancreatic diseases.
In an effort to identify the gene expression differences responsible for MIST1 regulating mature acinar properties. We generated a tamoxifen-inducible mouse model where MIST1 expression could be activated in vivoand performed gene expression arrays on wildtype, MIST1-null, and induced MIST1 pancreatic RNA.

Functional collaboration of the meningioma 1 (MN1) oncogene with MLL-fusions in pediatric leukemia

Expression of meningioma 1 (MN1) has been proposed to be a negative prognostic molecular marker in adult AML with normal cytogenetics, however its role in pediatric leukemia is unknown. We found elevated MN1 expression in 53 of 88 pediatric leukemia cases: significant amounts of MN1 were found in immature B-cell ALL and most cases of infant leukemia but no MN1 expression was detected in T-cell acute lymphoblastic leukemia (T-ALL). Interestingly 17 of 19 cases harboring MLL-X fusions showed also elevated MN1 expression. Lentiviral siRNA mediated MN1 knock-down resulted in cell cycle arrest and impaired clonogenic growth of 3 MLL-X-positive human leukemia cell lines overexpressing MN1 (THP-1, RS4;11, MOLM13). In a mouse MLL/ENL-induced leukemia MN1 overexpression resulted from retroviral provirus insertion. Strikingly co-expression of MN1 with MLL/ENL resulted in significantly reduced latency for induction of an AML phenotype in mice suggesting functional cooperation. MN1 overexpression in MLL/ENL-carrying cells resulted in expansion of the L-GMP population and facilitated disease induction in secondary recipients. Gene expression profiling allowed to define a number of potential MN1 hematopoietic targets. Up-regulation of CD34, FLT3, HLF, or DLK1 was validated in bone marrow transiently overexpressing MN1, in MN1-induced mouse leukemias, as well as in some cases of pediatric leukemias overexpressing MN1. Taken together, our work suggests that MN1 overexpression is essential for growth of leukemic cells, and that MN1 can act as a cooperating oncogene with MLL-X fusion genes most probably through modification of a distinct gene expression program that leads to expansion of a leukemia initiating cell population.

Nasopharyngeal carcinoma is an Epstein-Barr virus-associated epithelial cancer with high prevalence in Southeast Asia. mRNA expression levels were measured for essentially all human genes and all latent Epstein-Barr virus (EBV) genes in nasopharyngeal carcinoma tissue samples and normal nasopharyngeal tissues. Data were analyzed for differential gene expression between tumor and normal tissue and  for correlations with levels of viral gene expression. Primary publications: Sengupta et al, 2006, Cancer Research 66(16): 7999-8006. Dodd et al, 2006, Cancer Epidemiology, Biomarkers & Prevention 15(11): 2216-2225.
In subsequent studies using the same set of tissue samples, microRNA levels were measured in tumors and normal tissues and analyzed for correlations with differential target gene expression (Sengupta et al, 2008, Proc. Nat. Acad. Sci. USA 105: 5874-5878.)

Expression data from Pseudomonas aeruginosa exposed airway epithelium from C57Bl6 and MMP-7 and MMP-10 deficient mice.

Airway epithelium is the initial point of host-pathogen interaction in Pseudomonas aeruginosa infection, an important pathogen in cystic fibrosis and nosocomial pneumonia. We used global gene expression analysis to determine airway epithelial transcriptional responses dependent on matrilysin (MMP-7) and stromelysin-2 (MMP-10), two matrix metalloproteinases induced by acute P. aeruginosa pulmonary infection. Extraction of Differential Gene Expression (EDGE) analysis of gene expression changes in P. aeruginosa infected organotypic tracheal epithelial cell cultures from wildtype, Mmp7-/-, and Mmp10-/- mice identified 2,089 matrilysin-dependent and 1,628 stromelysin-2-dependent genes that were differentially expressed. Key node network analysis showed that these MMPs controlled distinct gene expression programs involved in proliferation, cell death, immune responses, and signal transduction, among other host defense processes. Our results demonstrate discrete roles for these MMPs in regulating epithelial responses to pseudomonas infection and show that a global genomics strategy can be used to assess MMP function.
Keywords: time course

Transcriptional response of rat cerebrocortical tissue following acute exposure to permethrin or deltamethrin.

Pyrethroids are neurotoxicants that disrupt nervous system function by interacting with a variety of membrane bound ion channels on neuronal plasma membranes.  This study is designed to investigate the transcriptional events downstream of pyrethroid-induced disruption of nervous system excitability.  Adult, male Long-Evans rats were orally dosed in vivo with a single dose of either permethrin (1, 10, or 100 mg/kg) or deltamethrin (0.3, 1, 3 mg/kg) at levels that produce only modest behaviroal effects in the whole animal (Wolansky et al. 2006).  Transcriptional profiles were obtained from frontal cerebrocortical tissue 6 hours after acute exposure.  The primary goals were 1) to identify dose-responsive biomarkers of effect for pyrethroids and 2) identify sensitive intracellular signaling or metabolic pathways sensitive to pyrethroid compounds.
Keywords: dose response

Expression data from conditional Klf5 knockout Pten-null mouse prostates

KLF5 is a basic transcription factor that regulates multiple biological processes, but its function in tumorigenesis appears contradictory in the current literature, with some studies showing tumor suppressor activity and others showing tumor promoting activity. In this study, we examined the function of Klf5 in prostatic tumorigenesis using mice with prostate specific deletion of Klf5 and Pten, both of which are frequently deleted in human prostate cancer. Histological and molecular analyses demonstrated that when one Pten allele was deleted, which causes mouse intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth and caused more severe morphological and molecular alterations, and homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Klf5 deletion clearly promoted tumorigenesis in luminal cells, it actually diminished the numbers of Ck5-positive basal cells in the Pten-null tumors. Klf5 deletion also increased the cell proliferation rate in tumors with Pten deletion, which involved extensive activation of the PI3K/AKT and MAPK mitogenic signaling pathways and inactivation of the p15 cell cycle inhibitor. Global gene expression and pathway analyses demonstrated that multiple mechanisms could be responsible for the tumor promoting effect of Klf5 deletion,
We used microarrays to detail the global programme of gene expression of Klf5-wildtype and Klf5-null mouse dorsal prostates under Pten-null context to figure out the differential expression profiling underlying tumorigenesis

Microarray Analysis of West Nile Virus infected Human Retinal Pigment Epithelium

Low-level infection is believed to play a role in the degradation of the outer blood retinal barrier, which is composed of retinal pigment epithelial (RPE) cells.
By investigating immunopathogenic West nile virus (WNV) infected RPE via microarray, we sought to find key genes involved in a low-level viral infection, which are vital for normal immune responses.

Gene Expression Analysis of ARC (NSC 188491) Treated  MCF7 cells

ARC (NSC 188491, SMA-491), 4-amino-6-hydrazino-7-beta-d-ribofuranosyl-7H-pyrrolo-(2,3-d)-pyrimidine-5-carboxamide, is a nucleoside analog with profound in vitro anti-cancer activity. First identified in a high-throughput screen for inhibitors of p21 mRNA expression, subsequent experiments showed that ARC also repressed expression of hdm2 and survivin, leading to its classification as a global inhibitor of transcription 1. The following Hu U133 plus 2.0 arrays represent single time point (24 hour) gene expression analysis of transcripts altered by ARC treatment. Arrays for the other compounds (sangivamycin and doxorubicin) are included as comparators.

Gene expression data from precision cut human liver slices treated to diclofenac

Drug-induced liver injury (DILI) is an important clinical problem. Here we used a genomics approach to establish the critical drug-induced toxicity pathways that act in synergy with the pro-inflammatory cytokine tumor necrosis factor  (TNF) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and Nrf2 antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ~80 DILI compounds in primary human hepatocytes. The ER stress was primarily related to PERK and ATF4 activation and subsequent expression of CHOP, which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision cut human liver slices. Targeted RNA interference studies revealed that while ER stress signaling through IRE1α and ATF6 acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNF-induced apoptosis. While inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNF cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNF-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing towards caspase-8-dependent TNF induced apoptosis.

Expression data from whole retina of 8-week old db/db diabetic mice and lean littermates

Diabetic retinopathy is one of the leading causes of blindness in diabetic patients. Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy, but the underlying causes of neuronal loss are unknown.
To unravel potential mechanisms underlying early retinal neurodegeneration in diabetic retinopathy, a gene expression profiling study was undertaken to compare the gene expression in retinas of 8-week db/db diabetic mice with that of lean non-diabetic littermates.

Over-expression of either MECP2_e1 or MECP2_e2 in neuronally differentiated cells (SK-N-SH)

Aim: to detect genes that are differentially transcribed in neuronal cells(SK-N-SH) over-expressing either of the two MECP2 isoforms, MECP2_e1 or MECP2_e2.
Methods: the human neuroblastoma cell line SK-N-SH was stably infected by lentiviral vectors over-expressing MECP2_e1, MECP2_e2, or eGFP, and were then differentiated into neurons. RNA extracted, and used for gene expression microarray analysis

Ectopic cerebellar cell migration causes maldevelopment of Purkinje cells and abnormal motor behaviour in Cxcr4 null mice

Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion

Cerebellum from post-natal day 11 L1 knockout mice on the 129Sv background were compared to wild type littermates.  The original goal of the study was to determine if there was compensation from other L1 family members or alterations in cell survival or apoptosis.  Interestingly no major changes were detected in those families or pathways.

Compensated and decompensated hypertrophy after monocrotaline injection

14 days after a single subcutaneous monocrotaline (MCT) injection we isolated the left and right ventricles from wistar rats.

Factor1: Comparison between control (CON), compensated hypertrophy (HYP), and decompensated hypertrophy (CHF).
Factor2: Comparison between left ventricle (LV) and right ventricle (RV).

Comparison of severely emphysematous tissue removed at lung volume reduction surgery to that of normal or mildly emphysematous lung tissue resected from smokers with nodules suspicious for lung cancer.
Data obtained from the 18 patients with severe emphysema and 12 patients with mild/no emphysema.
Research may provide insights into the pathogenetic mechanisms involved in chronic obstructive pulmonary disease (COPD).

Vastus lateralis biopsies from healthy trained and sedentary males

Needle biopsies were obtained from the vastus lateralis muscle of 6 healthy, sedentary, 67±2.5 year-old males before and after 3 months of training.

Hypothalamic gene expression profile indicates a reduction in G-protein signaling in the wfs1 mutant mice

WFS1 gene is coding protein with unknown function but its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study we aimed to find the functional networks influenced by the Wfs1 deficiency in the hypothalamus. We performed gene expression profiling (Mouse Gene 1.0 ST Arrays) in Wfs1 deficient mice (ko). Modified t-statistics was used for comparison of groups (wt vs ko). Functional annotation of the alterations in RNA levels was performed with Ingenuity Pathway Analysis. 305 genes were differentially expressed with nominal p-value less than 0.01. FDR adjusted p-values were significant (0.007) only for two genes – C4b (t=9.66) and Wfs1 (t=-9.03). However, several genes related to the G-protein signalling were very close to the FDR adjusted significance. For instance, Rgs4 (regulator of G-protein signalling 4) was down-regulated (-0.34, t=-5.4) in Wfs1 deficient mice. Changes in Rgs4 and C4B expression were confirmed by QRT-PCR. In humans, Rgs4 is in the locus for bipolar disease (BPD) and its expression is down-regulated in BPD. C4b is the gene related to the neurodegenerative diseases. In conclusion, hypothalamic gene expression profiling indicates alterations in some functionally relevant molecular pathways explaining the clinical syndrome in the Wolfram syndrome patients.

Microarray analysis of WT and Notch2-deficient classical DC subsets

Defense against attaching and effacing (A/E) bacteria requires the sequential generation of IL-23 and IL-22 to induce protective mucosal responses. While the critical source of IL-22 has been identified as CD4+ and Nkp46+ innate lymphoid cells (ILCs), the precise source of IL-23 is unclear. Here, we use genetic techniques to deplete specific classical dendritic cell (cDC) subsets and analyze immunity to the A/E pathogen Citrobacter rodentium. We find that Zbtb46+ cDCs, and specifically Notch2-dependent intestinal CD11b+ cDCs, but not Batf3-dependent CD103+ cDCs, are required for IL-23 production and immunity against C. rodentium. Notch2 controls cDC differentiation at a terminal step mediated by lymphotoxin signaling. Importantly, these results provide the first demonstration of a non-redundant function of CD11b+ cDCs in vivo.

Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line

Many heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the of toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays.
We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells.

Background.  The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells.  We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells.  CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.  Therefore, we propose CREB to be a potential target for therapy.  To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line.
Results.
By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes.  We performed extensive analyses on the top genes in this list as high confidence CREB targets.  We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes.  Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages.  The transcription factor Elk-1 was upregulated in response to CREB deletion.
Conclusions.
We have identified a high confidence list of CREB targets in K562 cells.  These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia.  In particular, we speculate that the regulation of histone genes may play an important role in this process, by possibly altering the regulation of DNA replication during the cell cycle.

Target genes of the transcription factors HNF1beta and HNF1alpha in the human embryonic kidney cell line HEK293

Hepatocyte nuclear factor 1beta (HNF1beta, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1beta overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1beta target genes whose activity may be deregulated in human patients we established a human embryonic kidney cell line (HEK293) expressing HNF1beta conditionally. Using Flp recombinase we introduced wildtype or mutated HNF1beta at a defined chromosomal position allowing a most reproducible induction of the HNF1beta derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1beta regulated genes. By an identical approach we identified that the closely related transcription factor HNF1alpha (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1beta target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1beta target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1beta overexpression in the ovarian cancer participates in the altered expression pattern
Keywords: cnditional expression

Profiling gene expression in HeLa cells following TFEB transient transfection

Total RNA samples from three biological replicates in which TFEB was transiently overexpressed in HeLa cells by transfection using a pcDNA3 vector. As negative control, we used total RNA samples from HeLa cells transfected with an empty pcDNA3 vector.

Glucocorticoid-Dependent Hippocampal Transcriptome in Male Rats: Pathway-Specific Alterations with Aging

Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the longstanding hypothesis that chronic GC exposure promotes brain aging/Alzheimer's disease. Here, we adrenalectomized male F344 rats at 15-months-of-age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid-receptor (GR)-activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between Intermediate- and Low-CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT-upregulated genes included learning/plasticity, differentiation, glucose metabolism and cholesterol biosynthesis, whereas processes overrepresented by CORT-downregulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC-transcriptome with a previously-defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same-direction, the majority were shifted in opposite directions by CORT and aging (e.g., glial inflammatory genes downregulated by CORT are upregulated with aging). These results contradict the hypothesis that GCs simply promote brain aging, and also suggest that the opposite-direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways while GC overstimulation develops in others, together generating much of the brain aging phenotype.

Recent work has shown that cytotoxic T cells play a central role in immune-mediated control of cancers1-3, and monoclonal antibodies that target inhibitory receptors on T cells can induce significant clinical benefit in patients despite advanced disease4-6. However, many of the regulatory pathways that result in loss of T cell function within immunosuppressive tumors remain unknown. Here we show that such regulatory mechanisms can be systematically discovered in vivo in the tumor microenvironment. We devised a pool shRNA screening approach aimed at identifying genes that block the function of tumor-infiltrating CD8 T cells. We postulated that shRNAs targeting key inhibitors would enable robust T cell infiltration and proliferation in tumors, despite multiple inhibitory signals. Candidate shRNAs were discovered by transfer of shRNA-transduced T cells into tumor-bearing mice, followed by deep sequencing to quantify the representation of all hairpins in tumors and lymphoid organs. A subset of shRNAs induced T cell accumulation in tumors but not the spleen, demonstrating feasibility of discovering shRNAs with differential action across tissues. One of the targets was Ppp2r2d, a regulatory subunit of the family of PP2A phosphatases7. Control shRNA-transduced T cells underwent apoptosis upon recognition of melanoma cells, while Ppp2r2d shRNA-transduced T cells accumulated in tumors due to enhanced proliferation and reduced apoptosis. Ppp2r2d shRNAexpressing T cells also significantly delayed tumor growth. This in vivo approach has wide applications to dissect complex immune functions in relevant tissue microenvironments.

Imatinib and Nilotinib induced molecular changes in Philadelphia chromosome-positive Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a malignant stem cell disease characterized by a reciprocal translocation between chromosome 9 and 22. The selective bcr-abl tyrosine-kinase inhibitor Imatinib has become the therapy of choice for patients with newly diagnosed CML including those previously considered candidates for allogeneic haematopoietic stem cell transplantation. The tyrosine-kinase inhibitor Nilotinib is a derivate of Imatinib with higher potency.
To examine the molecular and functional effects of Nilotinib and Imatinib in chronic myelogenous leukemia, we performed gene expression and functional analyses in K562 cells following treatment with the two tyrosine kinase inhibitors.

The unique cellular state of embryonic stem cells is maintained by pluripotency-associated transcription factors such as OCT4. The transcriptional regulatory circuitries between human and mouse pluripotent stem cells exhibit notable differences. Transposable elements have altered the pluripotency network by contributing new cis-regulatory elements to mammalian genomes. In our study, we found that the expression of HERVH is enriched in human embryonic stem cells (hESCs) and depletion of HERVH in hESCs induced differentiation. To confirm the differentiation phenotype and identify potential downstream target genes of HERVH, we conducted the microarray analysis on hESCs treated with control shRNA against Luciferase and shRNA against HERVH.

type I diabetes;cyclophosphamide;BDC2.5;NOD;pancreas;islets;time-course

Progression to Islet Destruction in a Cyclophosphamide-Induced Transgenic Model

BDC2.5/NOD mice were treated with cyclophosphamide to induce type 1 diabetes.  Their pancreatic islets were analyzed before treatment (Day 0) and as treatment progressed (Days 1 through 3)
Keywords = type I diabetes

Effective treatment of psoriasis with etanercept is linked to suppression of IL17 signaling, not immediate response TNF

The success of TNF inhibitors for treatment of psoriasis and other inflammatory diseases was previously attributed to blockade of innate immunity.  In a clinical trial using etanercept TNF blocking agent to treat psoriasis vulgaris, we used affymetrix gene arrays to analyze broad gene profiles in lesional skin at multiple timepoints during drug treatment (baseline, and weeks 1, 2, 4 and 12) compared to non-lesional skin.  This analysis created a temporal model of TNF-dependent gene regulation that informs molecular mechanisms of TNF-mediated inflammation.  We identified four gene clusters that were differentially down-modulated during etanercept treatment: the cluster down-regulated most rapidly contained mostly dendritic cell activation genes.  Culturing human keratinocytes with TNF, IFNg and IL-17 generated a list of keratinocyte genes regulated by each cytokine.  The IL-17 pathway genes were strongly down-modulated early, whereas IFNg pathway genes were not down-modulated until final disease resolution at week 12.  Finally, we show that TNF blockade rapidly inhibits IL-12/IL-23 p40 subunit expression, and that p40 neutralization inhibits psoriatic dermal émigré-mediated Th17 polarization.  We hypothesize that etanercept inhibits myeloid dendritic cell production of IL-23, a Th17 survival cytokine, resulting in rapid downregulation of IL-17 pathway genes.  This data links effects of TNF blockade on the innate immune system with the adaptive immune system.

2102Ep;Agilent oligo microarrays;carcinoma in situ;choriocarcinoma;development;developmental biology;differenciation;embryogenesis;embryonal carcinoma;homo sapiens;human;human development;intratubular germ cell tumor;nonseminoma;NTera2;pluripotency;pluripotent;retinoic acid;seminoma;teratocarcinoma;teratoma;testis;testicular germ cell tumor;testicular neoplasm;totipotency;totipotent;undifferentiated;universal human reference RNA (Stratagene);yolk sac tumor;other

Normal, premalignant and various histological subtypes of testicular germ cell tumor (TGCT) tissues were hybridized against Universal Human Reference RNA (Stratagene) onto Agilent 60mer oligo microarrays (GEO accession no GPL885). In vitro time series of two TGCT cell lines, NTERA2 and 2102Ep, treated with retinoic acid for 0, 3, and 7 days were also included. The data set (30 hybridizations) is particularly useful for comparisons between various histological subtypes of TGCT versus each other or versus normal testis.
Keywords = 2102Ep

Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation

Recurrent somatic mutations in TET2 and in other genes that regulate the epigenetic state have been identified in patients with myeloid malignancies and in other cancers.  However, the in vivo effects of Tet2 loss have not been delineated.   We report here that Tet2 loss leads to increased stem-cell self-renewal and to progressive stem cell expansion. Consistent with human mutational data, Tet2 loss leads to myeloproliferation in vivo, notable for splenomegaly and monocytic proliferation. In addition, haploinsufficiency for Tet2 confers increased self-renewal and myeloproliferation, suggesting that the monoallelic TET2 mutations found in most TET2-mutant leukemia patients contribute to myeloid transformation.  This work demonstrates that absent or reduced Tet2 function leads to enhanced stem cell function in vivo and to myeloid transformation.
These studies show that a ubiquitin ligase-substrate pair can orchestrate the molecular program of HSC differentitiation

Identification of Myelin-gene Regulatory Factor as a Critical Transcriptional Regulator Required for CNS Myelination

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named Myelin-gene Regulatory Factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, pre-myelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities, and die due to seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.
We used microarrays to compare cultured oligodendrocytes (differentiated in vitro for 4 days) from MRF conditional knockouts and control litteramates to look at the effects of MRF deficiency on myelin gene expression. Mouse OPCs grown in vitro in the presence of PDGF serve as a baseline for gene expression prior to differentiation.

Rosiglitazone Treatment Reduces Diabetic Neuropathy in STZ treated DBA/2J mice

Diabetic Neuropathy (DN) is a common complication of diabetes.  Currently, there is no drug treatment to prevent or slow the development of DN.  Rosiglitazone (Rosi) is a potent insulin sensitizer and may also slow the development of DN by a mechanism independent of its effect on hyperglycemia.  A two by two design was used to test the effect of Rosi treatment on the development of DN.  Streptozotocin-induced diabetic DBA/2J mice were treated with Rosi.  DN and oxidative stress were quantified, and gene expression was profiled using the Affymetrix Mouse Genome 430 2.0 microarray platform.  An informatics approach identified key regulatory elements activated by Rosi.  Diabetic DBA/2J mice developed severe hyperglycemia, DN and elevated oxidative stress.  Rosi treatment did not affect hyperglycemia but did reduce oxidative stress and prevented development of thermal hypoalgesia.  Two novel transcription factor binding modules were identified that may control genes correlated to changes in DN following Rosi treatment: SP1F_ZBPF and EGRF_EGRF.  Rosi treatment reduced oxidative stress and DN independent of its insulin sensitizing effects. Gene expression profiling identified two novel targets activated by Rosi treatment.  These targets may be useful in designing drugs with the same efficacy as Rosi in treating DN but with fewer undesirable effects.
Keywords: disease and treatment analysis

Analysis of HeLa cells following depletion of BRCA1 tumor supressor using RNAi against BRCA1. Results provide insight into the molecular mechanisms underlying loss of the BRCA1 function.

Gene expression profiles during in vivo human rhinovirus infection: insights into the host response.

RATIONALE: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases. OBJECTIVES: To define changes in gene expression profiles during in vivo rhinovirus infections. METHODS: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections. MEASUREMENTS AND MAIN RESULTS: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells. CONCLUSIONS: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.

DEHP activation of PPAR(alpha) and CAR regulartory pathway in mouse liver

Characterization of Peroxisome Proliferator-Activated Receptor alpha (PPAR(alpha)) - Independent Effects of PPAR(alpha) Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Activates the Constitutive Activated Receptor
data files in this series indicate the involvement of PPAR(alpha) and CAR regulatory pathway after DEHP treatment.

Comparative analysis of gene expression in Fra-1+/+ and Fra-1-/- mice lung

We hypothesized that gene expression in lungs of Fra-1+/+ and Fra-1-/- mice are divergent thus contributing fibrosis. More specifically, Fra-1-/- mice are increased susceptible to fibrosis. In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between Fra-1+/+ and Fra-1-/- mice at early time point.

Bronchial Epithelial Cells were isolated processed as described (Chu et al., 2002 and Zhao et al., 2011). The objective of the study was to identify differentially expressed genes between normal control (NC), mild-moderate asthmatic (MMA) and severe asthmatic (SA) patients.

Expression data of cystic renal epithelial tissue from mice deficient for fumarate hydratase.

Fumarate hydratase (FH) mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC). We have conditionally inactivated the murine ortholog (Fh1) in renal tubular epithelial cells in order to generate an in vivo model of HLRCC. Fh1 knockout mice recapitulates important aspects of HLRCC including the development of renal cysts that overexpress hypoxia inducible factor alpha (Hifa) and Hif-target genes.
We used microarrays to detail the global programme of gene  expression underlying cyst development in Fh1 knockout mice and identified distinct classes of up-regulated genes during this process.

YAP conveys an invasive phenotype to clear cell renal cell carcinoma cell lines via endothelin-2

To determine the biological mechanisms underlying the oncogenic properties of YAP in ccRCC the human ccRCC cell line MZ1774 was transduced with lentivirus containing a shRNA-cassette targeting YAP-mRNA. Expression profiles of MZ1774 YAP knockdown cells were compared to mock-transduced control cells.

Celecoxib pre-treatment in human colorectal adenocarcinoma patients.

Pharmacological inhibition of cyclooxygenase-2 (COX-2) is being explored as a chemotherapeutic option because COX-2 protein expression is often elevated in many cancers.  Cancer cells treated with COX-2 inhibitors, such as the selective COX-2 inhibitor celecoxib, show growth inhibition and the induction of apoptosis, through alterations in inflammatory processes, angiogenesis, cell adhesion and transforming growth factor-β signaling.  This study was conducted to determine if the same processes are relevant to celecoxib’s effects on human colorectal adenocarcinomas treated in vivo.  A cohort of 23 patients with primary colorectal adenocarcinomas was randomized to receive a 7-day course of celecoxib (400 mg b.i.d.) or no drug prior to surgical resection.  Gene expression profiling was performed on resected adenocarcinomas from patients with and without celecoxib pre-treatment.  Using fold change (>1.5) and p-value (<0.05) cut-offs, 190 genes were differentially expressed between adenocarcinomas from patients receiving celecoxib and those that did not.  Of the differentially expressed genes, multiple genes involved in cellular lipid and glutathione metabolism showed decreased expression levels in celecoxib pre-treated samples; changes associated with diminished cellular proliferation.  Other observed gene expression changes consistent with reduced proliferation include: altered expression of genes involved in cell adhesion (including collagen, laminin, von Willebrand factor and tenascin C), increased expression of inflammatory modulators (including inerleukin-6, S100 calcium binding protein A8, and several chemokines) and decreased expression of the pro-angiogenic gene, angiogenin.  Celecoxib pre-treatment for 7 days in vivo is associated with alterations in colorectal adenocarcinoma gene expression which are suggestive of diminished cellular proliferation.
Keywords: treatment outcome

All experiment was done according to the Affymetrix manufacturer’s protocol.  The resulting HGF gingiva expression profile(Hereditary gingival fibromatosis patient Gingiva replicate1 and replicate 2)was compared to normal gingiva control(Normal Gingiva replicate1 and replicate2). The data were collected and analyzed by GCOS 1.2 and GeneSpring 7.2 1-way T test.
Keywords: tissue specific expression profile

PGC1a is a transcriptional coactivator that regulates energy metabolism. PGC1a is highly expressed in a subset of melanoma tumors and cell lines. We generated gene-expression profile of control and PGC1alpha depleted A375P melanoma cells, a melanoma cell line that expresses very high levels of PGC1a to investigate the role of this gene in melanoma.

Epithelial organs including the lung are known to possess regenerative abilities through activation of endogenous stem cell populations but the molecular pathways regulating stem cell expansion and regeneration are not well understood.  Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation in Gata6 null lung epithelium.  This expansion of BASCs is the result of a dramatic increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the non-canonical Wnt receptor Fzd2 is down-regulated in Gata6 mutants and increased Fzd2 or decreased β-catenin expression rescues, in part, the lung epithelial defects in Gata6 mutants.  During lung epithelial regeneration, we show that canonical Wnt signaling is activated in the niche containing BASCs and forced activation of Wnt signaling leads to a dramatic increase in BASC numbers.  Moreover, Gata6 is required for proper lung epithelial regeneration and postnatal loss of Gata6 leads to increased BASC expansion and decreased differentiation.  Together, these data demonstrate that Gata6 regulated Wnt signaling controls the balance between stem/progenitor expansion and epithelial differentiation required for both lung development and regeneration.
Keywords: gene targets in knockout mouse model

Overexpression of high mobility group AT-hook 2 (HMGA2) associated with truncations of its 3’ untranslated region (UTR) with let-7 micro RNA-complementary sequences have been identified in patients with paroxysmal nocturnal hemoglobinuria (PNH). Here, we generated transgenic mice (∆Hmga2 mice) with a 3’UTR-trncated Hmga2 cDNA that overexpress Hmga2 mRNA and protein in hematopoietic organs. ∆Hmga2 mice showed proliferative hematopoiesis that mimicked a myeloproliferative neoplasm (MPN)-like phenotype with increased numbers of all lineages of peripheral blood cells, hypercellular bone marrow (BM), splenomegaly with extramedullary erythropoiesis, and erythropoietin-independent erythroid colony formation compared to wild-type mice. ∆Hmga2 BM-derived cells took over most of hematopoiesis in competitive repopulations during serial BM transplants. When we bred mice with circulating PNH cells (Piga- mice) with ∆Hmga2 mice, the lack of GPI-linked proteins did not add an additional clonal advantage to the ∆Hmga2+ cells. In summary, our results showed that the overexpression of a 3’UTR-truncated Hmga2 leads to a proliferative hematopoiesis with clonal advantage, which may explain clonal expansion in PNH or MPN at the level of HSC.

Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular Mortality

The type 2 diabetes medication, rosiglitazone, has come under scrutiny for possibly increasing the risk of cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiac transcriptional profiling  of a murine model of type 2 diabetes, the C57BL/KLS-leprdb/leprdb (db/db) mouse. We compared cardiac gene expression profiles from three groups: untreated db/db mice (db-c), db/db mice after rosiglitazone treatment (db-t), and non-diabetic db/+ mice.

Mx-Cre mediated deletion of SRF in murine hematopoietic LSK (Lin-Sca1+c-Kit+) cells

Analysis of hematopoietic LSK(Lin-Sca1+c-Kit+) cells lacking the Serum response factor (SRF) gene. Results provide insight into the role of SRF in regulating genetic programs important for hematopoietic stem cell development

TNF time course series of HelA tet off cells cultured in presence or absence of Dox

TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks.  Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown.  In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes.  A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed.

Methods:
Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23).  HeLa Tet-Off were grown in medium containing 90 % Dulbecco’s Modified Eagle’s Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2.   For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal.  Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest.

Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer’s recommendations and washed using both non-stringent (1 M NaCl, 25 °C) and stringent (1 M NaCl, 50  °C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final).  Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc).  For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA;  each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control.  The the Absolute Call [e.g., the gene is detected (“present”) or not (“absent”)] and the Signal Intensity (measure of mRNA abundance) is determined.

Four independent experiments were performed identically including control (0 h ), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium.
These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other.

Gene expression profiling of T47D breast cancer cells following progestin administration in presence or absence of Telapristone

Analysis of the effect of progesterone blockade at the gene expression level.  The hypothesis tested in the present study is that elapristone (CDB4124), an antiprogestin, will downregulate genes that are stimulated by R5020, a synthetic progestin, in the T47D breast cancer cell line.

Expression data of primary melanocytes from aryl hydrocarbon deficient mice and corresponding wild-type C57BL/6 mice

[original Title] Comparison of expression data of primary murine melanocytes from aryl hydrocarbon deficient mice and corresponding wild-type C57BL/6 mice

Melanin is produced exclusively by melanocytes and melanogenesis is the vital response to protect skin cells against Ultraviolet B (UVB)-induced DNA damage. The aryl hydrocarbon receptor (AhR) is a transcription factor, which may be involved in the physiological tanning response. Normal murine melanocytes express functional AhR. We tested gene expression in WT versus AhR-deficient mice primary murine melanocytes, isolated from the skin and cultivated for several passages.

Shifted Metabolic Bias in Livers of Mice Lacking Hepatocytic Thioredoxin Reductase-1 Protects Against Acetaminophen Toxicity

Genetic disruption of thioredoxin reductase 1 protects against acetaminophen (APAP) toxicity.
To determine the role of the thioredoxin system on xenobiotic metabolism we challeneged wildtype and txnrd1liver-null mice with acetaminophen.

Expression data from epidermal stem cells isolated from dorsal skin of P19 Per1-Venus mice and Bmal1 epidermal knockout mice

Epidermal stem cells ensure that skin homeostasis is maintained. In murine skin, epidermal stem cells cluster at specific niches where, under steady-state conditions, they undergo cycles of dormancy and activation1. When cellular replenishment is required, epidermal stem cells egress from the niche and proliferate for a limited number of times to subsequently feed into the differentiated compartment1-3. However, only a subset of stem cells becomes active during each round of morphogenesis, suggesting that stem cells coexist in heterogeneous responsive states within the same niche. Using a circadian clock fluorescent reporter mouse model, we show that the dormant epidermal stem cell niche contains two coexisting populations of stem cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. In dormant niches, the core molecular clock protein Bmal1 transcriptionally modulates the expression of stem cell regulatory genes, including modulators of Wnt and TGFb, to create two coexisting stem cell populations, one predisposed, and the other less prone, to activation. Unbalancing this equilibrium of epidermal stem cells, through conditional epidermal deletion of Bmal1, resulted in a long-term progressive accumulation of non-responsive stem cells, premature impairment of tissue self-renewal, and a significant reduction in the development of squamous cell carcinomas. Our results indicate that the molecular clock machinery fine-tunes the spatiotemporal behavior of epidermal stem cells within their niche, and that perturbation of this mechanism affects tissue homeostasis and the predisposition to neoplastic transformation. The goals of this study was to compare the transcriptome of epidermal stem cells according to their circadian rhythm phase. We isolated epidermal stem cells (bulge cells; alpha6bright/CD34+ population) from 19 days old Per1-Venus mice and separated them according to Venusbright (clock positive) and Venus dim (clock negative). The goals of this study was to compare the transcriptome of epidermal stem cells in which their circadian rhythm machinery has been perturbed by deleting the gene that encodes for Bmal1. We compared the transcriptomes of basal interfollicular epidermis cells (alpha6 integrin bright/CD34- cells) from the dorsal skin of 1 year old BmalKO mice and their respective control littermates. Each array corresponds to purified cells from approximately 5 mice.

Dysferlin deficiency modulates the proximal-distal muscle transcriptome

By comparing the transcriptome from proximal (quadriceps femoris, QF) and distal (tibialis anterior, TA)muscle groups in dysferlin deficient mouse muscle (the SJL mutation bred onto C57BL/10 to produces C57BL/10-SJL.Dysf) with proximal and distal muscle groups from control C57BL/10 mice of an equivalent age (3-weeks old, prior to the onset of overt pathology) we aim to address the issues of muscle selectivity in this this form of muscular dystrophy.

A novel approach to investigate tissue-specific trinucleotide repeat instability

In Huntington’s disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors.
Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue HD CAG instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability.
Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability.

Impact of IRF1 on transcriptome changes in Huh-7 human hepatoma cells and STAT1-deficient human fibroblasts

IRF1, and interferon-induced transcription factor, was found to have antiviral activity against a range of viruses when overexpressed. To determine which genes are associated with expression of IRF1, gene expression analysis was carried out in two different cell line expressing IRF1. The results show that more than 100 genes, many of which overlap with type I interferon-stimulated genes, are induced more than 3-fold by IRF1. A majority of the genes were induced in both cell types, while some genes were highly induced in a cell type-specific manner.

HMGA2/TET1/HOXA9 pathway regulates breast cancer growth and metastasis.

HMGA2, Tet1 and HOXA9 are all regulated human breast tumorigenesis. We use gene expression array analysis to identify and compare the genes regulated by HMGA2, Tet1 and/or HOXA9 in human breast cancer cells.

Comparison of bone-marrow mesenchymal stromal cells from multiple myeloma patients and healthy donors

It is now well established that bone marrow (BM) constitutes a microenvironment required for differentiation. Bone marrow mesenchymal stromal cells (BM-MSCs) strongly support MM cell growth, by producing a high level of Interleukin-6 (IL-6), a major MM cell growth factor. BM-MSCs also support osteoclastogenesis and angiogenesis. Previous studies have suggested that the direct (VLA-4, VCAM-1, CD44, VLA-5, LFA-1, syndecan-1,…) and indirect interactions (soluble factors) between MM plasma cells and BM-MSCs result in constitutive abnormalities in BM-MSCs. In particular, MM BM-MSCs express less CD106 and fibronectin and more DKK1, IL-1β and TNF-α as compared with normal BM-MSCs.  In order to gain a global view of the differences between BM-MSCs from MM patients and healthy donors, we used gene expression profiling to identify genes associated to the transformation of MM BM-MSCs.

Gene expression profiles of E14 ESCs treated with low levels of HDAC inhibitors for 16 hrs

Gene expression profiles of E14 embryonic stem cells (ESCs) before and after treatment with low levels of the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and sodium butyrate (NaBu).

Gene expression profiles of E14 ESCs treated with low levels of the HDAC inhibitor VPA for 4 hrs

Gene expression profiles of E14 embryonic stem cells (ESCs) before and after treatment with low levels of the histone deacetylase (HDAC) inhibitor valproic acid (VPA).

Gene expression profiling of primary mouse embryonic palatal mesenchymal cells in Tgfbr2 mutant mouse models

The overall goal of this project is to investigate the role of TGF-beta signaling in regulating the cellular metabolism of cranial neural crest (CNC) cells during palate development. Here, we conducted gene expression profiling of primary mouse embryonic palatal mesenchymal (MEPM) cells from wild type mice as well as those with a neural crest specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of cleft palate, which is among the most common congenital birth defects and observed in many syndromic conditions.

Expression data from TGF-beta treated Panc-1 pancreatic adenocarcinoma cell line

TGF-beta treatment of Panc-1 pancreatic adenocarcinoma cell line on Affymetrix HG_U133_plus_2 arrays; triplicate experiments.
The goal of the experiment is to profile temporal gene expression changes during TGF-beta-induced epithelial-mesenchymal transition (EMT). During EMT cancer cells lose their epithelial specifc proteins and gain mesenchymal proteins to acquire migratory and invasive phenotype essential for metastasis. Human Panc-1 pancreatic adenocarcinoma cell line was treated with 5 ng/mL TGF-beta for 48 h to induce EMT. The experiment was repeated 3 times. Samples were assayed using Affymetrix HG_U133_plus_2 arrays with 54675 probe-sets, using standard techniques.

Genome-wide expression profiling of Hic-5 knockdown in PshTert-AR human myofibroblasts

Identifying the effect of the co-regulator Hic-5 (TGFB1I1) on global androgen receptor transcriptional activity in PShTert-AR prostate fibroblast cells with view to further elucidating the broader biological role of Hic-5  on fibroblast spceific androgen signaling.
Knockdown of Hic5 for 48 hours in PShTert-AR cells significantly altered the androgen regulation of approximately 1347 genes. The effect of Hic-5 knockdown was to suppress androgen regulation of approximately 85% of those transcripts, by either reducing androgen mediated repression or activition of gene expression.

Defining the molecular response to trastuzumab, pertuzumab and combination therapy in ovarian cancer

The purpose of this study was to characterise the effects of trastuzumab and pertuzumab, either as single agents or as combination therapy on gene and protein expression in human ovarian cancer in vivo. Illumina BeadChips were used to profile the transcriptome after four days treatment of SKOV3 tumor xenografts. Although genes involved with HER2, MAP-kinase and p53 signaling pathways were commonly induced by all treatments, a greater number and variety of genes were differentially expressed by the complementary combination therapies compared to either drug on its own. The protein level of the CDK-inhibitors p21 and p27 were increased in response to both agents alone and further by the combination; pERK signaling was inhibited by all treatments; but only pertuzumab alone inhibited pAkt signaling. The expression of proliferation, apoptosis, cell division and cell cycle markers was distinct in a panel of primary ovarian cancer xenografts, suggesting heterogeneity of response in ovarian cancer and the need to establish biomarkers of response.
This first comprehensive study of the molecular response to trastuzumab, pertuzumab and combination therapy in vivo highlights that there are both common and distinct downstream effects to different HER2 antibodies and that pathways may be invoked more strongly or in a different manner by a combination of agents. Some of the in vivo results for ovarian tumors differ from previous in vitro studies in breast cancer cells, emphasizing that the molecular response to anti-cancer agents involves variable and complex disease-specific interactions of signaling mechanisms.

Human embryonic stem cells as a tool to study hepatocyte differentiation

The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluritpotent cells into cells with hepatocyte characteristics. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation toward a hepatocyte–like fate appeared to recapitulate many of the stages normally associated with the formation of hepatocytes in vivo. In the current study we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate i) that human ES cells express a number of mRNAs that characterize each stage in the differentiation process, ii) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses, and iii) that the nuclear hormone receptor HNF4a is essential for specification of human hepatic progenitor cells by establishing expression of the network of transcription factors that control hepatocyte cell fate.

Low-grade serous ovarian carcinomas are typically Ras-pathway mutated, TP53 wild-type, have limited chromosomal aberration, and are frequently associated with borderline tumors. By contrast, high-grade serous ovarian carcinoma lack Ras-pathway mutations, are invariably TP53 mutated, show widespread genomic change, and are commonly BRCA-pathway disrupted. We sought to identify differentially expressed genes between co-existing borderline and invasive components of serous carcinoma.

Whole genome profiling of fibroblasts from Diamond-Blackfan Anemia patients

Diamond-Blackfan Anemia (DBA) is a rare inherited red cell hypoplasia characterized by a defect in the maturation of erythroid progenitors and is in some cases associated to malformations. Patients have an increased risk of solid tumors. Mutations have been found  in several ribosomal protein (RP) genes. Studies in hematopoietic progenitors from patients show that the haploinsufficiency of an RP impairs rRNA processing and ribosome biogenesis. DBA lymphocytes and fibroblasts show reduced protein synthesis, and the latter display abnormal rRNA processing and impaired proliferation.
To evaluate the involvement of non-hematopoietic tissues in DBA, we have analyzed global gene expression in fibroblasts from DBA patients compared to healthy controls. Microarray expression profiling using Affymetrix GeneChip Human Genome U133A 2.0 Arrays revealed that 421 genes are statistically significantly differentially expressed in DBA patients' fibroblasts relative to controls. These genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes with roles in cell death, cancer and tissue development. Our results report for the first time into abnormal expression in a non-hematopoietic cell type in DBA, and support the hypothesis that DBA may be due to a defect in general or specific protein synthesis.

Resveratrol induces downregulation of DNA repair genes in MCF7 human breast cancer cells.

Breast cancer is a worlwide health problem. One of the factors involved in carcinogenesis is diet. Several studies indicate that resveratrol, a polyphenol present in diet, has several antitumor
properties. The purpose of the study is gain an insight of resveratrol effect in cancer by determining the gene expression profile of breast cancer cell lines treated with the compound.

Parkinson Phenotype in Aged PINK1-Deficient Mice Is Accompanied by Progressive Mitochondrial Dysfunction in Absence of Neurodegeneration

Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Thus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death. Transcriptome microarray data of Pink1-/- mouse brains in absence of a stressor, even at old age, show remarkably sparse dysregulations. See Gispert-S et al 2009 PLOS ONE.

Expression data from p27WT, p27CK and KO MEFs cells in quiescence

Low levels of the cell cycle regulator p27Kip1 are associated with a worse outcome in many tumor types. We report here a new regulatory role of p27Kip1 as a transcriptional regulator. In association with transcriptional factors such as ETS and E2F4 and co-repressors like p130, HDACs and mSin3A, p27 binds to promoters of multiple genes leading to their repression. The p27-target genes participate in RNA processing, translation, respiration and cell cycle. Remarkably, p27-target genes are over-expressed in different human tumors in tight association with a poor clinical prognosis. We also observed a clear correlation between low levels of p27 and over-expression of p27-target genes in tumors. Overall, our findings indicate new tumor suppressor roles of p271 as a transcriptional regulator of genes relevant for oncogenesis.

Clear cell sarcoma of the kidney (CCSK), the second most common renal tumor in children, poses significant diagnostic challenges.  No diagnostic positive markers are available, and the pathogenesis of CCSK remains an enigma.  To address these challenges, the gene expression patterns of fourteen CCSKs were compared to fifteen Wilms tumors (WT) and three fetal kidney samples using oligonucleotide arrays.

SK-MEL-2 cells treated with Ad-E2F-1, Ad-E2F-1 plus doxorubicn or Ad-LacZ plus doxorubicin

SK-MEL-2 cells treated with Ad-E2F-1 (MOI 2), Ad-E2F-1 (MOI 2)plus doxorubicin 0.1uM, or Ad-LacZ (MOI 2) plus doxorubicin 0.1uM
Keywords: ordered

Expression data from MCF7 cell line after silencing of Estrogen receptor

We propose the hypothesis that loss of estrogen receptor function which leads to endocrine resistance in breast cancer, also results in de-differentiation from an epithelial to a mesenchymal phenotype that is responsible for increased aggressiveness and metastatic propensity. siRNA mediated silencing of the estrogen receptor in MCF7 breast cancer cells resulted in estrogen/tamoxifen resistant cells (pII) with altered morphology, increased motility with rearrangement and switch from an actin to a vimentin based cytoskeleton, and ability to invade simulated components of the extracellular matrix. Phenotypic profiling using an Affymetrix Human Genome U133 plus 2.0 GeneChip indicated fold changes ≥ 3 in approximately 2500 identifiable unique sequences, with about 1270 of these being up-regulated in pII cells. Changes were associated with genes whose products are involved in cell motility, loss of cellular adhesion and interaction with the extracellular matrix. Selective analysis of the data also showed a shift from luminal to basal cell markers and increased expression of a wide spectrum of genes normally associated with mesenchymal characteristics, with consequent loss of epithelial specific markers. Over-expression of several peptide growth factors and their receptors are indicative of an increased contribution to the higher proliferative rates of pII cells as well as aiding their potential for metastatic activity. Signalling molecules that have been identified as key transcriptional drivers of epithelial to mesenchymal transition were also found to be elevated in pII cells. We suggest that these data support our hypothesis that induced loss of estrogen receptor in previously antiestrogen sensitive cells is a trigger for the concomitant loss of endocrine dependence and onset of a series of possibly parallel events that changes the cell from an epithelial to a mesenchymal type. Inhibition of this transition through targeting of specific mediators may be a useful supplementary strategy to circumvent the effects of loss of endocrine sensitivity.
We used microarrays to detail the global programme of gene expression underlying Epithelial to mesenchymal transition and identified distinct classes of regulated genes during this process.

Gene expression profiles of 39 human sarcoma samples (GSM 52571-GSM52609) and 15 control samples (GSM52556-GSM52570) were assessed using Affymetrix HG U133A oligonucleotide arrays.

siRNA-mediated inhibition compared to untreated cells and cells transfected with nonsense siRNA.
Loss of contact inhibition and anchorage-independent growth are hallmarks of cancer cells. In this context, frequent inactivation of the Hippo pathway and subsequent nuclear enrichment of the transcriptional coactivator yes-associated protein (YAP) uncouple cell proliferation and anti-apoptosis from contact inhibition, associated with uncontrolled tumor growth and tumor cell dissemination. However, general molecular mechanisms of tumor-supporting YAP activity remain unclear. In this study, we show that overexpression and nuclear accumulation of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells leads to an induction of the Notch pathways through transcriptional activation of the Notch ligand jagged-1 (Jag-1). This induction of Jag-1 strictly depends on binding of YAP to TEAD4 and does not rely on WNT/β-catenin pathway activity. Co-activation of YAP, TEAD4, Jag-1, and the Notch target gene Hes-1 was significantly higher in HCC from patients with poor prognosis. High-level expression and nuclear accumulation of YAP correlates with Jag-1/Notch activation not only in human HCC tissues, but also in colon and pancreatic cancer tissues. Thus, our data demonstrate that YAP-driven co-activation of the Jag-1/Notch pathway in part facilitates oncogenic properties of the oncogene YAP not only in HCC but also in different gastrointestinal malignancies.

Physiological defects associated with short hairpin RNA-mediated silencing of PGC-1-related coactivator (PRC)

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up regulated in proliferating cells. Here, we investigated its in vivo role by stably silencing PRC expression with two different short hairpin RNAs (shRNA#1 and shRNA#4) that were lentivirally introduced into U2OS cells. ShRNA#1 transductants exhibited nearly complete knockdown of PRC protein whereas shRNA#4 transductants expressed PRC protein at approximately 15 percent of the control level. Complete PRC silencing by shRNA#1 resulted in a severe inhibition of respiratory growth, reduced expression of respiratory protein subunits from complexes I, II, III and IV, markedly lower complex I and IV respiratory enzyme levels and diminished mitochondrial ATP production. Surprisingly, shRNA#1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA#4 transductants had normal respiratory subunit expression and a moderately diminished respiratory growth rate, both transductants showed markedly reduced growth on glucose accompanied by inhibition of G1/S cell cycle progression. Microarray analysis revealed striking overlaps in the genes affected by PRC silencing in the two transductants and the functional identities of these overlapping genes were consistent with the observed mitochondrial and cell growth phenotypes. The consistency between phenotype and PRC expression levels in the two independent transductant lines argues that the defects result from PRC silencing and not from off target effects. These results support a role for PRC in the integration of pathways directing mitochondrial respiratory function and cell growth.

Expression profiling of back skin of Get1 and control mice at e18.5

Defective permeability barrier is an important feature of many skin diseases and causes mortality in premature infants. To investigate the control of barrier formation, we characterized the epidermally expressed Grainyhead-like epithelial transactivator (Get-1)/Grhl3, a conserved mammalian homologue of Grainyhead, which plays important roles in cuticle development in Drosophila. Get-1 interacts with the LIM-only protein LMO4, which is co-expressed in the developing mammalian epidermis. The epidermis of Get-1(-/-) mice showed a severe barrier function defect associated with impaired differentiation of the epidermis, including defects of the stratum corneum, extracellular lipid composition and cell adhesion in the granular layer. The Get-1 mutation affects multiple genes linked to terminal differentiation and barrier function, including most genes of the epidermal differentiation complex. Get-1 therefore directly or indirectly regulates a broad array of epidermal differentiation genes encoding structural proteins, lipid metabolizing enzymes and cell adhesion molecules. Although deletion of the LMO4 gene had no overt consequences for epidermal development, the epidermal terminal differentiation defect in mice deleted for both Get-1 and LMO4 is much more severe than in Get-1(-/-) mice with striking impairment of stratum corneum formation. These findings indicate that the Get-1 and LMO4 genes interact functionally to regulate epidermal terminal differentiation.
Keywords: knockout and wildtype mice comparison

Global expression analysis of BAP1 knockdown in transfected 92.1 cells

Analysis of the effect that reduced BAP1 levels have on global gene expression.The hypothesis tested was that reduction in BAP1 levels would produce changes in gene expression similar to changes observed in class 2 uveal melanomas. Data provided insight into genes that are disrupted with reduced BAP1 levels.

Expression data from brain tissue of Rattus norvegicus treated with D-Serine

d-serine is naturally present throughout the human body.  It is also used as add-on therapy for treatment-refractory schizophrenia. d-Serine interacts with the strychnine-insensitive glycine binding site of NMDA receptor, and this interaction could lead to potentially toxic activity (i.e., excitotoxicity) in brain tissue.  The transcriptomic changes that occur in the brain after d-serine exposure have not been fully explored.
Affymetrix microarray technology was used to determine differential gene expression resulting from D-Serine exposure.

Gene expression profiling of L-540 Hodgkin lymphoma cell line after in vitro and in vivo treatment with perifosine in combination with sorafenib

Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice using tumor growth rates and survival as endpoints. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of cell death. In responsive cell lines, WB analysis showed that antiproliferative and pro-apototic events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. In addition, perifosine/sorafenib treatment had no effect on HDLM-2 nodules, but significantly reduced L-540 nodules with 50% tumor growth inhibition, compared to controls. CONCLUSIONS: Perifosine/sorafenib combination resulted in strong anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation of  perifosine/sorafenib combined-treatment in HL patients.

pool of thyroids from wild type, E7 and RET-PTC tg mice at 2, 6 and 10 months

The RET/PTC3 (RP3) fusion gene is the most frequent mutation found in radiation-induced papillary thyroid cancers (PTC). Several studies suggest that the RET/PTC rearrangement is an initiating event in tumorigenesis. E7 is an oncoprotein derived from the Human Papilllomavirus 16 (HPV16) responsible for most cervical carcinoma in women. We studied here the sequence of events leading to thyroid cancer in Tg-RP3 and Tg-E7 mice expressing the transgene exclusively in the thyroid under the control of thyroglobulin (Tg) promoter. Both transgenic mice develop thyroid hyperplasia followed by solid differentiated carcinoma in older animals. To understand the different steps leading to carcinoma, we analyzed thyroid gene expression in both strains at different ages (2, 6, 10 months) by microarray technology. Important biological processes were differentially regulated in the two tumor types. In E7 thyroids cell cycle was the most upregulated process; observation consistent with the huge size of these tumors. In RP3 thyroids immunity was the most significantly regulated process, as previously observed in microarray data on human PTC. Interestingly, other human PTC characteristics were also observed in RP3 but not in E7 mouse tumors: similar regulation of several human PTC markers, upregulation of many EGF-like growth factors and finally significant regulation of angiogenesis and extracellular matrix remodeling-related genes. In summary we showed that RP3 contrary to E7 mouse tumors share several important genotypic characteristics with human PTC, observation reinforcing the validity of this model to study human thyroid tumorigenesis.
Keywords: time course, genetic modification, disease state, cancer

CD3+ T cells derived from bone marrow or peripheral blood of aplastic anemia patients

CD3+ T cells derived from bone marrow aspirates and peripheral blood samples of newly diagnosed AA patients and healthy volunteers were  analysed using Affymetrix HG_U133A GeneChips. Additionally, two patients were studied after achieving a partial remission (post-Therapy).
Keywords: disease vs health vs response to therapy

Expression data from  the colon of wild-type and Slc9a3 (NHE3)-deficient mice

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. It has been considered as a target of proinflammatory cytokines and enteropathogenic bacteria and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea.
We used microarray analysis to detail the global programme of colonic gene expression in the absence of functional NHE3.

Mbd2 promotes Foxp3 demethylation and T-regulatory cell function

The proposed use of Foxp3+ T-regulatory (Treg) cells as potential cellular therapy in patients with autoimmune diseases, or post-hemopoietic stem cell or organ transplantation, requires a sound understanding of the transcriptional regulation of Foxp3 expression. Conserved CpG dinucleotides in the Treg-specific demethylated region (TSDR) upstream of Foxp3 are demethylated only in stable, thymic-derived Foxp3+ Tregs. Since methyl-binding domain (Mbd) proteins recruit histone-modifying and chromatin-remodeling complexes to methylated sites, we tested whether targeting of Mbd2 might promote demethylation of Foxp3 and thereby promote Treg numbers or function. Surprisingly, while ChIP analysis showed Mbd2 binding to the Foxp3-associated TSDR site in Tregs, Mbd2 targeting by homologous recombination or siRNA decreased Treg numbers and impaired Treg suppressive function in vitro and in vivo. Moreover, we found complete TSDR demethylation in WT Tregs but >75% methylation in Mbd2-/- Tregs, whereas re-introduction of Mbd2 into Mbd2-null Tregs restored TSDR demethylation, Foxp3 gene expression and Treg suppressive function. Lastly, Mbd2-/- Tregs had markedly binding of the DNA demethylase enzyme, Tet2, in the TSDR region. These data show that Mbd2 has a key role in promoting TSDR demethylation, Foxp3 expression and Treg suppressive function.

Morphine effects on striatal transcriptome in four inbred mouse strains

Chronic opiate use produces molecular and cellular adaptations in the nervous system, leading to tolerance, physical dependence and addiction. Genome-wide comparison of morphine-induced changes in brain transcription of mouse strains with different opioid-related phenotypes provides an opportunity to discover the relationship between gene expression and behavioral response to the drug.
Keywords: Drug response

Mepenzolate bromide displays beneficial effects in a mouse model of chronic obstructive pulmonary disease

For the clinical treatment of chronic obstructive pulmonary disease (COPD), it is important not only to improve the airflow limitation by bronchodilation but also to suppress emphysema by controlling inflammation.  In this study, we have screened for compounds that prevent elastase-induced airspace enlargement in mice from medicines already used clinically.  Mepenzolate bromide, a muscarinic antagonist used to treat gastrointestinal disorders was selected.  Intratracheal administration or inhalation of mepenzolate bromide decreased the severity of elastase-induced airspace enlargement, alteration of lung mechanics and respiratory dysfunction. While mepenzolate bromide showed bronchodilatory activity, most of other muscarinic antagonists tested did not improve the elastase-induced pulmonary disorders.  Mepenzolate bromide suppressed elastase-induced pulmonary inflammatory responses and production of superoxide anions, and reduced the level of cigarette smoke-induced airspace enlargement and alteration of lung mechanics.  Based on these results, we propose that this drug is therapeutically effective for COPD as a consequence of both its anti-inflammatory and bronchodilatory activities.

Transcriptional reprogramming during reloading of atrophied rat soleus muscle

The transcriptional modulator H2AFY marks Huntington's disease activity in men and mice

To accelerate the development of disease-modifying therapeutics for Huntington’s disease (HD), a dynamic biomarker of disease activity and treatment response is critically needed.

Gene silencing of BSK65-MONO1 (RNF185) and of its natural antisense RNA (RNF185-AS) using siRNAs

Microarray profiles of native HeLa cells and RNF185/BSK65 gene silenced HeLa cells using siRNAs LMG2 and LMG4 were monitored using Affymetrix U133 Plus 2.0 microarrays.

Effect of transgenic human IL8 on gene expression in mouse colon cancer tumors

mRNA expression in  colon cancer tumores
of CBA/C57BL/6 transgenic mice
bearing a BAC which contains the human IL8 gene and promoter
was compared to that of CBA/C57BL/6 controls.

Microarray Analysis of Differentially Expressed Genes in Placental Tissue of Severe Preeclampsia

Pre-eclampsia is one of the most serious pregnancy-associated disorders, and is defined by hypertension (systolic blood pressure higher than 140mmHg or diastolic blood pressure higher than 90mmHg) with proteinuria (more than 0.3g/day). It is not a simple complication of pregnancy, but is rather a syndrome of multiple organ failure involving the liver, kidney, and lung, as well as coagulatory and neural systems. There is now an emerging consensus that pre-eclampsia is a complex polygenetic trait in which maternal and fetal genes, as well as environmental factors, are involved, although the precise mechanism of the disorder has remained elusive. In this study, we have performed gene expression profiling to further elucidate the mechanisms underlying the development of the pre-eclamptic condition. We thus compared the expression profiles in placentas from women who underwent a normal pregnancy and from women suffering from severe pre-eclampsia. In addition, we compared the expression data between the early and late onset forms of pre-eclampsia and obtained a number of potential new prognostic biomarkers for this disease.
Keywords: disease state analysis

dendritic cell, A. fumigatus germ tubes, infection for 6h, affymetrix whole genome microarray analysis

Gene expression profile of human dendritic cells after infection with A. fumigatus

DCs are localized under the mucosa of the lungs and the gastrointestinal tract, and therefore come into close contact with A. fumigatus germ tubes during early steps of infection as soon as fungi become invasive. For a more detailed insight into differentially regulated genes, whole genome microarray analysis was performed.
On average, unstimulated DCs showed expression of about 13.500 genes (35% of all genes spotted on the chip). After 6h co-cultivation of DCs and live A. fumigatus germ tubes, 590 genes showed a 4fold altered gene expression, if normalizing data with VSN before. Therein, a wide range of immune response genes, including genes encoding for cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p40, TNF-α), chemokines (CCL5, CCL20, CXCL10), immunorelevant receptors (TLR2, TLR4, PTX-3), as well as costimulatory molecules (CD40, CD80, CD83, CD86) were differentially regulated.

Rat kidney in normo- and hypotensive animals.

Expression data from human primary fibroblasts (IMR90) stably expressing H-RasV12 and treated with Metformin or vehicle

Metformin reduces the incidence of cancer in diabetics or in animal models. At the cellular level, the effects of metformin include the inhibition of complex I of the mitochondrial electron transport chain, a reduction in ATP levels and the activation of the energy sensor AMP kinase. Metformin also prevents the production of reactive oxygen species in primary human cells expressing oncogenic ras and the DNA damage associated to the process.
We used microarrays to characterize the gene expression changes induced by metformin in primary human fibroblasts expressing oncogenic ras.

Microarray analysis of wildtype and Klf3 KO E14.5 fetal liver cells (TER119- and TER119+)

The aim of this experiment was to investigate the role of KLF3 in regulating gene expression at different stages throughout the erythroid maturation process.
Affymetrix microarrays were performed on fetal liver cells (both TER119- progenitor cells and TER119+ erythroblast cells) from E14.5 wildtype and Klf3 KO mice.

adenovirus, complement system, time course, innate immune response, toxicity

Replication-deficient adenovirus (Ad) type 5 capsids induce potent host innate immune responses.  One system pivotal in host innate immune defense is the complement system.  Intriguingly, excessive or inappropriate complement activation can result in extreme tissue damage and systemic inflammatory responses similar to those noted immediately after high dose systemic Ad vector injections.  To determine if the complement system has a significant role in intensifying Ad-associated acute toxicities, we compared complement deficient (C3-knockout (KO)) mice responses to those of wild type (WT) mice following systemic challenge with Ad vectors.  We noted not only thrombocytopenia and rapid increases in plasma IL-6, IFN-γ, TNF-α, and IL-12 levels previously reported after high dose Ad injections into WT mice, but also induction of cytokines IL-2, IL-4, IL-5 and IL-10, G-CSF and GM-CSF, and chemokines KC (CXCL1) and MIP-1.  This expanded innate response “profile” was significantly blunted in Ad injected C3-KO mice, with a near complete avoidance of thrombocytopenia.  Further validation for complement’s critical role in perturbing these innate responses against Ad were noted after comparison of gene specific RNA transcription levels in C3-KO to WT mice livers.  Finally, these disparities were not attributed to a diminished ability of the Ad vectors to transduce C3-KO mice hepatocytes.  These results suggest that Ad capsid interaction with the mammalian complement system may exacerbate the toxicity of systemic Ad injections, and thus represents a future target for manipulation in efforts to improve the efficacy and safety profile of Ad mediated gene delivery.
Keywords: adenovirus, complement system, time course, innate immune response, toxicity

We provide evidence that the Unc-51-like kinase 1 (ULK1) is phosphorylated and activated during engagement of the Type I IFN receptor. Our studies demonstrate that the function of ULK1 controls expression of key interferon stimulated genes (ISGs) that mediate important biological functions, including anti-viral and antineoplastic responses.

training induced gene expression in arrhythmogenic plakogloin (+/-) mice

Endurance training accelerated development of right ventricular dysfunction and arrhythmias in plakoglobin+/- mice. Histology and electron microscopy did not identify right ventricular abnormalities. To identify differences in the transcriptional response to training gene expression profiling was performed.
Keywords: expression response of plakoglobin (+/-) to training

Forty-four paired (from the same patient) samples of head and neck squamous cell carcinoma (HNSCC) and normal tissue were studied with Affymetrix U95A chips. A stringent multi-test approach, combining 7 traditional and microarray-specific statistical tests, was used to analyze the resultant data. Candidate genes were assigned to tiers of significance based on the number of statistical tests that each gene satisfied. Representative genes (both up-regulated and down-regulated) from each of the 3 tiers would be quantified with RT-PCR on both microarray-tested and new samples of HNSCC.
The goal of this study is to identify reliable differentially-expressed genes on HNSCC and to testify our hypothesis whether or not a combinatorial approach (multi-tests) to analyzing microarray data can really identify differentially-expressed genes with fewer false-positives.

comparison of  2 treatments, combination, and untreated at two time points

Differential Gene Induction by Type I And Type II Interferons and Their Combination

Type I and type II interferons (IFNs) bind to different cell surface receptors but activate overlapping signal transduction pathways.  We examined the effects of a type I IFN (IFN-acon1) and a type II iFN (IFN-g1b) on gene experession in A549 cells and demonstrate that there is a common set of genes modulated by both IFNs as well as a set of gene specifically regulated by each, reflecting the activation of different signaling pathways.  In particualr, IFN-g induced many more genes of the signaling pathways, apoptosis, and cytokine interactions than did IFN-a.  Even with genes induced by both IFNs there were distinctive quantitativive differences in expression. IFN-g1b plays a major role in the induction and regulation of the complement pathway.  Previous work has shown a synergistic antivral and antiproliferative effect of type I and type II IFNs in cell culture and in the treament of tumors in mice.  We demonstrate that a majority of genes showed and additive effect of IFN-acon1 and IFN-g1b, but a subset of gene is synergistically induced;  these incluce ISG10, MX2, OAS2, and other genes known to be involved in the antiviral response, TRAIL (TNFSF10) and caspases involved in apoptosis and chemokine genes RANTES, CXCL10, and CXCL11.  Greater than additive transcription of some of these genes in the presence of both IFNs was confirmed by real-time kinetic RT-PCR.  Elevated induction of many of these genes may be sufficient to explain the synergistic antiviral and antitumor effects of this combination of IFNS in vivo.
Keywords: comparison of  2 treatments, combination, and untreated at two time points

Analysis of livers from 74-day old male rats fed vitamin A sufficient or vitamin A deficient diet for 53 days. Either 10ug or 2ug of total RNA used in standard Affymetrix protocol

Cystic Fibrosis;Pseudomonas aeruginosa;CF;flagellin;alginate;lung;repeat sample

Pseudomonas aeruginosa infection of Calu-3 human lung epithelial cells

This series contains 5 groups of U133A arrays with targets from Calu-3 cell line post-infection with P aeruginosa strains. Calu-3 human lung epithelial cells were seeded onto 6-well plates and grown to about 80% confluency. Epithelial monolayers were infected with 108 CFUs per ml of each bacterial strain for 60 min and then washed extensively with phosphate-buffered saline. The cultures were subsequently incubated in fresh RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin. After 6 h, total RNA from the cells was prepared with RNAzol and further purified with RNEasy for microarray hybridization. All arrays processed and globally scaled to 500 using MAS 5.
CONTROL (4 arrays) - no infection
FRD1 (4 arrays)
FRD1234 (3 arrays)
FRD440 (4 arrays)
FRD875 (4 arrays)

Gene expression profile associated with p53R2 in human cancer cells

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In present design, we aimed to identify changes in gene expression profiles associated with p53R2 in cancer cells. We compared gene expression file of control KB cells with that of p53R2-attenuated KB cells by its specific siRNA by using cDNA microarray technology.
We used microarrays to display the global gene expression profile change, after knockdown of p53R2 expression in human oropharyngeal carcinoma KB cells, to find the gene set regulated by p53R2.

The Panc-1 human pancreatic cancer line (ATCC) was constructed to stably express control or GSK3B shRNA. Control or GSK3B shRNA expressing Panc-1 cells were implanted subcutaneously into the flanks of 3-5 week old, female, athymic nude mice. Tumors were grown to at least 250mm3. Tumor tissue was microdissected for further analysis.   We compared control shRNA (n=4) to GSK3B shRNA (n=3) Panc-1 xenografts. The array data with simple statistical calculations are also provided in a supplementary Excel workbook, with probe-set annotation that we used at the time (users may want newer annotation).

SIRT7 is a member of the mammalian sirtuin family of NAD+ dependent deacylases, and  interacts with RNA polymerase I and UBF to regulate rDNA transcription. Various  studies in mammalian cells and human clinical data have linked SIRT7 to cancer.  However studies differ as to whether SIRT7 is oncogenic or tumor suppressive. Here we  analyzed SIRT7 knockout mice and found SIRT7 deficiency caused sub-Mendelian birth  numbers and a reduction in body size. Moreover, at 18 month of age, roughly 60 % of  the SIRT7 knockout mice develop hepatocellular carcinoma (HCC), in many cases  leading to suspected metastasis. Several HCC associated genes were up-regulated in  livers of mice as young as 6 months of age, particularly targets of the proto-oncogene, c-  MYC. Indeed SIRT7 interacts with MYC at endogenous protein levels and also represses  MYC activity. Our findings thus show that SIRT7 acts as a tumor suppressor in vivo, and  may suggest novel strategies to treat liver cancer.

Temporal analysis of P15 and P30 hippocampi in kainate-induced seizures

tissue specificity, time course, development, kidney, metanephric mesenchyme, nephron, Lim1, knockout mice, conditional knockout

Gene expression profiles in developing nephrons using Lim1 metanephric mesenchyme-specific conditional mutant mice

BACKGROUND: Lim1 is a homeobox gene that is essential for nephrogenesis. During metanephric kidney development, Lim1 is expressed in the nephric duct, ureteric buds, and the induced metanephric mesenchyme. Conditional ablation of Lim1 in the metanephric mesenchyme blocks the formation of nephrons at the nephric vesicle stage, leading to the production of small, non-functional kidneys that lack nephrons.
METHODS: In the present study, we used Affymetrix probe arrays to screen for nephron-specific genes by comparing the expression profiles of control and Lim1 conditional mutant kidneys. Kidneys from two developmental stages, embryonic day 14.5 (E14.5) and 18.5 (E18.5), were examined.
RESULTS: Comparison of E18.5 kidney expression profiles generated a list of 465 nephron-specific gene candidates that showed a more than 2-fold increase in their expression level in control kidney versus the Lim1 conditional mutant kidney. Computational analysis confirmed that this screen enriched for kidney-specific genes. Furthermore, at least twenty-eight of the top fifty (56%) candidates (or their vertebrate orthologs) were previously reported to have a nephron-specific expression pattern. Our analysis of E14.5 expression data yielded 41 candidate genes that are up-regulated in the control kidneys compared to the conditional mutants. Three of them are related to the Notch signaling pathway that is known to be important in cell fate determination and nephron patterning.
CONCLUSIONS: Therefore, we demonstrate that Lim1 conditional mutant kidneys serve as a novel tissue source for comprehensive expression studies and provide a means to identify nephron-specific genes.

Gene expression profiles of RPMI8226 cells after knockdown of KDM3A and KLF2

Recent studies have implicated KDM3A, which catalyzes removal of H3K9 methylation, is associated with tumorigenesis. However, the biological role of KDM3A in multiple myeloma, has not been delineated. Here we identify KDM3A-KLF2-IRF4 axis dependence in multiple myeloma. We demonstrate that knockdown of KDM3A leads to apoptosis and significant growth inhibition in myeloma cells. Mechanistically, KDM3A directly regulates myeloma cell survival factor IRF4 expression through H3K9 demethylation at its promoter. We further show that KDM3A directly regulates KLF2 expression and that knockdown of KLF2 leads to growth inhibition in myeloma cells.
The goal of this analysis is to identify genes whose expression changes after shRNA-mediated knockdown of KDM3A and KLF2 using the human U133 plus 2.0 Affymetrix GeneChip in myeloma cell line (RPMI8226).

YWHAZ is an Invasion and Metastasis promoting genes of Lung cancer

Metastasis is a major cause leading to mortality for lung cancer patients. We identified YWHAZ as a potential metastasis-promoting candidate and found that overexpression of YWHAZ promotes lung cancer cell proliferation, anchorage-independent growth, migration, and invasion in vitro, as well as tumorigenesis and metastasis in vivo. It not only increases cell protrusions and branchings but also induces epithelial-mesenchymal transition. Most importantly, YWHAZ protein could prevent £]-catenin from ubiquitination via its association with £]-catenin and enhance slug transcriptional activity which is regulated by £]-catenin/TCF signaling pathway. Moreover, YWHAZ expression was higher in tumors than in adjacent normal tissues in 63 Non-small-cell lung cancer (NSCLC) patients. NSCLC patients with high YWHAZ expressing tumors had shorter overall survival than those with low-expressing tumors. We conclude that YWHAZ play a critical role in promoting NSCLC metastasis.

Mutations in TRPS1 cause trichorhinophalangeal syndrome types I and III, which are characterized by sparse scalp hair in addition to craniofacial and skeletal abnormalities.  Trps1 is a vertebrate transcription factor containing nine zinc-finger domains, including a GATA-type zinc finger through which it binds DNA.  Mice in which the GATA domain of Trps1 has been deleted (Trps1∆gt/∆gt) have a reduced number of pelage follicles and lack vibrissae follicles postnatally.
To identify the transcriptional targets of Trps1 in the developing vibrissa follicle, we performed microarray hybridization analysis comparing expression patterns in the whisker pads of wild-type versus Trps1∆gt/∆gt embryos.  We identified a number of transcription factors and Wnt inhibitors among transcripts downregulated in the mutant embryos, and several extracellular matrix proteins there were upregulated in the mutant samples.

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts [3p arrays]

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type.  Loss of Dicer leads to de-repression of let-7 targets at levels that exceed 10-100 fold with increases in transcription.  Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state.  Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 and the induction of let-7, upon differentiation, at embryonic day 10.5.  Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss.  In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor suppressive function.

Two prognostically significant subtypes of high-grade lung neuroendocrine tumors independent of small-cell and large-cell neuroendocrine carcinomas identified by gene expression profiles.

BACKGROUND: Classification of high-grade neuroendocrine tumors (HGNT) of the lung currently recognises large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung carcinoma (SCLC) as distinct groups. However, a similarity in histology for these two carcinomas and uncertain clinical course have led to suggestions that a single HGNT classification would be more appropriate. Gene expression profiling, which can reproduce histopathological classification, and often defines new subclasses with prognostic significance, can be used to resolve HGNT classification. METHODS: We used cDNA microarrays with 40386 elements to analyze the gene expression profiles of 38 surgically resected samples of lung neuroendocrine tumors and 11 SCLC cell lines. Samples of large-cell carcinoma, adenocarcinoma, and normal lung were also included to give a total of 105 samples analyzed. The data were subjected to filtering to yield informative genes before unsupervised hierarchical clustering that identified relatedness of tumor samples. FINDINGS: Distinct groups for carcinoids, large-cell carcinoma, adenocarcinoma, and normal lung were readily identified. However, we were unable to distinguish LCNEC from SCLC by gene expression profiling. Three independent rounds of unsupervised hierarchical clustering consistently divided SCLC samples into two main groups with LCNEC samples largely integrated with these groups. Furthermore, patients in one of the groups identified by clustering had a significantly better clinical outcome than the other (83% vs 12% survived for 5 years; p=0.0094. None of the highly proliferative SCLC cell lines subsequently analyzed clustered with this good-prognosis group. INTERPRETATION: Our findings show that HGNT of the lung can be classified into two groups independent of SCLC and LCNEC. To this end, we have identified many genes, some of which encode well-characterized markers of cancer that distinguish the HGNT groups. These results have implications for the diagnosis, classification, and treatment of lung neuroendocrine tumors, and provide important insights into their underlying biology.

Male Sprague-Dawley rats weighing 280-300 g were anesthetized with 1.75% to 2.5% isofluorane and instrumented with two arterial catheters, one externalized for blood withdrawal and the other (not externalized) attached to the Data Sciences International (DSI) PhysioTel (St. Paul, MN, model C50-PXT) monitoring device placed intraperitoneally and used in monitoring blood pressure, temperature, and electrocardiogram data via telemetry.  After 7 to 10 days of recovery, and only after the animals had begun to gain weight, the experiment was performed.  At the beginning of hemorrhage (H), blood was withdrawn manually through the externalized catheter with 1-ml syringes at l ml/min.  Total blood volume was estimated to be 6.5% of the body weight and 40% of this (2.6 ml/100g) was removed to effect the hemorrhage.
Control animals (Con) were uninstrumented and were not hemorrhaged or manipulated in any way. Sham (Sh) rats were instrumented, but not hemorrhaged, and physiologic measures were taken for 4 – 23 hrs 6 hrs prior to euthanasia. At 1, 3, 6, 16, 24, 48, or 72 hours (H) after hemorrhage animals were deeply anesthetized with Nembutal (50 mg/kg), and the post hemorrhage blood sample was collected by cardiac puncture and the animals were euthanized by exsanguination.  All tissues were collected within 10 minutes of death and 100 mg amounts of the following tissues were removed and placed in RNALater™  (Ambion, Austin, TX):  intestine, liver, lung, kidney, spleen, heart, skeletal muscle (gastrocnemius) skin, and brain.
Total RNA was isolated from each tissue from each animal with 2 rounds of TRI Reagent® (MRC, Cincinnati, OH) and its quality analyzed by gel electrophoresis.  A reference preparation consisting of equal amounts of RNA pooled from 10 organs (liver, lung, kidney, spleen, heart, skeletal muscle, skin, jejunum, and brain) of untreated control animals was used as reference RNA.  Five-µg samples exhibiting undegraded RNA from each rat lung were reverse transcribed with Superscript II™ (Invitrogen Corp., Carlsbad, CA) in the presence of a C-6 amine modified random hexamer and aminoallodeoxyuridine to produce fluorescent labeled cDNA.  RNA from the reference preparation was all labeled together.  Following reverse transcription, RNA was degraded with 0.1 M NaOH and neutralized with ph 7.0 Hepes buffer.  Following isolation on Qiaquick columns (Qiagen), elution and lyophilization, the lung-sample was labeled with Cy5 ester and reference cDNA samples with Cy3 ester (Amersham Biotech, Piscataway, NJ) After separation from unbound dye, the samples were again lyophilized and then reconstituted in hybridization solution (32% formamide, 5 X Denhardt's solutions, 3X SSC, 20 mM Hepes buffer, ph 7.0)  for 20 hrs at 42 degree. Following hybridization on a microarray (one per rat) and washing to remove unhybridized cDNA and scanning with an Axon 4000B (Axon Instruments, Union City, CA) at 10 micron resolution, the resulting 16-bit TIFF images were analyzed with GenePix 4.1 software (Axon Instruments) for calculation of Cy5 and Cy3 fluorescence intensities at each spot.

Cohesin is required for expression of the estrogen receptor-alpha (ESR1) gene

In this study we show that depletion of the cohesin subunit SMC3 or the Mediator subunit MED12 significantly impairs the ERα-regulated transcriptome. Surprisingly, SMC3 depletion appears to elicit this effect indirectly by rapidly decreasing ESR1 transcription and ERα protein levels. Moreover, we provide evidence that both SMC3 and MED12 colocalize on the ESR1 gene and are mutually required for occupancy as well as for transcriptional elongation. Finally, we show that extended proteasome inhibition decreases the mRNA expression of cohesin subunits which accompanies a decrease in ESR1 mRNA and ERα protein levels as well as estrogen-regulated transcription. Conclusions: These results identify the ESR1 gene as a cohesin/Mediator-dependent gene and indicate that this regulation may potentially be exploited for the treatment of estrogen-dependent breast cancer.

Expression data from MDA-MB-231 cells over-expressing RRP1B and MDA-MB-231 control cells with endogenous RRP1B levels

RRP1B is a breast cancer metastasis suppressor that interacts with various regulators of gene transcription
We examined the changes in gene expression caused by the stable over-expression of RRP1B using lentiviral transduction in the human breast cancer cell line MDA-MB-231.

Expression profiling of KSR1-/- MEFs expressing KSR1 and/or H-RasV12

We engineered KSR1-/- mouse embryonic fibroblasts (MEFs) to express GFP alone, KSR1 or RasV12 and GFP, or KSR1, H-RasV12 and GFP, and performed gene expression profiling on all 4 cell lines.

The goal of this study was to evalute the genetic regulations upon knok-down of VEGFR-2 in two different glioma cell lines in partiular focussing on genes related to glioma migration and invasion as well as resistance to chemotherapy.

Transcription profiling of human skin squamous cell carcinoma (SCC)

Skin squamous cell carcinomas are among the most frequent human cancers. In this study we compared the expression profiles of 10 skin SCCs with a set of 3 normal human epidermis controls.

Expression profiling of human DCIS and invasive ductal breast carcinoma

Human healthy tissue samples, DCIS and invasive mammary tumors were analyzed in order to identify marker genes which show enhanced expresssion in DCIS and invasive ductal carcinomas.
Using this approach, we were able to identify a set of genes which might allow a better detection of DCIS and invasive carcinomas in the future.

Polycomb Group Protein Bmi1 Promotes Hematopoietic Cell Development from ES Cells

Bmi1 is a component of the Polycomb-repressive complexes (PRC) and essential for maintaining the pool of adult stem cells. PRC are key regulators for embryonic development by modifying chromatin architecture and maintaining gene repression. To assess the role of Bmi1 in pluripotent stem cells and upon exit from pluripotency during differentiation, we studied forced Bmi1 expression in mouse embryonic stem cells (ESC). We found that ESC do not express detectable levels of Bmi1 RNA and protein and that forced Bmi1 expression had no obvious influence on ESC self-renewal. However, upon ESC differentiation Bmi1 effectively enhanced development of hematopoietic cells. Global transcriptional profiling identified a large array of genes that were differentially regulated during ESC differentiation by Bmi1. Importantly, we found that Bmi1 induced a prominent up-regulation of Gata2, a zinc finger transcription factor, which is essential for primitive hematopoietic cell generation from mesoderm. In addition, Bmi1 caused sustained growth and a more than 100-fold expansion of ESC-derived hematopoietic stem/progenitor cells within 2-3 weeks of culture. The enhanced proliferative capacity was associated with reduced Ink4a/Arf expression in Bmi1-transduced cells. Taken together, our experiments demonstrate distinct activities of Bmi1 in ESC and ESC-derived hematopoietic progenitor cells. In addition, Bmi1 enhances the propensity of ESC in differentiating towards the hematopoietic lineage. Thus, Bmi1 could be a candidate gene for engineered adult stem cell derivation from ESC.

Rod-derived Cone Viability Factor (RdCVF, alias nxnl1) is a retina-specific protein identified for its therapeutic potential in supporting cone survival during retinal degeneration.
A nxnl1 knockout mouse model was created and the transcriptome used to demonstrate that the retina is compromised by the absence of nxnl1.

Comparative gene expression profile of Hes1-overexpressing cultured hippocampal neurons vs the corresponding control populations (neurons expressing GFP)

Homologue of Enhancer-of-split 1 (Hes1) is a transcription factor that regulates neuronal plasticity, promoting the growth of dendrites and increasing the GABAergic input. A higher expression of Hes1 also results in neuronal resistance against the noxious activity of amyloid beta, the main agent in the advent and progression of the Alzheimer's disease. As a transcription factor, Hes1 controls de expression of many genes. Using the microarray technology we have detected that the expression of one secreted synaptic protein, cerebellin 4 (Cbln4) was particularly increased upon overexpression of Hes1. We also present evidence that Cbln4 plays an essential role in the formation and maintenance of inhibitory GABAergic connections and that either overexpression of Cbln4 in cultured hippocampal neurons or the application of recombinant Cbln4 to the cultures increased the number of GABAergic varicosities and rescued neurons from amyloid beta induced cell death.

Differential gene expression in mouse kidney following 72 hours of colistin exposure by Illumina WG-6_V2 array

Colistin sulfate (polymixin E) is an antibiotic prescribed with resurging frequency for multidrug resistant gram negative bacterial infections. It is associated with nephrotoxicity in humans in up to 33% of cases.  Little is known regarding genes involved in colistin nephrotoxicity. A murine model of colistin-mediated kidney injury was developed.  C57/BL6 mice were administered saline or colistin at a dose of 16mg/kg/day in 2 divided doses. An Illumina gene expression array was performed on kidney RNA harvested 72 hours after first colistin dose to identify differentially expressed genes early in drug treatment. Array platform was MouseWG-6, 48,000 probes. Drug given intraperitoneal.

Murine inner medullary collecting duct cells response to aldosterone

Murine inner medullary collecting duct cells were treated for 1 hour with vehicle (control) or aldosterone. Total RNA was isolated and used as template to generate the eventual cRNA target. The experiment was repeated a total of three times. Six cRNA samples, three control and three treated, were generated and used in a total of six hybridizations.

The mineralocorticoid aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. Although the long-term effects of aldosterone have been extensively studied, the early aldosterone-responsive genes remain largely unknown. Using DNA array technology, we have characterized changes in gene expression after 1 h of exposure to aldosterone in a mouse inner medullary collecting duct cell line, mIMCD-3. Results from three independent microarray experiments revealed that the expression of many transcripts was affected by aldosterone treatment. Northern blot analysis confirmed the upregulation of four distinct transcripts identified by the microarray analysis, namely, the serum and glucose-regulated kinase sgk, connective tissue growth factor, period homolog, and preproendothelin. Immunoblot analysis for preproendothelin demonstrated increased protein expression. Following the levels of the four transcripts over time showed that each had a unique pattern of expression, suggesting that the cellular response to aldosterone is complex. The results presented here represent a novel list of early aldosterone-responsive transcripts and provide new avenues for elucidating the mechanism of acute aldosterone action in the kidney.

Affymetrix high-density oligonucleotide microarray analysis was performed to analyse cisplatin-induced gene expression changes in A549 NSCLC cells. Cells were treated with 50 µM of cisplatin for 1 hour and incubated for a further 10 hours in drug-free media before the gene expression changes were investigated. Results show that cisplatin induced changes in the expression of genes involved in apoptosis, cell cycle control, DNA repair and transcription.
Keywords: drug response

Expression data from lungs of Vldlr-/- mice and wild type (WT) B6129SF2/J mice that had received intranasal house dust mite (HDM) challenges to induce experimental asthma or saline, as a control.

The very low density lipoprotein receptor (VLDLR) is a multi-ligand receptor that mediates pleiotropic biological processes, such as brain development.
In this dataset, we include the expression data obtained from lungs from mice that had been challenged with house dust mite to induce experimental asthma or saline, as a control. These data are used to obtain genes that are differentially expressed in response to VLDLR signaling.

Gene expression from pre- and post- Cediranib treated patients with metastatic Alveolar Soft Part Sarcoma (ASPS)

Gene expression analysis of RB1 knockdown in bone in response to radiation

The role of RB1 in response to radiation was examined in human osteoblasts.  We demonstrate that RB1 induced SASP genes, a response which was attenuated in RB1 knockdown osteoblasts.

KMT1E Mediated H3K9 Methylation Is Required for the Maintenance of Embryonic Stem Cells by Repressing Trophectoderm Differentiation

Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1- null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome- wide defect. Here we show that conditional deletion of the Setdb1 gene in ES cells results in the upregulation of lineage differentiation markers, especially trophectoderm-specific factors, similar to effects observed upon loss of Oct3/4 expression in ES cells. We demonstrate that KMT1E deficiency in ES cells leads to a decrease in histone H3K9 methylation at and derepression of trophoblast-associated genes such as Cdx2. Furthermore, we find genes that are derepressed upon Setdb1 deletion to overlap with known targets of polycomb mediated repression, suggesting that KMT1E mediated H3K9 methylation acts in concert with polycomb controlled H3K27 methylation. Our studies thus demonstrate an essential role for KMT1E in the control of developmentally regulated gene expression programs in ES cells.

Data present the expression analysis of different mouse ES cell line with altered expression of GTF2I.

The response to a 4 hour treatment with TGFbeta (4 ng/ml) was evaluated in lung fibroblasts derived from three controls (normal periphery of resected tumor), open lung biopsies from three patients with idiopathic pulmonary fibrosis (usual interstitial pneumonia pattern on biopsy) and from three patients with fibrosing alveolitis associated with systemic sclerosis (fibrotic non specific interstitial pneumonia pattern on biopsy). Lung fibroblasts were grown to confluence in DMEM with 10% fetal calf serum. At confluence, lung fibroblasts were serum-deprived overnight, and exposed to either 4 ng/ml of activated TGF-ß1 (R&D Systems) or serum-free culture medium with 0.1% BSA for four hours.

Protection from obesity and diabetes by blockade of TGF-beta/Smad3 signaling

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-beta/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3 deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3-/- white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3-/- adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-alpha1 expression. We observe significant correlation between TGF-beta1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-beta1 signaling protects mice from obesity, diabetes and hepatic steatosis. Together, these results demonstrate that TGF-beta signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-beta1 activity might be an effective treatment strategy for obesity and diabetes.

Effect of wt versus mutant hsa-miR-122 overexpression on spontaneous hESC differentiation

We aimed to determine whether overexpression of endoderm-specific miRNA may affect hESC differentiation. To this end, we analyzed the effect of lentiviral-based overexpression of liver-specific miR-122 on hESC differentiation, using genomewide gene microarrays. Stable overexpression of endoderm-specific miR-122 in hESC resulted in increased expression of a few endodermal markers in spontaneously-differentiating hESC, but had no clear effect on directing differentiation towards an endodermal fate; rather, it delayed the general differentiation of hESC.

Gene expression in human umbilical cord endothelial cells following FoxP1 knockdown

To search for genes regulated by the transcription factor Forkhead box Protein P1 (FoxP1)  in endothelial cells, we transfected human umbilical cord endothelial cells (HUVECs) with a combination of three siRNA oligonucleotides directed against human FoxP1 . Human umbilical vein endothelial cells (HUVECs) were isolated from donated umbilical cords, pooled from two donors and cultivated up to passage 5. For transfection with FoxP1-siRNA cells were cultured to 70% confluence and transfected with a combination of three FoxP1-targeting siRNAs or an irrelevant control oligonucleotide (all from invitrogen) using Lipofectamin RNAiMax (Invitrogen) according to the manufacturers instructions. Total RNA was isolated 48h after transfection.

HNF4a is an important liver transcription factor that regulates at least a thousand genes in the liver. Here we used expression profiling in HepG2 cells, a hepatocellular carcinoma cell line, in which HNF4a was knocked down by RNAi to identify some of those target genes. This dataset accompanies the article in Hepatology 2010 Feb;51(2):642-53. Integrated approach for the identification of human hepatocyte nuclear factor 4alpha target genes using protein binding microarrays by Bolotin E, Liao H, Ta TC, Yang C, Hwang-Verslues W, Evans JR, Jiang T, Sladek FM.

Cellular responses to 4-hydroxy-2-nonenal (HNE)--dose response and time course--three replicate experiments for each treatment

Gb5 is a divergent, evolutionarily-conserved, member of the heterotrimeric G protein b subunit family that is expressed principally in brain and neuronal tissue.  Among Gb isoforms, Gb5 is unique in its ability to heterodimerize with members of the R7 subfamily of the regulator of G protein signaling (RGS) proteins that contain G protein-g like (GGL) domains.  Previous studies employing Gb5 knockout mice have shown that Gb5 is an essential stabilizer of GGL domain-containing RGS proteins and regulates the deactivation of retinal phototransduction and the proper functioning of retinal bipolar cells.  The purpose of this study is to better understand the functions of Gb5 in the brain outside the visual system by employing molecular biology, immunohistochemistry and confocal imaging technologies.  We show here that mice lacking Gb5 have a markedly abnormal neurologic phenotype that includes neurobehavioral developmental delay, wide-based gait, motor learning and coordination deficiencies, and hyperactivity.  Using immunohistochemical analysis and a green fluorescent reporter of Purkinje cell maturation we show that the phenotype of Gb5-deficient mice includes, in part, delayed development of the cerebellar cortex, an abnormality that likely contributes to the neurobehavioral phenotype.  Multiple neuronally-expressed genes are dysregulated in cerebellum of Gb5 KO mice.

Sex-specific effects of prenatal stress in 5-Htt deficient mice: towards molecular mechanisms of gene x environment interactions

prenatal stress response, genetic modification
Background: Prenatal stress (PS) exposure has been shown to increase the risk for emotional disorders in later life. Furthermore, the serotonin transporter (5-HTT) genotype is suggested to exert a modulating effect on the association between early life stress and the risk for depression. In the present study, we use a 5-HTT x PS paradigm to investigate whether the effects of PS are dependent upon the 5-HTT genotype.  Methods: The effects of PS on cognition, anxiety- and depression-related behaviour were examined using a maternal restraint stress paradigm of PS in C57BL6 wild-type (WT) and heterozygous (+/-) 5-HTT knockout mice. Additionally, in the female offspring, a genome-wide hippocampal gene expression screening was performed.  Results: 5-HTT +/- offspring showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. Conversely, exposure of 5-HTT +/- mice to PS was associated with altered stress-responsivity and increased depressive-like behaviour, particularly in female offspring. Further, 5-HTT genotype, PS and their interaction differentially affected the expression of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signalling were regulated by both the 5-HTT +/- genotype and PS exposure, whereas cytokine and Wnt signalling were affected in a 5-HTT genotype x PS manner, indicating a gene x environment interaction at the molecular level.  Conclusions: The long-term behavioural effects of PS in C57BL6 mice are partly dependent on the 5-HTT genotype. Further, hippocampal gene expression profiles suggest that distinct molecular mechanisms mediate the behavioural effects of the 5-HTT genotype, PS exposure, and their interaction.

Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis

Interferon Regulatory Factor-8, an Integral Determinant of Myeloid-Derived Suppressor Cell Subset Development.

Myeloid-derived suppressor cells (MDSC) are a major barrier to anticancer responses. Although much is known about how MDSC promote tumor progression, little is known about how they develop. We hypothesized that MDSC develop as a consequence of tumor-induced downregulation of interferon regulatory factor-8 (IRF-8), a key myeloid developmental transcription factor. We showed that: 1) IRF8-deficiency in mice generated myeloid populations highly homologous to tumor-induced MDSC; 2) IRF-8 overexpression in mice reduced MDSC accumulation and retarded tumor growth; 3) MDSC-inducing factors, G-CSF or GM-CSF, facilitated IRF-8 downregulation via STAT3- or STAT5-dependent pathways, respectively; and 4) IRF-8 levels in MDSC-like subsets of breast cancer patients were depressed compared to healthy donors. Altogether, our data implicate IRF-8 as a novel MDSC-dependent transcription factor.

MicroRNAs from lymphoma and leukemia cell lines with or without the 13q31 amplicon were quantitated using a custom microarray platform.  5ug total RNA from each line was labeled with cy3 and hybridized to a microarray that contains probes to 238 mouse and human mature microRNAs.  Four data sets per cell line are provided.  Data is presented as log (base 2) values median center normalized.

Total pancreatic RNA was isolated from 3 week old NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid animals by GITC method. Targets were produced using standard Affymetrix procedures from about 10ug total RNA. The data from NOD.scid, NOD, BDC2.5/NOD and BDC2.5/NOD.scid Affymetrix MGU74Av2 cel files was converted into Robust Multi Array (RMA) text file for analysis using GeneSpring 6.1

Differential expression of genes after 48 hrs, 10 d, and 3 wks of TAC

Microarray analysis of gene expression after transverse aortic constriction in mice: comparison of TAC vs. sham group at 48 hours, 10 days, and 3 weeks.

prostate cancer; gene profile; primary cultures; prognosis; molecular pathology.

The histopathological and molecular heterogeneity of prostate cancer and the limited availability of human tumor tissue make unraveling the mechanisms of prostate carcinogenesis a challenging task. Our goal was to develop an ex vivo model that could be reliably utilized to define a prognostic signature based on gene expression profiling of cell cultures that maintained the tumor phenotype. To this end, we derived epithelial cultures from tissue explanted from 59 patients undergoing radical prostatectomy or cistoprostatectomy because of Prostate Benign Hyperplasia/Prostate Cancer or Bladder Carcinoma. Patient selection criteria were absence of hormonal neo-adjuvant treatment before surgery and diagnosis of clinically localized disease. Using this unique experimental material we analyzed expression of 22.500 transcripts on the Affymetrix Human U133A Gene Chips platform. Cultures from normal/hyperplastic tissues with a prevalent luminal phenotype, and from normal prostate epithelial tissue with basal phenotype (PrEC) served as controls.
We have established a large number of prostate primary cultures highly enriched in the secretory phenotype. From them we derived an epithelial-restricted transcriptional signature that: 1) differentiated normal from tumor cells; and 2) clearly separated cancer derived lines into two distinct groups which correlated with indolent and aggressive clinical behavior of the disease.
Our findings provide:1) a method to expand human primary prostate carcinoma cells with a luminal phenotype; 2) a powerful experimental model to study primary prostate cancer biology; and 3) a novel  means to characterize these tumors from a molecular genetic standpoint for prognostic and/or predictive purposes.

Duplicate analysis of pooled healthy and carious pulpal tissue gene expression using Affymetrix HG_U133A arrays
Keywords: repeat sample

Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders. MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knockout mice using brain tissue homogenates have revealed only subtle changes in gene expression. Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes up-regulated following loss of MeCP2 are biased toward longer genes but this is not true for down-regulated genes, suggesting MeCP2 may selectively repress long genes. Since genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome.

Expression data from cultured human ovarian carcinoma cell lines with and without Cisplatin treatment

Chemo-resistance to platinum such as cisplatin is critical in the treatment of ovarian cancer. Recent evidences have linked epithelial-mesenchymal transition (EMT) with the drug resistance as a contributing mechanism. The current study explored the connection between cellular responses to cisplatin with EMT in ovarian cancer.

The mitochondrial superoxide dismutase (SOD2) is a major antioxidant protein which detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973).  We designed a model of oxidative stress-induced anemia caused by SOD2-deficiency (Friedman et al. J. Exp. Med. 2001).  Our previous work showed that mice reconstituted with SOD2-deficient hematopoietic stem cells develop an anemia with striking similarity to human sideroblastic anemia (SA) (Friedman et al. Blood 2004; Martin et al. Exp Hematol 2005).  Our overall goal was to define early events in the pathogenesis of SOD2-deficiency SA and, in particular, to identify genes involved in the response of erythroid progenitors to oxidative stress.  We compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2-/- ('KO') versus Sod2+/+ ('WT') hematopoietic stem cell recipients using cDNA microarrays.
Samples used in this study were re-hybridized with GLYCOv2 oligonucleotide arrays.  GLYCOv2 oligonucleotide arrays are custom Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA) designed for the Functional Glycomics Gateway, collaboration between the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/) and Nature Publishing Group.  A complete description of the array can be found at http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml .  Hybridization results and quality control details can be found at https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp , by clicking on the ‘Raw Data’ icon link for microarray experiment ‘Jeff Friedman 1: Sod2 KO anemic mice’. Briefly but importantly, one Sod2-/- sample, which was prepared separately, and whose GAPDH 3'/5' ratio was off range, was legitimately removed from the analysis of the GLYCOv2 and 430 2.0 arrays.

Expression data from B220+ Hspa9+/+ and Hspa9+/- CFU-PreB colonies isolated on Day 7 of culture

CFU-PreB colonies are reduced in number and size in Hspa9+/- mice compared to wildtype littermates. We compared the expression profiles of these colonies to gain insight into the mechanism driving this difference.
HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndromes (MDS).  HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. While homozygous knockout of Hspa9 is embryonic lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/-) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/- mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/- mice have a cell-intrinsic reduction in bone marrow CFU-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9+/- B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNAi and observe reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B-lymphopoiesis in vivo. Knockdown of Hspa9 in an IL-7 dependent mouse B-cell line reduced Stat5 phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B-cells. Collectively, these data implicate a role for Hspa9 in B-lymphopoiesis and Stat5 activation downstream of IL-7 signaling.

Gene expression profiling in the lung and liver of low and high dose Perfluorooctanoic Acid exposed mouse fetuses

Exposure to PFOA during gestation altered the expression of genes related to fatty acid catabolism in both the fetal liver and lung.  In the fetal liver, the effects of PFOA were robust and also included genes associated with lipid transport, ketogenesis, glucose metabolism, lipoprotein metabolism, cholesterol biosynthesis, steroid metabolism, bile acid biosynthesis, phospholipid metabolism, retinol metabolism, proteosome activation, and inflammation.  These changes are consistent with activation of PPAR alpha.  Non-PPAR alpha related changes were suggested as well.

The transcriptome of TRPV1-positive sensory neurons revealed by subgroup-elimination transcriptomics

We developed an approach to rapidly eliminate the subgroup of sensory neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia using agonist treatment followed by density centrifugation. To identify transcripts predomintly expressed in TRPV1-positive neurons, we compared the transcriptome of all cells within sensory ganglia versus all cells without TRPV1 expressing neurons using RNA-Seq.

A combined RNAi and localization approach for dissecting long noncoding RNAs reveals a function of Panct1 in ES cells

Long non-coding RNAs (lncRNAs) regulate diverse biological pathways. Unlike protein coding genes, where methods to comprehensibly study their functional roles in cellular systems are available, techniques to systematically investigate lncRNAs have largely remained unexplored. Here, we report a technology for combined Knockdown and Localization Analysis of Non-coding RNAs (c-KLAN) that merges phenotypic characterization and localization approaches to study lncRNAs. Using a library of endoribonuclease prepared short interfering RNAs (esiRNAs) coupled with a pipeline for synthesizing labeled riboprobes for RNA fluorescence in situ hybridization (FISH), we demonstrate the utility of c-KLAN by identifying a novel transcript Panct1 (Pluripotency associated non-coding transcript 1) that regulates embryonic stem cell identity. We postulate that c-KLAN should be generally useful in the discovery of lncRNAs implicated in various biological processes.

Antisense miRNA-221/222 (si221/222) and control inhibitor (GFP) treated fulvestrant-resistant breast cancer cells

Full title: Expression data from antisense miRNA-221/222 (si221/222) and control inhibitor (GFP) treated fulvestrant-resistant breast cancer cells
The expression of miR-221/222 were found to be upregulated in fulvestrant resistant breast cancer cells MCF7-FR compared to its drug-sensitive counterpart MCF7.
To investigate the role of miR-221/222 in acquired resistance to fulvestrant, we lowered the level of miR-221/222 in MCF7-FR cells using miRNA inhibitors (antagomirs), and compared gene expression profiles before and after treatment.

NESG1-mediated differential expression genes in nasopharyngeal carcinoma (NPC)

Analysis of differential expression genes in NESG1-overexpressed and negative nasopharyngeal carcinoma. NESG1 is a candidate tumor suppressor in NPC. Results provide insight into the molecular pathogenesis of NESG1 in NPC.

Tuberous sclerosis complex 1 (Tsc1) enforces quiescence of naive T cells to promote immune homeostasis and function

The mechanisms that regulate T cell quiescence are poorly understood. We report that tuberous sclerosis complex 1 (Tsc1)   establishes a quiescent program in naïve T cells by controlling cell size, cell cycle entry, and responses to T cell receptor stimulation. Loss of quiescence predisposed Tsc1-deficient T cells to apoptosis that depleted conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells exhibited increased mTORC1 but diminished mTORC2 activities, with mTORC1 activation essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in establishing naïve T cell quiescence to facilitate adaptive immune function.

Retinal gene expression of low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice

Retinal gene expression was assessed from postnatal day (P) 8 old low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice with a Illumina mouse-WG6 expression BeadChip.

Novel Roles for ERK5 and Cofiin as Critical Mediators Linking Estrogen Receptor α-Driven Transcription, Actin Reorganization and Invasiveness in Breast cancer

Cancer cell motility and invasiveness are fundamental characteristics of the malignant phenotype and are regulated through diverse signaling networks involving kinases and transcription factors. In this study, we identify a nuclear hormone receptor (ERα)-protein kinase (ERK5)-cofilin (CFL1) network that specifies the degree of breast cancer cell aggressiveness through coupling of actin reorganization and hormone receptor-mediated transcription. Using dominant negative and constitutively active forms, as well as small molecule inhibitors of ERK5 and MEK5, we show that hormone activation of estrogen receptor-α determines the nuclear versus cytoplasmic localization of the MAPK family member ERK5, which functions as a coregulator of ERα-gene transcription.
Notably, ERK5 works with the actin remodeling protein, CFL1, and upon hormone exposure both became localized to transcription factories in the nucleus, verified by immunofluorescence and proximity ligation assays. Both factors facilitated PAF1 recruitment to the RNA Pol II complex and both ERK5 and CFL1 were required for regulation of gene transcription. By contrast, in cells lacking ERα, ERK5 and CFL1 localized to cytoplasmic membrane regions of high actin remodeling, promoting cell motility and invasion, thereby revealing a mechanism likely to contribute to the generally poorer prognosis of ERα-negative breast cancers. Our study uncovers the dynamic interplay of nuclear receptor-mediated transcription and actin reorganization in phenotypes of breast cancer aggressiveness, and highlights new prognostic biomarkers and suggests novel approaches for developing targeted therapies to moderate cancer aggressiveness.

Gene expression profiling in acute myeloid leukemia with mutated NPM

Approximately one third of acute myeloid leukemias (AMLs) are characterized by aberrant cytoplasmic localization of Nucleophosmin (NPMc+ AML), consequent to mutations in the NPM putative nucleolar localization signal.  These events are mutually exclusive with the major AML-associated chromosomal rearrangements, and are frequently associated with normal karyotype, Fms-like tyrosine kinase (FLT3) mutations and multilineage involvement. We report the gene expression profiles of 78 de novo AMLs (72 with normal karyotype; 6 with non-major chromosomal abnormalities) that were characterized for the subcellular localization and mutation status of NPM. Unsupervised clustering clearly separated NPMc+ from NPMc- AMLs, regardless of the presence of FLT3 mutations or non-major chromosomal rearrangements, supporting the concept that NPMc+ AML represents a distinct entity. The molecular signature of NPMc+ AML includes up-regulation of several genes putatively involved in the maintenance of a stem cell phenotype, suggesting that NPMc+ AML may derive from a multipotent hematopoietic progenitor.

Gene Expression Changes in Non-Dysplastic Mucosa from Patients with Ulcerative Colitis Harboring Remote Neoplastic Lesions

Background and Aims: Individuals with ulcerative colitis (UC) are at increased risk for colorectal cancer, although underlying mechanisms are incompletely understood. We sought to identify a potential gene expression signature in non-dysplastic distal mucosa that as a “genetic field effect” could be a marker for remote neoplastic lesions.

Results: 468 genes were significantly up-regulated and 541 genes were significantly down-regulated >2-fold in UC patients with neoplasia compared to UC patients without neoplasia. Nine genes (ACSL1, BIRC3, CLC, CREM, ELTD1, FGG, S100A9, THBD, and TPD52L1) were progressively and significantly up-regulated from controls to quiescent non-dysplastic UC to UC with neoplasia. Immunostaining of proteins revealed increases in tissue expression of S100A9 and REG1 in UC-associated cancer and in non-dysplastic tissue from UC patients harboring remote neoplasia, compared to UC patients without dysplasia and normal controls.

Conclusions: Gene expression changes occur as a field effect in UC patients without active inflammation who harbor a remote dysplastic lesion. Further characterization of these genes and proteins might elucidate pathways of carcinogenesis in IBD and lead to the development of more accurate, less invasive markers of dysplasia in those at increased risk.

Affymetrix GeneChip Human Gene 1.0 ST Array was applied to compare the expression profiles in peripheral blood mononuclear cells(PBMC) between  healthy controls and  multiple sclerosis patients(MS pt).
It suggested that certain genes involved in apoptosis pathway have been changed regulated in PBMC from MS pt.

Response of A673-cells to transfection with interleukin 2 (IL-2). Ewing family tumor (EFT) cell line A673 was transfected with an expression vector for IL-2 (cell line SBVGA1) or empty control vector (cell line SBVGA3). Cells were harvested at 3 different time points and processed for DNA-microarray analysis using Affymetrix HG-U95A microarrays. Primary image analysis was performed using MAS 5.0. In addition, primary analysis was performed with MAS 4.0 and data were scaled to an target intesity of 200. These data have been analysed together with other published data that have been scaled to the same target intensity (see Staege et al. Pediatr Blood Cancer 2004 Jul;43(1):23-34. See also Staege et al. Cancer Res. 2004).

Response of HEK293-cells after transfection with EWS-FLI1. HEK293 cells were transfected with the expression vector pIRES2-EGFP containing type I EWS-FLI1 or empty control vector. For transient transfection standard DEAE dextran method was used and RNA was isolated 48h post transfection. For stable transfection cells were transfected using FuGENE 6 (Roche, Mannheim, Germany) and cells were selected with 400 ug/mL G418. DNA-microarray analysis was performed using Affymetrix HG-U133A microarrays.(see Staege et al. Cancer Res. 2004)
Keywords = HEK293

Effect of ablation of Sfrp5 on gene expression in gonadal white adipose tissue

We identified secreted frizzled-related protein-5 (Sfrp5) as a transcript that is upregulated during adipocyte differentiation and that is increased in white adipose tissue (WAT) of obese mice, compared to lean mice. To investigate the function of sFRP5 in adipose tissue biology, we studied sFRP5Q27stop mice, in which ENU mutagenesis was used to create a premature stop codon at Gln27, thereby creating a likely null allele.

Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress

The infant leukemia-associated gene, Ott1 (Rbm15), has broad regulatory effects on embryonic development and hematopoiesis. Embryonic deletion of Ott1 results in defects to the placenta, spleen and heart. Conditional deletion within the adult hematopoietic compartment demonstrates a requirement in pre-B development and inhibitory roles in myeloid progenitor and megakaryocyte populations. Ott1-deleted bone marrow has an expansion of the Lin- Sca-1+ c-Kit+ (LSK) population which includes the hematopoietic stem cell (HSC) population. Functional HSC testing through competitive repopulation of irradiated recipients demonstrated however, a severe defect in Ott1-deficient HSCs, despite adequate numbers of immunophenotypically identified long term HSCs. Although mice deleted in situ for Ott1 are able to maintain hematopoiesis in steady state over a normal lifetime, but when subjected to proliferative stress, the HSC population loses the self-renewing, G0 fraction and undergoes bone marrow failure.
Therefore, Ott1 is required for maintaining HSC quiescence during proliferative stress. To investigate the downstream effects of Ott1 loss, LSK cells were sorted from Ott1-deleted mice and controls then global gene expression profiles were generated through hybridization of total RNA onto microarrays. Gene set enrichment analysis showed significant enrichment with genes altered in aged HSCs. Ott1-deficient HSCs were found to have other aging-associated features such as increased DNA damage, NF kappa B activation and p38Mapk activation, suggesting Ott1 controls pathways also involved in the aging process.

Whole-Tissue Gene Expression Study of Pancreatic Ductal Adenocarcinoma

Expression analysis of 36 pancreatic ductal adenocarcinoma tumors and matching normal pancreatic tissue samples from pancreatic cancer patients of the Clinical Institute Fundeni (ICF) using Affymetrix U133 Plus 2.0 whole-genome chips.

IFNb-1a in-vivo treatment induces the expression and signaling of IFNAR1 and inhibits Th17 responses in PBMCs derived from CIS patients

IFNb has been used as a first line therapy for relapsing remitting multiple sclerosis (RRMS).  Since only a few studies have addressed the role of endogenous IFNb in the pathogenesis of the disease, our objective was to characterize its role in the transcriptional regulation of pathogenic Th17 cytokines in patients with RRMS.  In-vitro studies have demonstrated that IFNb inhibited IL-17A, IL-17F, IL-21, IL-22 and IFN-b secretion in CD4+ lymphocytes through the induction of suppressor of cytokine secretion (SOCS)1 and 3.  We found that patients with RRMS have increased serum and cerebrospinal fluid (CSF) Th17 (IL-17A and IL-17F) cytokine levels in comparison to the control subjects, suggesting that deficient endogenous IFNbeta secretion and/or signaling may contribute to the dysregulation of those pathogenic cytokines in CD4+ cells.  We identified that the endogenous IFNb from serum of RRMS patients induced a significantly lower IFN-inducible gene expression in comparison to healthy controls (HCs).  In addition, in-vitro studies have revealed a deficient endogenous and exogenous IFNb signaling in CD4+ cells derived from MS patients. Interestingly, upon inhibition of the endogenous IFNb signaling by silencing interferon regulatory factor (IRF)7 gene expression, the resting CD4+ T cells secreted significantly higher level of IL-17A, IL-17F, IL-21, IL-22 and IL-9, suggesting that endogenous IFNb suppresses the secretion of these pathogenic cytokines. In-vivo recombinant IFNb-1a treatment induced IFNAR1 and its downstream signaling molecules’ gene expression, suggesting that treatment may reconstitute a deficient endogenous IFNbeta regulation of the CD4+ T-cells’ pathogenic cytokine production in MS patients.

Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. 21 of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.

In this study, we examined C57BL/6J and AJ mice who received either sham surgery or cholestatic intestinal injury.
Mice were anesthetized by inhaled isoflurane anesthesia. The abdomen was clipped and then prepared in sterile fashion with 70% ethyl-ethanol followed by betadine. A transverse upper abdominal incision was performed. The CBD was dissected away from the portal vein and was ligated near its junction with the duodenum using aneurysm clips engineered with a precisely standardized opening/closing mechanism. The abdominal wall was then closed in a two-layer fashion using absorbable sutures. Sham-operated mice were treated identically but without dissection or ligation of the CBD.

Insulin is a potent pleiotropic hormone that affects processes such as cellular growth, differentiation, apoptosis, ion flux, energy expenditure, and carbohydrate, lipid, and protein metabolism.
We used microarrays to detail the global programme of gene expression underlying the influence of insulin in human skeletal muscle collected from different human individuals including 20 insulin sensitive, 20 insulin resistant and 15 diabetic patients. We identified distinct classes of up-regulated and down-regulated genes during these processes.
The pathophysiology of obesity represents an imbalance between a high energy intake and/or low energy expenditure. Resting energy expenditure (REE) comprises 60-75% of total energy expenditure. The respiratory quotient (RQ) is used to estimate fuel partitioning between fat and carbohydrate as preferred substrates for energy generation, and fuel preferences to generate REE also exhibit individual variation. Genes influencing REE and RQ could represent candidate genes for obesity, Metabolic Syndrome, and Type 2 Diabetes due to the involvement of these traits in energy balance and substrate oxidation.
We used microarrays to explore the molecular bases for individual variation in REE and fuel partitioning as reflected by RQ. We performed microarray studies in human vastus lateralis muscle biopsies from 40 healthy subjects with measured REE and RQ values. We identified genes significantly correlated with REE and RQ, respectively.

Expression data from biopsies of Nasopharyngeal carcinoma (NPC) and non-malignant controls

Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and South East Asia where more than 50,000 new cases are diagnosed each year.
We used microarrays to identify down or upregulated genes in NPC compared with non-malignant controls.

Gene expression profiling after induction of WTX in HEK293 cells

WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in beta-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53 and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell cycle arrest, apoptosis, and p53-target gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.

Transcriptional profiling in facioscapulohumeral muscular dystrophy to identify candidate biomarkers

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. In order to develop mRNA-based biomarkers of affected muscles, we used GeneChip Gene 1.0 ST arrays for global analysis of gene expression in muscle biopsy specimens obtained from FSHD subjects and their unaffected first degree relatives.

We hypothesized that microarray analyses of whole blood gene expression would identify patterns of gene expression useful in the diagnosis for sacroidosis and identify inflammatory mediators relevant to the underlying pathophysiology.

Dmrt1 (doublesex and mab-3 related transcription factor 1) conditional knockout expression analysis of P28 testes

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates.  This study examines the result of conditional removal of Dmrt1 from Sertoli cells in P28 testis tissue.

Examination of CD4+ T cells from Foxp3-GFP knock-in mice. Aim is to understand the genetic program governed by Foxp3 in T cells by comparison of CD4 T cells subdivided into four groups based on expression of Foxp3 and CD25.
Keywords = regulatory T cells

A novel role of stem cell factor SALL4 as an oncofetal protein in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Treatment of HCC remains abysmal; discovery of novel pathways implicated in hepatocarcinogenesis is needed for more effective therapeutics. SALL4 is a stem cell factor essential for the maintenance of embryonic stem cell self-renewal properties and expressed in fetal liver, but silenced in normal adult liver. We observed that expression of SALL4 in murine liver in a transgenic model led to development of HCC. In humans, SALL4 is re-expressed as an oncofetal protein in a subgroup of HCC patients with unfavorable prognoses. Loss-of-function studies demonstrated that SALL4 is essential for human HCC cell survival and tumorigenicity.
We demonstrated that a peptide can block the oncogenic function of SALL4 in HCC by modulating the PTEN/AKT pathway. Our findings reveal a novel role of SALL4 in HCC with important implications for understanding disease mechanisms and development of innovative therapeutics.

Comparison between gene expression in heart from Lmna H222P heterozygous and control mice

The present research is devoted to the identification of gene(s) severely affected by LMNA mutations, leading to striated muscle laminopathies and more specifically the cardiomyopathy. For this purpose, we developped a large-scale gene expression approach on heart and skeletal tissues from Lmna H222P heterozygous Knock-In mouse model.
Keywords: disease state modification

Comparison between gene expression in heart from Emd KO and control mice

The present research is devoted to the identification of gene(s) severely affected by EMD mutations, leading to striated muscle laminopathies and more specifically the cardiomyopathy. For this purpose, we developped a large-scale gene expression approach on heart and skeletal tissues from Emd KO mouse model.

Expression data of inducible stable cell lines overexpressing Ero1alpha WT and  Ero1alpha C104A/C131A

The overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian endoplasmic reticulum (ER) is poorly characterized. Here, we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α.
Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the unfolded protein response. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.

Gene-expression profiling of ZNF217-overexpressing MDA-MB-231 cells

To obtain an overview of the cellular functions regulated by ZNF217 signaling in breast-cancer cell lines, we performed global gene-expression profiling on MDA-MB-231-pcDNA6 and MDA-MB-231-ZNF217 cells

Curcumin is a potent modulator of the inflammatory transcriptome in microglia

Role of retinoblastoma protein (Rb) in mouse Sertoli cells function

Gene expression profiling study of Sertoli cells (Sc)-specific Retinoblastoma (Rb) knockout (KO) mice. We generated a Sc-specific Rb knockout (Sc-RbKO) line and characterised its testicular phenotype. Sc-RbKO showed a progressive germ cell depletion leading to infertility and Sc dysfunction. We observed that up to 10 days old, Sc-RbKO mice present a phenotype similar to that of their WT siblings. This age was choosen to study the gene expression changes that would eventually lead to the described phenotypical changes. Sc-RbKO testis gene expression was compared to WT and to Sc-Rb+/- samples. Our Microarray results show changes of many genes involved in cell cycle control, DNA repair, apoptosis and misregulation of genes that may be important for proper Sertoli cell function such as tight junction formation, cell membrane and tissue remodeling.

Comparison of gene expression in the epidermis of Tcfap2c mutant and control skin at embryonic day 16.5

The development of the epidermis, a stratified squamous epithelium, is dependent on the regulated differentiation of keratinocytes. Differentiation begins with the initiation of stratification, a process tightly controlled through proper gene expression. AP-2γ is expressed in skin and previous research suggested a pathway where p63 gene induction results in increased expression of AP-2γ which in turn is responsible for induction of K14. This study uses a conditional gene ablation model to further explore the role of AP-2γ in skin development.  Mice deficient for AP-2γ exhibited delayed expression of p63, K14, and K1, key genes required for development and differentiation of the epidermis. In addition, microarray analysis of E16.5 skin revealed delayed expression of additional late epidermal differentiation genes: filaggrin, repetin and secreted Ly6/Plaur domain containing 1, in mutant mice. The genetic delay in skin development was further confirmed by a functional delay in the formation of an epidermal barrier. These results document an important role for AP-2γ in skin development, and reveal the existence of regulatory factors that can compensate for AP-2γ in its absence.
Keywords: genetic modification

Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

Illumina Beadchip array analyses of mouse embryonic stem cell gene expression profiles in the presence of or upon knockdown of Aurka. An ES complementation 'rescue' system was employed to measure the effects of knockdown (minus Dox). Aurka was rescued to wildtype levels in the presence of Dox (plus Dox). A control rescue ES cell line, with a Luciferase-targeting shRNA, was also assessed.

Classical Hodgkin’s Lymphoma: Molecular evidence for specific alterations of circulating T lymphocytes

Gene expression profiles were performed from CD3+ T cells isolated from peripheral blood samples of untreated patients with cHL versus 2 control groups consisting of healthy donors and patients with sarcoidosis.
Keywords = Hodgkin’s Lymphoma

The Cdk7/cyclin H/ménage-à-trois 1 (MAT1) heterotrimer has proposed functions in transcription as the kinase component of basal transcription factor TFIIH and is activated in adult hearts by hypertrophic pathways. Using cardiac-specific Cre, we ablated MAT1 in myocardium. Despite reduced Cdk7 activity, MAT1-deficient hearts grew normally. However, fatal heart failure ensued at 6-8 weeks. By microarray profiling, quantitative RT-PCR, and Western blotting at 4 weeks, genes for energy metabolism were found to be suppressed selectively, including targets of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1). Cardiac metabolic defects were substantiated in isolated perfused hearts and isolated mitochondria. In culture, deleting MAT1 with Cre disrupted PGC-1 function: PGC-1α failed to activate PGC-1-responsive promoters and nuclear receptors, GAL4-PGC-1α was functionally defective, and PGC-1β likewise was deficient. PGC-1 was shown to interact with MAT1 and Cdk7, in co-precipitation assays. Thus, we demonstrate an unforeseen essential role for MAT1 in operation of the PGC-1 family of co-activators.
Keywords: conditional knockout in mice

Recently global gene expression profiling of patients samples lead to a molecular definition of Burkitt Lymphoma (BL) with lymphocyte enhancer-binding factor 1 (LEF1) as a signature gene. Here we report the discovery of nucleic LEF1 in a very high proportion of BL cases (15/18) and LEF1 target genes. Germinal center B cells were devoid of detectable nuclear LEF1 expression as mantle cell lymphoma (0/5), marginal zone lymphoma (0/6), follicular lymphoma (0/12) or diffuse large B cell lymphoma (DLBCL) (1/31). Using whole genome gene expression profiling after transient knockdown of LEF1 in BL cell lines, new LEF1 target genes were identified. The joint expression of these genes in primary BL samples shows that LEF1 is not only expressed aberrantly in BL but also transcriptionally active. Our study identified aberrantly expressed LEF1 and its target genes suggesting an important functional role in BLs.

A molecular profile of chronic cocaine abuse includes differential expression of genes regulating transcription, chromatin and dopamine cell phenotype

Midbrain dopamine (DA)-synthesizing neurons play a key role in the addiction process, providing a compelling rationale for determining drug-induced molecular changes arising in these cells. This microarray-based study determined the profiles of midbrain gene expression in chronic cocaine abusers (n = 10) and well-matched drug-free control subjects (n = 10). Array-related procedures were performed in triplicate for each subject. Data analysis revealed that 98 array probes (corresponding to 91 genes) exhibited robust, statistically significant expression differences between chronic cocaine abusers and matched control subjects (p ≤ 0.05, FDR = 5%; with ≥ 1.4 fold-change cut-off applied). Changes in transcript abundance identified by microarray were validated by qPCR analysis in every instance examined (n = 11), regardless of the direction or magnitude of change, supporting the validity of the larger dataset of genes differentially expressed in cocaine abusers. Many of the genes exhibiting robust differential expression were associated with the regulation of transcription, chromatin function, or dopamine cell phenotype. For approximately one-half of these genes, transcript abundance was significantly predictive for subject assignment to the cocaine-abusing versus control cohort. The findings suggest that there is a molecular signature associated with core pathophysiological changes in the DA neurons of chronic cocaine abusers that can be exploited for the development of potential biomarkers and novel therapeutic targets for addiction.

D-2-Hydroxyglutarate produced by mutant IDH2 causes cardiomyopathy and neurodegeneration in mice

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) have been discovered in several cancer types and cause the neurometabolic syndrome D2-Hydroxyglutaric aciduria  (D2HGA). The mutant enzymes exhibit neomorphic activity resulting in production of D2- hydroxyglutaric acid (D-2HG). To study the pathophysiological consequences of the  accumulation of D2-HG, we generated transgenic mice with conditionally activated IDH2R140Q and IDH2R172K alleles. Global induction of mutant IDH2 expression in adults  resulted in dilated cardiomyopathy, white matter abnormalities throughout the central nervous system (CNS), and muscular dystrophy. Embryonic activation of mutant IDH2  resulted in more pronounced phenotypes, including runting, hydrocephalus, and shortened life span—recapitulating the abnormalities observed in D2HGA patients. The  diseased hearts exhibited mitochondrial damage and glycogen accumulation with a concordant upregulation of genes involved in glycogen biosynthesis. Notably, mild  cardiac hypertrophy was also observed in nude mice implanted with IDH2R140Q expressing xenografts, suggesting that 2HG may potentially act in a paracrine fashion.  Finally, we show that silencing of IDH2R140Q in mice with an inducible transgene restores heart function by lowering 2HG levels. Together, these findings indicate that inhibitors of  mutant IDH2 may be beneficial in the treatment of D2HGA and suggest that 2HG produced by IDH mutant tumors has the potential to provoke a paraneoplastic condition.

OH-PBDE-induced gene expression profiling in H295R adrenocortical carcinoma cells

Polybrominated diphenyl ethers (PBDEs) are commonly used as flame retardants in a variety of commercial and household products. They have been detected in the environment and accumulate in mammalian tissues and fluids. PBDE toxicity is thought to be associated with endocrine disruption, developmental neurotoxicity and changes in fetal development. Although humans are exposed to PBDEs, our knowledge of the effects of PBDE metabolites on human cells with respect to health risk is insufficient. Two hydroxylated PBDEs (OH-PBDEs), 2-OH-BDE47 and 2-OH-BDE85, were investigated for their effects on cell viability/proliferation, DNA damage, cell cycle distribution and gene expression profiling in H295R adrenocortical carcinoma cells. We show that the two agents are cytotoxic in a dose-dependent manner only at micromolar concentrations, with 2-OH-BDE85 being more toxic than 2-OH-BDE47. However, no DNA damage was observed for either chemical, suggesting that the biological effects of OH-PBDEs occur primarily via non-genotoxic routes. Furthermore, no evidence of aryl hydrocarbon receptor (AHR)-mediated, dioxin-like toxicity was observed. Instead, we report that a micromolar concentration of OH-PBDEs induces transcriptional changes associated with endoplasmic reticulum stress and the unfolded protein response. We discuss whether OH-PBDE bioaccumulation could result in impairment of the adrenocortical secretory function.

Gene expression changes in mice overexpressing amyloid precursor protein with the Swedish mutation (Tg2576; APPSw).  The gene expression levels in male 12 month-old APPSw mice (GSM26708, GSM26709) were compared to those in male nontransgenic littermate control mice (GSM26706, GSM26707).  Data were analyzed with MAS 5.0 and scaled to 2500.

MyD88-mediated signaling prevents development of adenocarcinomas of the colon via interleukin-18

Inflammation has pleiotropic effects on carcinogenesis and tumor progression. Signaling through the adaptor protein MyD88 promotes carcinogenesis in several chemically induced cancer models. Interestingly, we observed a protective role for MyD88 in the development of AOM/DSS colitis-associated cancer. The inability of Myd88-/- mice to heal ulcers generated upon injury creates an inflammatory environment that increases the frequency of mutations and results in a dramatic increase in adenoma formation and cancer progression. Susceptibility to colitis development and enhanced polyp formation were also observed in Il18-/- mice upon AOM/DSS treatment, suggesting that the phenotype of MyD88 knockouts is in part due to their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that differentially impact tissue homeostasis and carcinogenesis.

Muscle biopsies from biceps and deltoid were taken from 5 patients with FSHD, 5 asymptomatic carriers and 5 normal controls. The genome-wide expression patterns were compared using Affymetrix U133 Plus 2.0 chips.

Gene Expression of ApoE Null and ApoE Null/RAGE Diabetic and Non-diabetic Mice

The multi-ligand Receptor for AGE (RAGE) contributes to atherosclerosis in apolipoprotein (ApoE) null mice in both the non-diabetic and diabetic states. Previous studies using soluble RAGE, the extracellular ligand-binding domain of RAGE, or homozygous RAGE null mice showed that blockade or deletion of RAGE resulted in marked reduction in atherosclerotic lesion area and complexity compared to control animals. In parallel, significant down-regulation of inflammatory mediators and matrix metalloproteinases was evident in ApoE null mice aortas devoid of RAGE compared to those of ApoE null RAGE-expressing mice. Although these findings suggested that RAGE triggered pro-atherogenic mechanisms via regulation of inflammatory gene expression, these studies did not reveal the broader pathways by which RAGE contributed to atherosclerosis in ApoE null mice.
Therefore, we performed Affymetrix gene expression arrays on aortas of non-diabetic and diabetic ApoE null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but, that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis. The comparisons were as follows: 1. diabetic ApoE null relative to non-diabetic ApoE null; 2. non-diabetic ApoE null / RAGE null relative to non-diabetic ApoE null; 3. diabetic ApoE null / RAGE null relative to non-diabetic ApoE null /  RAGE null; and 4. diabetic ApoE null / RAGE  null relative to diabetic ApoE null aorta.
Our data reveal that there is very little overlap of the genes which are differentially expressed both in the onset of diabetes in ApoE null mice, and in the effect of RAGE deletion in diabetic ApoE null mice. We next performed a Pathway-Express analysis to determine the pathways that were most associated with the onset of diabetes in ApoE null mice and the effect of RAGE gene deletion in diabetic ApoE null mice. Rigorous statistical analysis was undertaken and revealed that the transforming growth factor-β pathway (tgf-β) and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE null mice, and the mechanism by which deletion of RAGE ameliorates this effect. We focused on three genes of the tgf-β family which were found to be up-regulated in diabetic vs. non-diabetic ApoE null aorta, and which were reduced by deletion of RAGE. These included: thrombospondin1 (Thbs1), transforming growth factor-β2 (tgf-β2) and rho-associated kinase (ROCK1). Real-time quantitative polymerase chain reaction and Western blotting experiments were performed, as well as ROCK1 activity assays in mouse aorta, and validated the findings of the Affymetrix gene array. Further, confocal microscopy revealed that a principal cell type in the ApoE null aorta expressing these factors was the vascular smooth muscle cell. Our data suggest the novel finding that the observed reduction of accelerated atherosclerosis in diabetic ApoE null / RAGE null vs. diabetic ApoE null mice occurs, all or in part, through the ROCK1 branch of the TGF-β pathway. We have inferred a detailed mechanism for this process.
Taken together, these data suggest that suppression of ROCK1 activity in the atherosclerosis-vulnerable vessel wall, especially in diabetes, but in non-diabetes as well, may underlie the beneficial effects of RAGE antagonism and genetic deletion in murine models. These findings highlight logical and novel targets for therapeutic intervention in cardiovascular disease and diabetes.

Imprints of atherosclerosis are present in circulating T cells of patients with Familial Hypercholesterolemia

Atherosclerosis is the major cause of death in industrialized countries. This disease has initially been characterized as a lipid disorder, but current concepts argue for an inflammatory disease, which develops in the background of hypercholesterolemia and other risk factors. In response to initial events of atherosclerosis formation, such as LDL-deposition in the subendothelial space, monocytes and T cells interact with the vessel wall. However, little is known about the properties and the behavior of these cells in this context. Using familial hypercholesterolemia (FH) as a model we demonstrate substantial differences in the gene expression of freshly isolated human monocytes and T lymphocytes. In FH monocytes we found an increased uptake of oxidized LDL, elevated amounts of scavenger receptors and adhesion molecules, and differences in the regulation of intracellular lipoprotein metabolism compared to monocytes from healthy individuals. Furthermore, the monocyte subpopulation of CD14+/CD16+ cells is less frequent in FH but exhibits significantly higher levels of CD11c and CD29 which increases the likelihood for their transmigration through the endothelial layer. The presence of increased amounts of CD69 in T lymphocytes from FH patients suggests that these cells are more activated than control cells. Our results indicate that some important steps of atherosclerosis formation already take place in circulating blood cells which extends current atherosclerosis models to the plasma compartment.
Keywords: atherosclerosis, T cells, Familial Hypercholesterolemia

Time series of diabetes and exercise training induced expression changes in skeletal muscle of mice

Experiment protocol:

Experiment was performed on 10 to 15 weeks old male NMRI mice (Harlan, Holland) housed in standard conditions (temperature 22°C, humidity 60 ± 10 %, artificial light from 8.00 am to 8.00 pm, normally 5 animals per cage). Animals had free access to tap water and food pellets (R36, Labfor, Stockholm, Sweden). Animals were randomly divided into healthy and diabetic groups. The diabetic group received a single peritoneal injection of streptozotocin (STZ, Sigma-Aldrich, France, 180 mg/kg) dissolved in sodium citrate buffer solution (0.1 mol/l, pH 4.5) to induce experimental diabetes similar to type 1. The other group received injection of an equal volume of buffer. Diabetes was confirmed 72 hours after the injection by urine glucose testing (Glukotest(r), Roche, Germany), and mice were characterized diabetic when urine glucose values were greater than 200 mg/dl. Diabetic and healthy animals were randomly assigned into 12 groups (n = 5 per group), which were sedentary or trained for one, three or five weeks. Training groups performed 1 hour per day of treadmill running at 21 m/min and 2.5° incline. After one day of familiarization on a rodent treadmill, the mice ran as described above 5 days per week. Mice were sacrificed 24 hours after the last training bout (respective sedentary controls at the same time) by cervical dislocation followed by decapitation. Calf muscles were removed, dissected free of fat and connective tissue, weighed, snap frozen in liquid nitrogen and stored at -80°C for further analysis.

Total RNA extraction and sample preparation:

Total RNA was extracted from the left calf muscle complex (soleus + gastrocnemius + plantaris) with Trizol Reagent (Invitrogen, Carlsbad, CA) and further purified with RNeasy columns (Qiagen, Valencia, CA) according to the manufacturers' protocols. Concentration and purity of RNA was determined by measuring absorbances at wavelengths 260 and 280 nm. Integrity of the RNA was checked with agarose gel electrophoresis. RNA samples were pooled within each group for microarray analyses. Concentration, purity and integrity of pooled RNA samples were checked as described above.

Micro array analysis:

Pooled RNA samples were analyzed with Affymetrix Gene Chip MG U74Av2 (Affymetrix , Inc., Santa Clara, CA) representing 6000 known genes and 6000 ESTs. Microarray analyses were performed according to the instructions of Affymetrix. Briefly, 5 µg of total pooled RNA was reverse transcribed using T7-(dT)24-primers and SuperScript II RT enzyme (Invitrogen). Single stranded cDNA was turned to double stranded cDNA using T4 DNA polymerase (Invitrogen). Produced cDNA was then purified and transcribed in vitro to biotin labeled cRNA using Enzo Bioarray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY). RNA was purified and its quality was checked. Purity and quantity of RNA was measured with Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Montchanin, DE) and integrity was tested with agarose gel electrophoresis. Sample was then fragmented and hybridized to Affymetrix test chip to check the function of the hybridization cocktail and to ensure adequate representation of both 5´and 3´ends of the RNA. After testing, samples (15 µg) were hybridized to expression chips. After hybridization, chips were washed and stained in Affymetrix Fluidics Station 400. Arrays were stained first with R-Phycoerythrin Streptavidin (Molecular Probes, Eugene, OR), then with biotinylated anti-streptavidin antibody (Vector Laboratories, Burlingame, CA) and again with R-Phycoerythrin Streptavidin for signal enhancement. The chip was scanned with GeneArray Scanner G2500A (Agilent, Palo Alto, CA)

Scaling and normalization of data:

The array images were analyzed with Microarray Suite 5.0 (Affymetrix) software. All chips were scaled (global scaling) to target intensity of 50 to minimize differences between chips caused by physical differences in chips, hybridization efficiencies and manual laboratory work. The data was subjected to robust normalization that reduces the errors, which are caused by binding capacity and linearity differences between probe sets.

Obesity is often associated with a low-grade systemic inflammation state that contributes to the development of insulin resistance and atherosclerotic complications. This is usually coupled with increased macrophage infiltration in the adipose tissue and a defect in adipocyte differentiation that results in accumulation of hypertrophic fat cells characterized by a deregulated pattern of adipokine expression. Here we show that knockdown of histone demethylase lsd1 in 3T3-L1 preadipocytes results in defective adipogenesis and derepression of an inflammatory program in these cells.

Expression profiles of extremely low gestational age newborns as predictors of BPD

One third to one half of all infants born before the 28th wek of gestation develop BPD bronchopulmonary dysplasia.  Our objective is to evaluate the feasibility of using expression profiling in umbilical cord tissue to discover molecular signatures for developmental staging and for risk of BPD.

Effect of H19 Knockdown by siRNA on gene expression in C2C12 cells

We used microarrays to characterize the global changes in gene expression in C2C12 cells due to siRNA knockdown of long non-coding RNA H19

Comparison of transcriptional profiles of CD4+ and CD8+ T cells from HIV-infected pateints and uninfected control group

We examined the gene expression profiles in ex vivo human CD4+ and CD8+ T cells from untreated HIV-infected individuals at different clinical stages and rates of disease progression. Profiles of pure CD4+ and CD8+ T cells subsets from HIV-infected nonprogressors who controlled viremia were indistinguishable from HIV-uninfected individuals. Similarly, no gene clusters could distinguish T cells from individuals with early from chronic progressive HIV infection, whereas differences were observed between uninfected or nonprogressors versus early or chronic progressors. In early/chronic HIV infection, three characteristic gene expression signatures were observed: (1) CD4+ and CD8+ T cells showed increased expression of interferon stimulated genes (ISGs).  However, some ISGs including CXCL9, CXCL10, and CXCL11, and the IL15Rα in both CD4+ and CD8+ T cells and the anti-HIV ISG APOBEC3G in CD4+ T cells, were not upregulated. (2) CD4+ and CD8+ T cells showed a cluster similar to that observed in thymocytes, and (3) more genes were differentially regulated in CD8+ T cells than in CD4+ T cells, including a cluster of genes downregulated exclusively in CD8+ T cells. In conclusion, HIV infection induces a persistent T cell transcriptional profile, early in infection, characterized by a dramatic but potentially aberrant interferon response, and a profile suggesting an active thymic output.

TSLC1/IGSF4, an immunoglobulin superfamily molecule, is predominantly expressed in the brain, lung, and testis and plays important roles in epithelial cell adhesion, cancer invasion, and synapse formation.  We generated Tslc1/Igsf4-deficient mice by disrupting exon 1 of the gene and found that Tslc1-/- mice were born with the expected Mendelian ratio but Tslc1-/- male mice were infertile.  In adult Tslc1-/- mice of 11-week of age, the weight of the testis was 90% of that of the Tslc1+/+ mice, and the number of sperm in the semen was approximately 0.01% of it.  Histological analysis revealed that the round spermatids and the pachytene spermatocytes failed to attach to the Sertoli cells in the seminiferous tubules and sloughed off into the lumen with apoptosis in the Tslc1-/- mice.  On the other hand, the spermatogonia and the Sertoli cells, as well as the interstitial cells, were essentially unaffected.  In the Tslc1+/+ mice, TSLC1/IGSF4 expression was observed in the spermatogenic cells from the intermediate spermatogonia to the early pachyten spermatocytes and from step-7 or later spermatids.  These findings suggest that TSLC1/IGSF4 expression is indispensable for the adhesion of spermatocytes and spermatids to Sertoli cells and for their normal differentiation to mature spermatozoa.
Keywords: Tslc1 Igsf4 knock out mice testis

Gene expression profiles during the development of compensated or decompensated hypertrophy during pressure overload

We recently introduced a modification of the established monocrotaline (MCT) model for pulmonary hypertension (PH) and subsequent right ventricular (RV) hypertrophy, which allows for the selective induction of either a compensate or decompensated RV hypertrophic phenotype within four weeks after a single subcutaneous MCT injection. Both doses of 30 or 80 mg/kg body weight lead to an intermediate phase of compensated RV hypertrophy (day 14-19), while the former dose leads to a stable compensated phenotype (HYP) and the latter dose progresses towards decompensated ventricular hypertrophy and RV failure (CHF) around day 25-28 (Buermans et. al, Physiological Genomics 2005). This model provides the unique opportunity to characterize the development of either hypertrophic phenotype during the very early stages after imposition of RV pressure overload remodeling, well before phenotypical differences have become apparent. This experiment series describes gene expression profiles, generated by spotted oligonucleotide microarrays, to characterize and compare the expression of ~4800 genes from the RV of HYP, CHF and time matched control rats at 10, 19 and 25 days after injections. RV samples were hybridized against a common reference pool, prepared from total RNA isolated from the interventricular septum wall from animals at day 19 and 25.
Keywords: dose response x time course

Expression data of embryonic stem (ES) cells from both control and Prmt6 overexpressed population

ES cells are able to self-renew and remain pluripotent. These characteristics are maintained by both genetic and epigenetic regulators. Protein arginine methyltransferase (PRMT) 4 and 5 are shown to be important in early embryonic development and in ES cells. PRMT6-mediated di-methylation of histone H3 at arginine 2 (H3R2me2) can antagonize the tri-methylation of histone H3 at lysine 4, which marks active genes. However, it is unclear whether PRMT6 and PRMT6-mediated H3R2me2 play crucial roles in early embryonic development and ES cell identity. In this study, we investigate their functions using mouse ES cells as the model.
We used microarray (Affymetrix GeneChip Mouse Gene 1.0ST) to examine the global change of gene expression in mouse ES cells when Prmt6 was overexpressed and identified distinct classes of genes that are up-regulated and down-regulated during this process.

Accumulating mitochondrial DNA mutations drive premature hematopoietic aging phenotypes molecularly distinct from physiologic stem cell aging

Somatic stem cells mediate tissue maintenance for the lifetime of an organism. Despite the well-established longevity that is a prerequisite for such function, accumulating data argue for compromised stem cell function with age. Identifying the mechanisms underlying age-dependent stem cell dysfunction is therefore key to understand the aging process.
Using a model that carries a proofreading defective mitochondrial DNA polymerase, we demonstrate hematopoietic defects reminiscent of premature HSC aging including anemia, lymphopenia and myeloid lineage skewing. However, in contrast to physiologic stem cell aging, rapidly accumulating mitochondrial DNA mutations displayed little involvement of the hematopoietic stem cell pool but rather with distinct differentiation blocks and/or disappearance of downstream progenitors.

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (Treg cells). To gain insights into the molecular mechanisms of Foxp3 mediated gene expression we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multi-protein complexes of 400-800 kDa or larger and identified 361 associated proteins ~30% of which are transcription-related. Foxp3 directly regulates expression of a large proportion of the genes encoding its co-factors.  Reciprocally, some transcription factor partners of Foxp3 facilitate its expression. Functional analysis of Foxp3 cooperation with one such partner, Gata3, provided further evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of Treg cell biology.
Gene expression profile of Treg specific knock-out of Gata3 vs. their littermate controls were analyzed to gain insight into Gata3 dependendent genes in Treg cells.

we analyzed the gene expression profiles of 7 PTC samples compared to 7 paired normal samples using Affymetrix tools and dChip software. The objective was to find potential molecular markers for this disease
Keywords: gene expression analysis

Gene expression profile of peripheral blood lymphocytes: comparison between melanoma patients and healthy controls

We focused on the major peripheral blood lymphocyte populations that may be involved in anti-tumor responses and negatively impacted by cancer, specifically CD8 T cells, CD4 T cells, B cells and CD56dim natural killer cells. The pure cell subsets were stringently sorted by flow cytometry from PBMC samples. Gene expression profiles of these cell populations from melanoma patients were compared to healthy controls.
Keywords: gene expression profile

shRNA-mediated knock down of Bmi-1 and Mel-18 in medulloblastoma cells

Bmi-1 and Mel-18 are close structural homologues that belong to the polycomb group (PcG) of transcriptional regulators of homeotic gene expression in development. They are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters.  A number of clinical and experimental observations have also implicated Bmi-1 in tumorigenesis and stem cell maintenance.  Bmi-1 overexpression or amplification has been observed in a number of human malignancies, particularly in B-cell lymphomas, medulloblastomas and breast cancer. We report here that shRNA-mediated knock-down of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival and anchorage-independent growth, and suppression of xenograft tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increased clonogenic survival of Rat1 fibroblasts.  In contrast, stable downregulation of Bmi-1 or Mel-18 alone did not affect the growth of SK-OV-3 or U2OS cancer cell lines or normal human WI38 fibroblasts.  Gene expression analysis of shRNA-expressing DAOY cells has demonstrated a significant overlap in the Bmi-1- and Mel-18-regulated genes and revealed novel gene targets under their control.  Taken together, these results suggest that Bmi-1 and Mel-18 might have overlapping functions in human tumorigenesis.
Keywords: shRNA knock-down

To investigate the function of CITED1 in melanoma, its expression was transiently down regulated using CITED1-targeting siRNA. The HT144 melanoma cell line was chosen as it had a relatively high level of detectable CITED1 mRNA and protein expression.

Transcription Profiling of Lung Adenocarcinomas of c-Myc-Transgenic Mice

The transcriptional regulator c-Myc is the most frequently deregulated oncogene in human tumors. Targeted overexpression of this gene in mice results in distinct types of lung adenocarcinomas. By using microarray technology, alterations in the expression of genes were captured based on a female transgenic mouse model in which, indeed, c-Myc overexpression in alveolar epithelium results in the development of bronchiolo-alveolar carcinoma (BAC) and papillary adenocarcinoma (PLAC).
In this study, we analyzed exclusively the promoters of induced genes by different in silico methods in order to elucidate the c-Myc transcriptional regulatory network.

Cellular pathways involved in the adaptation and progression of motor neuron injury in the mouse model of familial ALS

Microarray analysis has been applied to the study of ALS in order to investigate gene expression in whole spinal cord homogenates of SOD1 G93A mice and human ALS cases, although the massive presence of glial cells and inflammatory factors has made it difficult to define which gene expression changes were motor neuron specific. Recently, laser capture microdissection (LCM), combined with microarray analysis, has allowed the identification of motor neuron specific changes in gene expression in human ALS cases.
The aim of the present study is to combine LCM and microarray analysis to study how motor neurons in the spinal cord of transgenic SOD1 G93A mice and transgenic SOD1 WT respond to stimuli determined by the presence of the human mutant protein throughout the evolution of the stages in motor neuron injury

Unique Transcriptional Profile in Wrist Muscles From Cerebral Palsy Patients

Cerebral palsy is caused be an upper motor neuron lesion which casues spasticity as well as secondary effects on muscle .  Muscle from cerebral palsy patients is has been shown to be smaller, with more ECM and longer sarcomere lengths
We used microarrays to globally investigate the transcriptional adaptations of cerebral palsy muscle and research which muscle pathways are altered in the diseased state

Parkinson's disease-associated DJ-1 is required for the expression of GDNF receptor Ret in human neuroblastoma cells

DJ-1 is an atypical peroxiredoxin-like peroxidase that may act as a redox-dependent chaperone and a regulator of transcription. To explore DJ-1-mediated transcriptional control in Parkinson’s disease (PD), we generated human neuroblastoma cells with inducible knock-down of DJ-1 expression. We then used functional genomic techniques to identify novel pathways dysregulated by loss of DJ-1 function. Using microarray gene expression profiling, we found that DJ-1 silencing alters the expression of 26 genes, with 10 down-regulated and 16 up-regulated transcripts. Among the down-regulated genes we found Ret, tyrosine kinase receptor for the neurotrophic factor GDNF. Taking advantage of Ingenuity Pathways Analysis, we identified hypoxia inducible factor 1 alpha (Hif1a) as a possible mediator of the interplay between DJ-1 and Ret. We show that Hif1a is stabilized in the absence of DJ-1, and that loss of DJ-1 generates hypoxia and accumulation of free radical species (ROS). Overexpression of wt DJ-1, but not of C106A and L166P mutants deficient in ROS scavenger activity, rescues Ret expression in neuroblastoma cells. These findings reveal novel players in PD pathogenesis and provide evidence for additional pathways involved in DJ-1-mediated neurodegeneration.

GATA3 Reprograms Basal Breast Cancer Cells towards a Luminal Subtype and Inhibits Metastases through Suppression of Lysyl Oxidase

The transcription factor GATA3 is essential for luminal cell differentiation during mammary gland development and critical for formation of the luminal subtypes of breast cancer. Ectopic expression of GATA3 promoted global alterations of the transcriptome of basal triple-negative breast cancer cells resulting in molecular and cellular changes associated with a more differentiated, luminal tumor subtype and a concomitant reduction in primary tumor growth, lung metastasis, and macrophage recruitment at the metastatic site.  Importantly, we demonstrate that the inhibition of metastases by GATA3 results from the suppression of lysyl oxidase (LOX) expression, a metastasis promoting matrix protein that affects cell proliferation, cross-linking of extracellular collagen types, and establishment of the metastatic niche.

Expression profiling of human medulloblastoma cell line ONS76 upon siRNA-mediated knockdown of KDM5A/LSD1

KDM5A/LSD1 is an important epigenetic regulator in medulloblastoma, the most frequent brain tumor of childhood. Here, the response of ONS76 medulloblastoma cells upon siRNA-mediated knockdown of KDM5A is analysed.

Murine skin gene expression analysis of skin-specific Scd1-deficient and control mice

To help elucidate the metabolic changes in the skin that contribute to the obesity resistance and skin pathology in mice lacking Scd1, we performed microarray analysis of skin gene expression in male skin Scd1 knockout (SKO) and Scd1 flox/flox control (Lox) mice fed a standard rodent diet.
We identified an extraordinary number of differentially expressed genes that support the previously documented histological observations of sebocyte atrophy, inflammation and epidermal hyperplasia in SKO mice. Additionally, transcript levels were reduced in skin of SKO mice for genes involved in fatty acid synthesis, elongation and desaturation, which may be attributed to decreased abundance of key transcription factors including SREBP1c, ChREBP and LXRα. Conversely, genes involved in cholesterol synthesis were increased, suggesting an imbalance between skin fatty acid and cholesterol synthesis. Unexpectedly, we observed a robust elevation in skin retinol, retinoic acid and retinoic acid-induced genes in SKO mice. These results highlight the importance of monounsaturated fatty acid synthesis for maintaining retinol homeostasis and point to disturbed retinol metabolism as a novel contributor to the Scd1 deficiency-induced skin pathology.

Expression data from C57BL/6 and MRL/MpJ heart left ventrical with LAD ligation

Previous studies have suggested that the heart may be capable of limited repair and regeneration in response to a focal injury while other studies indicate that the mammalian heart has no regenerative capacity.  To further explore this issue, we performed a series of superficial and transmural myocardial injuries in C57BL/6 and MRL/MpJ adult mice.  At defined time intervals following the respective injury (Days 3, 14, 30 and 60) we examined cardiac function using echocardiography, morphology, FACS cell sorting for BrdU positive cells and molecular signature using microarray analysis.  We observed complete restoration of myocardial function in the superficial MRL cryoinjured heart and significantly less scar formation as compared to the injured hearts of C57BL/6 mice.  Following a severe transmural myocardial injury, the MRL mouse has increased survival and decreased ventricular remodeling compared to the C57BL/6 mouse but without evidence of significant regeneration.  The cytoprotective program observed in the severely injured MRL heart is in part due to increased vasculogenesis and decreased apoptosis that limits the extension of the injury.  We conclude that C57BL/6 and MRL injured hearts have evidence of limited myocardial regeneration, in response to superficial injury, but the improved function and survival observed in the MRL mouse heart following severe injury is not due to significant regenerative processes.

The discoidin domain receptor 1 (DDR-1) deficient mice exhibit a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) already at early age. Young DDR-1 knock-out mice show typical histological signs of OA, like, surface fissures, loss of proteoglycans, cluster formation of the chondrocytes, altered collagen types as well as atypical arrangement of the collagen fibrils. The isolated chondrocytes from the TMJ exhibit an osteoarthritic character with high amounts of Runt-related transcription factor 2 (runx-2) and collagen type I compared to low levels of SRY (sex determining region Y)-box 9 (sox-9) and aggrecan. Especially, the amount of discoidin domain receptor 2 (DDR2) is increased, which is key player of OA in this model. The gene expression as well as the proteins of the DDR-1-deficient chondrocytes from the TMJ could be influenced by a three dimensional matrix, combined with a knockdown of runx-2 or the stimulation with components of the extracellular matrix, for example nidogen-2. These manipulations caused the osteoarthritic chondrocytes of DDR-1-deficient mice to change their gene expression towards a signature of more physiological cartilage and will open new possibilities for future regenerative treatment options of temporomandibular disorders like OA of the TMJ.

Murine 3T3-L1 progenitor adipocytes cell cultures, treated and untreated (Control) with resveratrol before the induction of differentiation and the effects on adipogenesis and insulin signaling was investigated.
Keywords: Treatment response

pancreas regeneration;STZ;diabetes;tungstate;insulin-like agents;beta cell plasticity;neogenesis;ordered

Gene expression profile of tungstate-induced beta cell mass increase in STZ rats

Background and Aims: It is well demonstrated that in the beta cell population of the pancreas there is a dynamic turnover, which results from the net balance of several processes; beta cell replication, apoptosis and neogenesis. These processes have been studied in partial pancreatectomy and glucagon-like peptide 1 treated animals, where an increase in pancreas regeneration has been observed. Similarly, sodium tungstate, which decreases hyperglycemia in several animal models of diabetes, promotes a rise in the beta cell mass of nSTZ and STZ animals. However, the molecular mechanisms underlying this pancreas regeneration remain unknown. Therefore the objective of this study is to identify which genes are up or down regulated in the increase of the beta cell population of STZ rats treated with sodium tungstate.
Materials and methods: Adult male Wistar (225-250 g) rats were kept under a constant 12-hour light-dark cycle and rats were kept under a constant 12-hour light-dark cycle and were allowed to eat and drink freely. Diabetes was induced by a single i.p. injection of streptozotocin (STZ) (70 mg/Kg body weight) in 0.9% NaCl with 100 mmol/L sodium citrate buffer (pH 4.5). Diabetes was confirmed by determination of its hyperglycaemia (>500mg/dL [Reflotron, Roche Diagnostic]). Healthy rats received an i.p. injection of the vehicle. Treatment started 7 days after the STZ or vehicle injection. Diabetic and healthy rats were divided into two groups. In the first (untreated), rats received deionized drinking water; in the second (treated) group, they were given a solution of sodium tungstate. During the first week of treatment, the rats received a solution of 0.7 mg/mL and in the next 4-5 weeks, the concentration was increased to 2 mg/mL. At the end of the experiment, the animals were sacrificed and pancreatic RNA isolated. Three chips (Affymetrix RAE-230A) were hybridized for each of the four experimental groups (untreated and treated healthy rats and untreated and treated diabetic rats). The raw intensity data obtained from the microarrays was normalized and summarized using the Bioconductor package RMA.

Expression data from D3, siSCR, siPRMT1 and siPRMT8 ES cell derived neurons

PRMT1 and PRMT8 knockdown D3 embryonic stem cells were generated (siPRMT) or as a control, scrambled sequence was introduced (siSCR).
We used microarray to investigate the effect of PRMT1 and PRMT8 in early embryonic neurogenesis.

IKKbeta is a subunit of the IkB kinase (IKK) complex required for NF-kB activation in response to pro-inflammatory signals. NF-kB regulates the expression of many genes involved in inflammation, immunity and apoptosis, and also controls cell proliferation and differentiation in different tissues; however, its function in skin physiopathology remains controversial. We here report the alterations caused by increased IKKbeta activity in basal cells of the skin of transgenic mice.

Expression data of substantia nigra from postmortem human brain of Parkinson's disease patients (PD)

We used microarrays to detail the global program of gene expression underlying Parkinson's disease
Keywords: normal versus diseased

Gene expression changes in human hepatocytes exposed to VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate)

Organophosphorus compounds induce hepatotoxicity through currently unknown mechanisms, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis.
We used microarrays to study gene expression changes in human hepatocytes after exposure to VX, and identified pathways underlying these changes.

The transcriptome of the hypothalamoneurohypophyseal system in euhydrated and dehydrated rats

Abiraterone acetate and di-n-butyl phthalate (DBP) response in a human fetal testis xenograft bioassay

The goal of this study was to determine the effects of a well-characterized anti-androgen, abiraterone acetate, and a suspected human anti-androgen, di-n-butyl phthalate (DBP) on the androgenic function of human fetal testis. Human fetal testis was xenografted into the renal subcapsular space of castrated male athymic nude mice.  Hosts were treated with hCG to stimulate testosterone production in the xenografts, and were concurrently treated with either abiraterone acetate or DBP.  While abiraterone acetate (14 d, 75 mg/kg/d p.o.) dramatically reduced testosterone and the weights of androgen-sensitive host organs, DBP (14 d, 500 mg/kg/d p.o.) had no effect on androgenic endpoints.
Three paired analyses were performed using the LIMMA package in R (commands lmfit and eBayes), with the Benjamini-Hochberg correction for multiple comparisons (Smyth 2005):  vehicle-treated xenografts vs. unimplanted testis (n=5), abiraterone-treated xenografts vs. matched control xenografts (n=3), and DBP-treated xenografts vs. matched control xenografts (n=3).  There were significant differences in gene expression between grafted and ungrafted samples, including dramatic upregulation of microRNAs.  Gene expression analysis also showed that abiraterone decreased expression of genes related to cell differentiation, while DBP induced expression of oxidative stress response genes and decreased expression of factors related to embryonic development.

SIRT3 is a mitochondrial NAD(+)-dependent protein deacetylase, which regulates the enzymatic activity of several mitochondrial proteins.
We used microarrays to identify the differences in gene expression as a result of loss of SIRT3, under standard and high-fat diet conditions

Differential gene expression in mouse skNAC knockout heart at E11.5

Targeted deletion of skNAC in mice resulted in early embryonic lethality with cardiac defects.  In order to investigate the molecular mechanism of the cardiac defect, we designed the microarray comparing gene expression of the mutant E11.5 heart to wild type E11.5 heart.
Keywords: genetic modification in mouse

Expression data from cultured human esophageal squamous cell carcinoma cell lines and cultured human fibroblasts.

Cancer cells express different sets of receptor type tyrosine kinases. These receptor kinases may be activated through autocrine or paracrine mechanisms. Fibroblasts may modify the biologic properties of surrounding cancer cells through paracrine mechansms.
We used microarrays to obtain the global gene expression of human esophageal squamous cell carcinoma cell lines and various esophageal fibroblasts.

The inflammasome is a central player in the induction of obesity and insulin resistance

Inflammation plays a key role in the pathogenesis of obesity. Chronic overfeeding leads to macrophage infiltration in the adipose tissue, resulting in pro-inflammatory cytokine production. Both microbial and endogenous danger signals trigger assembly of the intracellular innate immune sensor Nlrp3 [NLR family, pyrin domain containing 3] resulting in caspase-1 activation and production of pro-inflammatory cytokines interleukin (IL)-1beta and IL-18. Here, we showed that mice deficient in Nlrp3, ASC [apoptosis-associated speck-like protein containing a CARD; a.k.a PYCARD (PYD and CARD domain containing)] and caspase-1 were resistant to the development of high fat diet-induced obesity, which correlated with protection from obesity-induced insulin resistance. Detailed metabolic and molecular phenotyping demonstrated that the inflammasome controls energy expenditure and adipogenic gene expression during chronic overfeeding. These findings reveal a critical function of the inflammasome in obesity and insulin resistance and suggest inhibition of the inflammasome as a potential therapeutic strategy.

Comparisons of hippocampi of Syracuse High Avoidance (SHA) and Syracuse Low Avoidance (SLA) learning rats

total prefrontal cortex RNA in female FVB/N mice (control) versus transgenic female mice overexpressing the human synaptic AChE

Analysis of gene expression in skin of rats subjected to electron irradiation and the effect of vitamin A supplements.

HUMAN PMN CHRONIC GRANULOMATOUS DISEASE NEUTROPHILS;parallel sample

Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals (Healthy1, Healthy2, Healthy3, and Healthy4) or patients with X-linked chronic granulomatous disease (XCGD1, XCGD2, XCGD3, XCGD4, XCGD5, and XCGD6). The studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases, and the Institutional Review Board for Human Subjects at the University of Iowa. All studies were conducted according to Declaration of Helsinki principles.
PMNs (107) were combined with or without IgG and C3bi-coated latex beads (8 x 107) in wells of a 12-well tissue culture plate (pre-coated with 20% normal human serum) and centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis. For the purpose of these studies, activated or "Stimulated" PMNs are defined as those that have been stimulated by phagocytosis of IgG- and C3bi-coated latex beads. "Control" PMNs are defined as resting cells or those left unstimulated throughout the time-course. Following centrifugation, plates were incubated at 37 deg. C in a CO2 for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target (12  g) was performed as described in Methods. Labeling of samples, hybridization of cRNA with Hu95Av2 oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols. Gene expression in healthy control and XCGD individuals was always compared at the same time points after phagocytosis

Comparison of control wild-type and Rb-/- prostate epithelial cell lines under untreated and serum-free conditions
Keywords = prostate

Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism

Hepatic metabolic derangements are key components in the development of fatty liver, insulin resistance, and atherosclerosis. SIRT1, a NAD+-dependent protein deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. Here we demonstrate that hepatic SIRT1 regulates fatty acid metabolism by positively regulating PPAR-alpha. Hepatocyte-specific deletion of SIRT1 impairs PPAR-alpha signaling and decreased fatty acid beta-oxidation in the liver. When challenged with a high-fat diet, liver-specific SIRT1 knockout mice develop hepatic steatosis, hepatic inflammation, and endoplasmic reticulum stress. Taken together, our data indicate that SIRT1 plays a vital role in the regulation of hepatic lipid homeostasis.

Gallstone disease is a major contributor to health care costs in the United States.  Approximately 12 % of the U.S. population has gallstones.  As a result, more than 700,000 cholecystectomies are performed in this country each year.  Many of these patients are obese and have a positive family history; but surprisingly, little is known about the link between obesity, genetics and gallstone formation.   Obese individuals have been shown to have supersaturated bile, larger gallbladder fasting volumes and impaired gallbladder emptying. We have recently demonstrated that leptin plays a role in gallbladder motility.  In an effort to understand the genetic basis for these observations, we tested the hypothesis that leptin would alter gallbladder gene expression.  Methods:  Affymetrix oligonucleotide microarrays 430 2.0 were used to compare gallbladder gene expression profiles from 12 week old control saline-treated leptin-deficient (Lep ob) and from leptin-treated Lep ob female mice.  Analyses were performed on pooled RNA (n=4) from the gallbladders of 12 saline-treated Lep ob mice and from 12 Lep ob mice which were administered daily IP 5 ug/g of recombinant murine leptin for 4 weeks.  Resulting data were analyzed utilizing Gene Chip Operating Software or MAS 5.0.  Results:  Of the genes analyzed 314 were upregulated and 108 were downregulated by leptin administration.  Numerous genes related to gallstone pathogenesis, gallbladder absorption/secretion, inflammatory cytokines, and insulin resistance were altered by leptin.
Keywords: comparitive response to leptin

Cardiac and skeletal muscle gene expression profiles in dysferlin deficient mice

Dysferlin is expressed in skeletal and cardiac muscle. However, dysferlin deficiency, namely limb girdle muscular dystrophy 2B (LGMD2B) and Myoshi myopathy, results in skeletal muscle weakness and spares the heart. This dichotomy could be caused by differential regulation of protective mechanisms. Therefore, we compared intraindividual mRNA expression profiles between cardiac and skeletal muscle in dysferlin-deficient SJL/J mice  and normal C57BL/6 mice.

In order to examine the consequences of human miR-34a induction on the transcriptome, HCT116 cells (a colon cancer cell line) were infected with a retrovirus that produces miR-34a.  Gene expression profiles were then monitored using Affymetrix microarrays.
Affymetrix microarrays were used to examine the transcriptomes of HCT116 cells infected with an empty retroviral vector (pMSCV-PIG) or a retroviral vector that expresses human miR-34a.

Transcriptional Profiling of Untreated, HIV-1 Vector-Infected and TNFalpha-treated Jurkat T cells

Jurkat T cells (in triplicate per treatment group) were left untreated in culture, infected with VSV-G-pseudotyped HIV-based vector (which transduces Tat and eGFP) or treated with TNFalpha.

normal skin (no), actinic keratosis (ak), and squamous cell carcinoma (scc) of the skin were examined:

Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. The type and number of genes involved in non-melanoma skin cancer (NMSC) are still unclear. This study examined and identified different expressed genes in NMSC. Fifteen snap-frozen biopsies of five immunosuppressed organ-transplanted recipients each normal skin, actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) were analysed. Total RNA was extracted reverse transcribed and biotin-labeled cRNA was used for hybridization with Affymetrix HG-U133A gene expression cDNA-microarrays containing 22,283 known genes. Confocal scanner evaluated the signals twice and data analysis was performed by predicition analysis of microarrays (PAM) using nearest shrunken centroids with the threshold 3.5 to identify genes best characterizing normal skin and skin cancer. ANOVA analysis was performed as a second independent method to identify differentially expressed genes (p<0.05, and p<0.15). Quantitative verification of 13 up- or down-regulated genes was performed by real-time RT-PCR and genes were confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. Some of the genes (e.g. annexin, lamin, and metalloproteinases) up-regulated in carcinomas in our study have previously reported as over-expressed in NMSC. In total 42 genes were up-regulated and 76 genes were down-regulated in non-melanoma skin cancer. The majority of genes identified have not been previously reported different expressed during non-melanoma skin carcinogenesis.

Gene Expression Profiling in Peripheral Blood in Untreated New Onset Systemic Juvenile Idiopathic Arthritis

Systemic Juvenile Idiopathic Arthritis (sJIA) has been strongly associated with macrophage activation syndrome (MAS).  To better understand the pathogenesid of sJIA and to facilitate the search for MAS biomarkers, we examine gene expression profiles in untreated new onset sJIA.
17 new onset sJIA patients were included in the study.  5 of the 17 patients showed evidence of subclinical MAS and 2 eventually developed overt MAS.

The molecular consequences of Nix ablation on apoptosis and erythropoiesis

Normal erythropoiesis requires a critical balance between proapoptotic and antipaoptotic pathways. Bcl-xl, an antiapoptotic protein is induced at end-stages of differentiation of erythroid precursors in response to erythropoietin. The details of the proapoptotic pathway and the critical proapoptotic proteins inhibited by Bcl-xl in erythropoiesis are not well understood. We employed gene targeting to ablate Nix, a proapoptotic BH3-domain only Bcl2 family protein, which is known to be transcriptionally induced during erythropoiesis. Nix null mice exhibited reticulocytosis and thrombocytosis in the peripheral blood; and profound splenomegaly with erythroblastosis in the spleen and bone marrow despite normal erythropoietin levels and blood oxygen tension. In vivo apoptosis was diminished in erythroblast precursors from Nix null spleens. To define the molecular consequences of Nix ablation on apoptosis and erythropoiesis, we conducted a detailed comparative analysis of gene expression in spleens from 8 week old Nix null mice and wild type controls. Of 45,101 genes analyzed, 514 were significantly upregulated and 386 down-regulated in Nix-/- splenocytes. Functional cluster analysis delineated the ten most highly regulated gene sets, revealing increased levels of cell cycle and erythroid genes, with decreased levels of cell death and B-cell genes.
Keywords: strain comparison

Recent studies point to a pivotal role of polycomb repressive complex 2 (PRC2) in stem cell function and cancer. Loss of function approaches targeting individual PRC2 subunits have however generated findings that are difficult to reconcile.
Here, we prevent assembly of both Ezh1- and Ezh2-containing PRC2 complexes by conditional deletion of Eed, a core subunit, and assess glodbal gene expression changs in LT-HSCs.

Sox4 is upregulated in adenoid cystic carcinoma (ACC). The ACC3 cell line is the most widely available cell culture model system of this tumor type. RNAi mediated knockdown of Sox4 in ACC3 cells induces apoptosis. Since Sox4 is a transcription factor, it is hypothesized that this apoptotic phenotype is due to changes in transcription from genes under Sox4 control.This experiment was designed to find candidate genes whose expression levels change after Sox4 knockdown that could contribute to the pro-apoptotic phenotype.
Keywords: gene knockdown

TAK1-dependent checkpoint in dendritic cell survival regulates immune homeostasis and function

Homeostatic control of dendritic cell (DC) survival is crucial for a productive adaptive immune response, but the molecular mechanism is not well defined. Moreover, how DCs influence homeostasis of the immune system under steady state remains unclear. Combining DC-specific and inducible deletion systems, we report here that the kinase TAK1 is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c+ cells diminished DC populations, especially the CD8+ and CD103+ DC subsets in the lymphoid and non-lymphoid organs, respectively. This was associated with increased apoptosis of DCs, whereas DC proliferation and differentiation from precursors appeared largely normal. In addition, acute deletion of TAK1 caused DC apoptosis, indicating a direct role of TAK1 in actively maintaining DC survival. TAK1 deficiency impaired activities of the pro-survival NF-kB and AKT pathways but upregulated expression of the pro-apoptotic molecule Bim. Under steady state, loss of TAK1 in DCs resulted in a myeloid proliferative disorder, and altered homeostasis of T cells. In response to antigen stimulation, TAK1-deficient DCs were impaired for T cell priming and regulatory T cell generation. Therefore, TAK1 orchestrates a pro-survival checkpoint in DCs that affects the homeostasis and function of the immune system

c-Myc or v-Src retroviral vector-infected immortalized mouse embryonic fibroblasts.

Expression data from retroviral vector-infected immortalized mouse embryonic fibroblasts (MEFs)

Cultured cancer cells exhibit substantial phenotypic heterogeneity when measured in a variety of ways such as sensitivity to drugs or the capacity to grow under various conditions.  Among these, the ability to exhibit anchorage-independent cell growth (colony forming capacity in semisolid media) has been considered to be fundamental in cancer biology because it has been connected with tumor cell aggressiveness in vivo such as tumorigenic and metastatic potentials, and also utilized as a marker for in vitro transformation.  Although multiple genetic factors for anchorage-independence have been identified, the molecular basis for this capacity is still largely unknown.  To investigate the molecular mechanisms underlying anchorage-independent cell growth, we have used genome-wide DNA microarray studies to develop an expression signature associated with this phenotype.  Using this signature, we identify a program of activated mitochondrial biogenesis associated with the phenotype of anchorage-independent growth and importantly, we demonstrate that this phenotype predicts potential for metastasis in primary breast and lung tumors.

Purpose:The goals of this study are to understand the mechanisms underlying reduced self-renewal and loss of pluripotency by depletion of Rif1.
Methods: We performed global gene expression analysis of Rif1 knockdown ES cell lines using Affymetrix 430 2.0 arrays, compared to shRNA controls.

Cardiac hypertrophy was induced by treadmill running. Sedentary animals served as controls. Gene expression was determined by Affymetrix RGU34A GeneChip's to identify genes with differential expression in an adaptive model of cardiac hypertrophy.

Attenuation of miR-126 activity expands HSC in vivo without exhaustion

Life-long blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell cycle progression of HSC in vitro and in vivo. miR-126 knockdown using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell cycle entry leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway thus attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

Activation of MAPK pathways links LMNA mutations to cardiomyopathy in Emery-Dreifuss muscular dystrophy

Molecular-genetic correlates of fatigue in cancer patients receiving localized external beam radiation therapy

The etiology behind cancer-related fatigue (CRF) is currently unknown. The physiological mechanisms of CRF are based on limited evidence that genetic factors, energy expenditure, metabolism, aerobic capacity, and the individual's immune response to inflammation are responsible for the experience of CRF. Gene expression profiling using microarray analysis from white blood cells of men with non-metastatic prostate cancer shows significant, differential expression of 463 probesets during localized external beam radiation therapy (EBRT). Pathway analysis shows a central role of SNCA (alpha-synuclein gene) among these differentially expressed probesets. Significant expression of SNCA was confirmed by qPCR (p<.001) and ELISA (p<.001) over time during EBRT. A significant correlation was noted between averaged fatigue scores and delta CT values of SNCA expression using confirmatory qPCR over time during EBRT (R=-.90, p=.006). Development of fatigue experienced by these men during EBRT may be mediated by SNCA expression. Pathways related to alpha-synuclein may serve as useful biomarkers to understand the mechanisms behind the development of fatigue.

Global gene expression patterns associated with early stage endometrial cancer have been reported, but changes in molecular expression associated with tumor grade, depth of myometrial invasion, and non-endometrioid histology have not been previously elucidated.  Our group hypothesized there are unique genetic events underlying early endometrial carcinogenesis.  Ninety-two samples of pathologically reviewed stage I endometrial cancers (80 endometrioid and 12 serous) with a heterogeneous distribution of grade and depth of myometrial invasion (i.e. 9 IAG1, 14 IAG2, 7 IAG3, 14 IBG1, 12 IBG2, 13 IBG3, 7 ICG1, 10 ICG2, and 6 ICG3)  were examined in relation to 12 samples of atrophic endometrium from postmenopausal women.  Specimens were analyzed using oligonucleotide microarray analysis and a subset of the differentially expressed transcripts was validated using quantitative PCR.  Comparison of early stage cancers with normal endometrium samples by the univariate t-test with 10,000 permutations identified 900 genes that were differentially regulated by at least 4-fold at a p value of <0.001.   Unsupervised analysis revealed that when compared to normal endometrium, serous and endometrioid stage I cancers appeared to have similar expression patterns.  However, when compared in the absence of normal controls, they were distinct.   Differential expression analysis revealed a number of transcripts that were common as well as unique to both histologic types.  This data uncovers previously unrecognized, novel pathways involved in early stage endometrial cancers and identifys targets for prevention strategies that are inclusive of both endometrioid and serous histologic subtypes.

Expression data from STAG2 deficient and proficient human cancer cells

We have discovered frequent genetic inactivation of the STAG2 gene in diverse human cancers including glioblastoma, Ewing's sarcoma, and melanoma.  STAG2 encodes a subunit of the sister chromatid cohesion complex called the "cohesin complex" that is responsible for the cohesion of sister chromatids following DNA replication and is cleaved at the metaphase to anaphase transition to enable chromosome segregation into daughter cells.  Interestingly, the cohesin complex has also been implicated as a regulator of chromatin architecture and transcription.  To determine the functional significance of STAG2 inactivation in cancer pathogenesis, we used somatic cell gene targeting to correct the endogenous mutations of STAG2 in two aneuploid human glioblastoma cell lines, H4 and 42MGBA.  Similarly, somatic cell gene targeting was also used to introduce a nonsense mutation into codon 6 of the endogenous wild-type allele of STAG2 in HCT116 cells, a near-diploid human colorectal cancer cell line with stable karyotype.  Expression profiling of these three paired sets of STAG2-proficient and deficient cells demonstrated that STAG2 does not play a global role in transcriptional regulation nor does it recurrently modulate the expression of specific tumor-promoting or suppressing genes.
For further details, see Solomon et al., Mutational inactivation of STAG2 causes aneuploidy in human cancer.

Overcoming cetuximab resistance in HNSCC: The role of AURKB and DUSP proteins

Unraveling the underlying mechanisms of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is of major importance as many tumors remain non-responsive or become resistant. Out microarray results suggest that resistant cells still exhibit RAS-MAPK pathway signaling contributing to drug resistance, as witnessed by low expression of DUSP 5 and DUSP6, negative regulators of ERK1/2, and increased expression of AURKB, a key regulator of mitosis. Therefore, interrupting the RAS-MAPK pathway by an ERK1/2 inhibitor  (apigenin) or an AURKB inhibitor (barasertib) might be  a new strategy for overcoming cetuximab resistance in HNSCC

Primary cultures of astrocytes from rat optic nerve heads were treated with EGFR ligand, EGF. Two cell lines from two different rat donors were used. The sister cell cultures were set as control and EGF treated groups.
Keywords: rat optic nerve head astrocytes

differential expression profiling from transcription factor depletion

mRNA expression profiling of p63-depleted versus wildtype ME180 cells

We investigated the transcriptional effects of p63 binding by analyzing ME180 cells depleted for all p63 isoforms via expression of a small hairpin RNA (shRNA) targeting the p63 oligomerization domain.
Keywords: differential expression profiling from transcription factor depletion

Suppression of Progenitor Differentiation Requires the Long Non-Coding RNA ANCR [HG-U133_Plus_2]

Long non-coding RNAs (lncRNAs) regulate diverse processes, yet a potential role for lncRNAs in maintaining the undifferentiated state in somatic tissue progenitor cells remains uncharacterized. We used transcriptome sequencing and tiling arrays to compare lncRNA expression in epidermal progenitor populations versus differentiating cells. We identified ANCR (anti differentiation ncRNA) as an 855 bp lncRNA down-regulated during differentiation. Depleting ANCR in progenitor-containing populations, without any other stimuli, led to rapid differentiation gene induction. In epidermis, ANCR loss abolished the normal exclusion of differentiation from the progenitor-containing compartment. The ANCR lncRNA is thus required to enforce the undifferentiated cell state within epidermis.

Microarray analysis of gene expression changes in human A549 lung cancer cells upon siRNA knockdown of FAM60A and SDS3

The Sin3 histone deacetylase (HDAC) complex is a 1.2 MDa chromatin modifying complex that can repress transcription by binding to gene promoters and deacetylating histones.  The Sin3/HDAC complex can affect cell cycle progression through multiple mechanisms and is among the targets of anticancer drugs, called HDAC inhibitors.  We describe the identification of a new subunit of the Sin3 complex named family with sequence similarity 60 member A (FAM60A).  We show that FAM60A/Sin3 complexes normally suppress the epithelial-to-mesenchymal transition (EMT) and cell migration.  This occurs through transcriptional repression of genes that encode components of the TGF-beta signaling pathway.  This work reveals that FAM60A and the Sin3 complex are upstream repressors of TGF-beta signaling, EMT and cell migration and extends the known biological roles of the Sin3 complex.  This experiment investigates the role of FAM60A in gene expression by comparing A549 lung cancer cells treated with or without siRNA against FAM60A.
The Sin3 histone deacetylase (HDAC) complex is a 1.2 MDa chromatin modifying complex that can repress transcription by binding to gene promoters and deacetylating histones. SDS3 is a core component of the Sin3 complex. The Sin3/HDAC complex can affect cell cycle progression through multiple mechanisms and is among the targets of anticancer drugs, called HDAC inhibitors.  We describe the identification of a new subunit of the Sin3 complex named family with sequence similarity 60 member A (FAM60A).  We show that FAM60A/Sin3 complexes normally suppress the epithelial-to-mesenchymal transition (EMT) and cell migration.  This occurs through transcriptional repression of genes that encode components of the TGF-beta signaling pathway.  This work reveals that FAM60A and the Sin3 complex are upstream repressors of TGF-beta signaling, EMT and cell migration and extends the known biological roles of the Sin3 complex. As a base line to better understand the relationship between FAM60A and the Sin3 complex, this experiment investigates the gene expression changes which occur in A549 lung cancer cells when the Sin3 complex is perturbed by knockdown of a core component via siRNA against SDS3.

Differential Gene Expression in Granulosa Cells from Polycystic Ovary Syndrome Patients with and without Insulin Resistance:  Identification of Susceptibility Gene Sets through Network Analysis

Polycystic ovary Syndrome (PCOS) is a heterogeneous endocrine disorder that shows evidence of genetic predidposition among affected individuals. We have utilized the Microarray data from  granulosa cells of normal and PCOS women for network construction.

TRNP1 regulates expansion and folding of the mammalian cerebral cortex by control of radial glial fate

Evolution of the mammalian brain encompassed a remarkable increase in size of cerebral cortex, including tangential and radial expansion, but the mechanisms underlying these key parameters are still largely unknown. Here, we identified the novel DNA associated protein TRNP1 as a regulator of cerebral cortical expansion in both these dimensions. Gain and loss of function experiments in the mouse cerebral cortex in vivo demonstrate that high Trnp1 levels promote neural stem cell self-renewal and tangential expansion, while lower levels promote radial expansion resulting in a potent increase in the generation of intermediate progenitors and outer radial glial cells resulting in folding of the otherwise smooth murine cerebral cortex. Remarkably, TRNP1 expression levels exhibit regional differences also in the cerebral cortex of human fetuses anticipating radial or tangential expansion respectively. Thus, the dynamic regulation of TRNP1 is critical to regulate tangential and radial expansion of the cerebral cortex  in mammals.

Gene expression changes induced by overexpression of MN1 and MN1VP16  in murine bone marrow cells

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. MN1 is overexpressed in some AML patients and confers resistance to all-trans retinoic acid (ATRA)-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like Irf8 and Ccl9. As MN1 is a co-factor of MEIS1 and RARA, we compared chromatin occupancy between MN1, MEIS1 and RARA. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of  Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of MN1-induced leukemia. Our data show that MN1 prevents activation of the immune response pathway, and suggest that restoration of Irf8 signalling as a novel therapeutic target in AML.

Expression data from Adriamycin-treated Emu-myc; Suv39h1-/- B-cell lymphoma

Oncogene-induced senescence (OIS), a terminal cell cycle block countering (pre)neoplastic lesions, is characterised on the molecular level by trimethylated histone H3 lysine 9 (h3K9me3), a transcriptionally repressive chromatin mark linked to silencing of S-phase-promoting genes. Whether H3K9-governed chromatin remodelling influences anticancer treatment-induced senescence (TIS) and whether functional control of this mark impacts on treatment outcome is not known. We used global gene expression profiling by microarrays to gain insight into the molecular responses of Emu-myc; Suv39h1-/- B-cell lymphoma cells to senescence-inducing anticancer agent Adriamycin (ADR).

Identification and validation of the pathways and functions regulated by the ROR alpha1 in skeletal muscle

Identification and validation of the pathways and functions regulated by the orphan nuclear receptor, ROR alpha1, in skeletal muscle

The retinoic acid receptor-related orphan receptor (ROR) alpha has been demonstrated to regulate lipid metabolism. We were interested in the physiologically relevant roles, and pathways regulated by RORalpha1 action in skeletal muscle. This major mass organ accounts for ~40% of the total body mass, and significant levels of lipid catabolism, glucose disposal and energy expenditure. We utilized the strategy of targeted muscle specific expression of a truncated (dominant negative) RORalpha∆DE in transgenic mice, to investigate RORalpha1 signalling (and function) in this peripheral tissue. Expression profiling and pathway analysis indicated that RORalpha regulated genes involved in: (i) lipid and carbohydrate metabolism, cardiovascular and metabolic disease; and (ii) the LXR nuclear receptor signaling pathway and, (iii) the Akt and AMPK signaling cascades. This analysis was extensively validated by rigorous qPCR analysis using TaqMan Low Density Arrays, coupled to rigorous statistical analysis (with Empirical Bayes, and Benjamini-Hochberg). Moreover, westerns and metabolic profiling were utilized to validate the genes, proteins and pathways (lipogenic, Akt, AMPK and fatty acid oxidation) involved in the regulation of metabolism by RORalph1. The identified genes and pathways were in concordance with the demonstration of hyperglycemia, glucose intolerance, attenuated insulin stimulated phosphorylation of Akt, and impaired glucose uptake in the transgenic heterozygous Tg-RORalpha∆DE animals. In conclusion, we propose that RORalpha1 is involved in regulating the Akt2-AMPK signalling pathways in the context of lipid homeostasis in skeletal muscle.

Expression data from peripheral blood mononuclear cells in multiple sclerosis patients and controls

Multiple sclerosis (MS) is a neurodegenerative disease with a presumed autoimmune component. Expression profiling in immune cells can therefore be used in order to identify genes and pathways involved in MS pathogenesis.
We conducted a genome-wide expression study in peripheral blood mononuclear cells (PBMC) from 12 MS patients and 15 controls in order to identify differentially expressed genes and pathways in MS.

The transcription factor Ets-1 is over-expressed in a wide variety of cancers, and has been strongly associated with the upregulation of factors involved in metastasis and angiogenesis. In this study, we created an inducible Ets-1 over-expressing ovarian cancer cell line from parental 2008 ovarian cancer cells that do not express detectable levels of Ets-1 protein. This microarray compares the stable over-expressing Ets-1 ovarian cancer cell line (2008-Ets1) to their parental 2008 cells to examine differences in gene expression beyond metastasis and angiogenesis.

Expression data of MCF-7 cells treated with gamma tocotrienol (g-T3)

Gamma tocotrienol induces apoptosis in breast cancer cells however, the molecular mechanisms are not completely understood.
We used microarrays to detail the global programme of gene expression underlying the effects of gamma tocotrienols on MCF-7 cells and identified distinct classes of up-regulated genes during this process.

Cultured A549 lung cancer cells treated with actinomycin D and sapphyrin PCI-2050

The goal of this project was to characterize changes in gene expression in response to the anti-cancer agent sapphyrin PCI-2050.  Cultured A549 human lung cancer cells were treated with sapphyrin PCI-2050 or actinomycin D, a known transcripitonal inhibitor.  The gene expression profiles of drug-treated and control A549 cultures were determined using Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA).  Further details are provided in our published manuscript: <http://www.molecular-cancer.com/content/6/1/9>.
Keywords: responses to treatments with anti-cancer agents

The Hay Wells Syndrome-Derived TAp63alphaQ540L Mutant Has Impaired Transcriptional and Cell Growth Regulatory Activity

p63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC syndrome, ADULT  syndrome and AEC  syndrome . The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. We report a study on the TAp63a isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63a wt, DeltaNp63 alpha or the TAp63 alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63a and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.
Keywords: Hay Wells syndrome, TAp63alpha, genetic alteration, pp63 Q540L

BACKGROUND: Previous genomic studies with human tissues have compared differential gene expression between 2 conditions (ie, normal versus diseased) to identify altered gene expression in a binary manner; however, a potentially more informative approach is to correlate the levels of gene expression with quantitative physiological parameters. METHODS AND RESULTS: In this study, we have used this approach to examine genes whose expression correlates with arterial stiffness in human aortic specimens. Our data identify 2 distinct groups of genes, those associated with cell signaling and those associated with the mechanical regulation of vascular structure (cytoskeletal-cell membrane-extracellular matrix). Although previous studies have concentrated on the contribution of the latter group toward arterial stiffness, our data suggest that changes in expression of signaling molecules play an equally important role. Alterations in the profiles of signaling molecules could be involved in the regulation of cell cytoskeletal organization, cell-matrix interactions, or the contractile state of the cell. CONCLUSIONS: Although the influence of smooth muscle contraction/relaxation on arterial stiffness could be controversial, our provocative data would suggest that further studies on this subject are indicated.

colon cancer;gene expression;DNA microarrays;beta-catenin;familial adenomatous polyposis;ordered

Transcriptional Profiling of the Transition from Normal Intestinal Epithelia to Adenomas and Carcinomas in the APC(Min/+) Mouse.

Samples used in analysis:
* GSM6191-GSM6196 (WT): Ilea epithelial cells from C57/BL6 wild-type samples
* GSM6197-GSM6201 (Adenoma): Epithelial cells from crypts of adenomas of APC(Min/+) mice
* GSM6202-GSM6206 (Carcinoma): Epithelial cells from crypts of carcinomas of APC(Min/+) mice

Using a PixCell IIe instrument (Arcturus), ~30,000 laser firings per sample were used to collect cells of interest. RNA was extracted from captured cells by PicoPure (Arcturus) technique, followed by 2 rounds of RiboAmp amplification (Arcturus), with incorporation of biotinylated nucleotides (Enzo) in the IVT of round 2. All protocols were as per manufacturers instructions. 15ug of labeled cRNA from individual samples were hybridized to respective MG_U74Av2 chips (Affymetrix) and washed/stained using the standard EukGEWS2v4 protocol (Affymetrix). Chips were scanned and analyzed using MAS 5 (Affymetrix) and data scaled to TI=500.

Pairwise comparisons using the Mann-Whitney test were performed in DMT 3 (Affymetrix), comparing:
* WT vs Adenoma (n=6 x n=5; 83.3% concordance)
* WT vs Carcinoma (n=5 x n=5; 84% concordance)
* Adenoma vs Carcinoma (n=5 x n=5; 84% concordance)

This resulted in the identification of differentially expressed transcripts. Fold changes were calculated from signal-log-ratios. Identified transcripts were clustered based on functional information which was publicly available at time of analysis, obtained through the NetAffx web portal (Affymetrix).

The RSK2 gene is responsible for Coffin-Lowry syndrome, an X-linked monogenic disease associating severe learning deficit andassociated to typical facial and digital abnormalities and skeletal changes. Craniofacial and dental anomalies encountered in this rare disease have been poorly characterized.

A single intradermal injection of IFN-γ induces a psoriasis-like state in both non-lesional psoriatic and healthy skin

Psoriasis is a chronic, debilitating, immune-mediated inflammatory skin disease. As IFN-γ is involved in many cellular processes, including activation of T cells and dendritic cells (DCs), antigen processing and presentation, cell adhesion and trafficking, and cytokine and chemokine production, IFN-γ-producing Th1 cells were proposed to be integral to the pathogenesis of psoriasis. Recently, IFN-γ was shown to enhance IL-23 and IL-1 production by DCs and subsequently induce Th17 cells, important contributors to the inflammatory cascade in psoriasis lesions.  To determine if IFN-γ indeed induces the pathways leading to the development of psoriasis lesions, a single intradermal injection of IFN-γ was administered to an area of clinically normal, non-lesional skin of psoriasis patients and biopsies were collected 24 hours later. Although there were no visible changes in the skin, IFN-γ induced molecular and histological features characteristic of psoriasis lesions.  IFN-γ increased a number of differentially expressed genes in the skin, including many chemokines concomitant with an influx of T cells and inflammatory DCs. Furthermore, inflammatory DC products TNF, iNOS, IL-23, and TRAIL were present in IFN-γ-treated skin. Thus, IFN-γ, which is significantly elevated in non-lesional skin compared to healthy skin, appears to be a key pathogenic cytokine that can induce the inflammatory cascade in psoriasis.

Affymetrix U133A expression levels for 12 symptomatic and 5 presymptomatic Huntington's disease patients versus 14 healthy controls.

Effect of Tet1-knockdown on gene expression in mouse ES cells cultured in ES and TS cell culture conditions

TET-family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA.  Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem (ES) cells.  ES cells depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1, and display hyperactive Nodal signalling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro.  In Fgf4- and heparin-supplemented culture conditions that favor derivation of trophoblast stem (TS) cells, Tet1-depleted ES cells activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in mid-gestation embryo chimeras.  Consistent with these findings, Tet1-depleted ES cells form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm and ectopic appearance of trophoblastic giant cells. Thus Tet1 functions to regulate the lineage differentiation potential of ES cells. Here, we performed whole-genome transcriptome profiling of ES cells stably depleted of Tet1 by shRNA knockdown (Tet1-kd) cultured in either standard ES cell or in TS cell culture conditions. Gene expression changes in Tet1-kd ES cells were fairly modest compared to control (GFP-kd) cells, although gene ontology (GO) analysis of differentially expressed genes yielded many terms related to embryonic development and cell cycle regulation. In TS cell culture conditions, a core set of genes defining trophectodermal cell differentiation, including Cdx2, Eomes and Tead4, was enriched in Tet1-kd compared to GFP-kd cells.

Genome-wide analysis of gene expression in Krt1+/+ versus Krt1-/- epidermis and full-thickness skin.

Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) constitute the intermediate filament cytoskeleton of suprabasal skin keratinocytes. They participate in formation of the epidermal barrier, which protects against dehydration and inflammation. Mutations in KRT1 cause keratinopathic ichthyosis with erythema, recurrent inflammation, and barrier defects. Here, we show that genetic deletion of Krt1 in mice causes a defective inside-out epidermal barrier, pre- and postnatal increases in Mrp8/Mrp14, interleukin (IL) 18, IL-33, and thymic stromal lymphopoietin (TSLP) in skin extracts, and systemic release of IL-18 into newborn serum. Perinatal lethality was partially rescued by treatment with glucocorticoids to promote barrier repair or with IL- 18-blocking antibodies in utero. In human keratinocytes, IL-18 release was cellautonomous and caspase-1-dependent, indicating KRT1-dependent inflammasome activation. Our data reveal a novl function of KRT1 in controlling inflammasome activity and stimulating barrier formation, thereby integrating the keratin cytoskeleton into the epidermal immune response. In view of their widespread expression, keratins merit investigation of their functions in inflammatory conditions, including asthma and inflammatory bowel disorders.

Gene Expression Profiles of Nondiabetic and Diabetic Obese Mice--Adipose tissue, Liver, Muscle and Islets

Obesity is a strong risk factor for the development of type 2 diabetes. We have previously reported that in adipose tissue of obese (ob/ob) mice, the expression of adipogenic genes is decreased. When made genetically obese, the BTBR mouse strain is diabetes susceptible and the C57BL/6J (B6) strain is diabetes resistant. We used DNA microarrays and RT-PCR to compare the gene expression in BTBR-ob/ob versus B6-ob/ob mice in adipose tissue, liver, skeletal muscle, and pancreatic islets. Our results show: 1) there is an increased expression of genes involved in inflammation in adipose tissue of diabetic mice; 2) lipogenic gene expression was lower in adipose tissue of diabetes-susceptible mice, and it continued to decrease with the development of diabetes, compared with diabetes-resistant obese mice; 3) hepatic expression of lipogenic enzymes was increased and the hepatic triglyceride content was greatly elevated in diabetes-resistant obese mice; 4) hepatic expression of gluconeogenic genes was suppressed at the prediabetic stage but not at the onset of diabetes; and 5) genes normally not expressed in skeletal muscle and pancreatic islets were expressed in these tissues in the diabetic mice. We propose that increased hepatic lipogenic capacity protects the B6-ob/ob mice from the development of type 2 diabetes. Diabetes 52:688–700, 2003
Keywords: Genetic modifications

A constitutively activated form of the p110 beta isoform of PI3-kinase induces prostatic intraepithelial neoplasia in mice

The global gene expression profiles of ventral prostates of wild type mice and p110 beta transgenic mice.

We are investigating the response of human lymphoblastoid cells to low-dose exposure of environmental metals
We used microarrays to detail the global programme of gene expression upon response to low-dose metals

Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain

Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Additional cellular activities, unrelated to cell death seem to be influenced by these enzymes. Identification of genes co-regulated with caspases could help to ascertain new biological roles for these proteases.To identify genes and pathways under the influence of caspase-2 we silenced its expression in U87MG glioblastoma cell line. Transcriptional expression profiles of cells transfected with caspase-2 siRNA or control siRNA were compared.

We define a pathogenic role for a ß-catenin-activated genetic pathway in murine renal dysplasia. Cre-mediated stabilization of ß-catenin in the ureteric cell lineage prior to the onset of kidney development increased ß-catenin levels and caused renal aplasia or severe hypodysplasia. A genome-wide analysis of mRNA expression in dysplastic tissue identified down-regulation of genes required for ureteric branching and up regulation of Tgfß2 and Dkk1.

Gene expression profile in CD4+ and CD8+ T cells from identical twins discordant for Multiple sclerosis

To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS.
We studied 4 monozygotic twin pairs discordant for disease, with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip®

time course, cell type comparison, tissue engineered cartilage; osteoarthritis; Hyaff-11 scaffold; human chondrocytes; gene expression profiling; regenerative medicine; differentiation potential

Chondrogenic differentiation potential of OA chondrocytes and their use in autologous chondrocyte transplantation

Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR.   Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated.   Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT.

The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regu- latory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.
We used microarrays to detail the global programme of gene expression after knockdown of HIRA and CABIN1 in 3 replicates

SIRT6 has been implicated in a range of biological processes including inflammation, aging and the control of metabolism. Hence inhibitors or activators of SIRT6 have the potential to be therapeutics for a number of indications.
Genome wide expression studies were used to investigate the effect of overexpression of SIRT6 and mutant SIRT6 on a wide range of NFκB dependent gene expression

VGLL4 is a novel regulator of survival in human embryonic stem cells

Human embryonic stem cells (hESCs) are maintained in a self-renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes by using a gain-of-function screen of a large collection of human open reading frames. We identified Vestigial-like 4 (VGLL4), a co-transcriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain-of-function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process.
The goal of this experiment was to compare the global transcriptional profile of hESCs overexpressing VGLL4 to wild-type hESCs in order to understand the transcriptional differences behind the phenotype exhibited by VGLL4-overexpressing cells. This list was then used for subsequent analysis where genes were filtered individually, but also per sample, such that only genes with a pvalue of detection of less than 0.05 on at least 5 of the samples were kept. Next, the SAM statistical suite was used to generate a list of genes that are expressed at a significantly higher level in the 4 Vgll4 samples compared to the 4 WT samples using an FDR of 7% for discovery purposes.

Hmga2 is required for canonical WNT signaling during lung development

The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. Here we deciphered the role of the high mobility group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2 deficient mice (Hmga2-/-).We found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2-/- mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout (KO) led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA6 (GATA binding protein 6) and FZD2 (frizzled homolog 2).
Comparison of Hmga2-/- with Hmga2+/+ mice by Affymetrix microarray-based expression analysis of embryonic lung revealed an increased expression of genes whose products participate in cell cycle and canonical Wnt signaling.

e tested the responses of normal and AIS genital fibroblasts to dihydrotestosterone (DHT), under culture conditions similar to those that were reported to produce aromatase induction in these cells [8]. Cells were treated with DHT (100-1,000 nM) both at confluency (G0) and during exponential growth, and transcript levels were assessed using DNA microarrays.
A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed.

Gene expression in liver tissue from Ghrh-KO and normal (wild-type) mice

The hypothalamus has recently emerged as a key regulator of metabolism and aging in mammals. We have examined the impact of targeted disruption of hypothalamic hypophysiotropic peptide: Growth Hormone-releasing Hormone (GHRH) in mice on longevity, and the putative mechanisms of delayed aging. GHRH knockout (KO) mice are remarkably long-lived and in comparison to genetically normal (wild type) animals exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of GH. While these effects overlap with those of caloric restriction (CR), we show that effects of CR and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity.

Posterior Second Heart Field (pSHF) transcriptomes of wild-type and Shh mutant mouse embryos

To understand Sonic hedgehog homolog (Shh)-mediated molecular networks in the posterior second heart field (pSHF), we have employed whole genome microarray expression profiling as a discovery platform to identify genes with Shh-dependent expressional changes.  We microdissected the pSHF from E9.5 embryos and compared the Shh mutant samples with than of wild-type using Agilent 4x44k Mouse Whole Genome Arrays (n = 4 WT pools and 4 Shh - /- pools).

Myelomonocytic cells (i.e., monocytes or macrophages) play a key role in tumor progression as revealed by numerous mice tumor model studies. However, their contribution in human tumor progression is not well-studied. Using  Renal Cell Carcinoma (RCC) as a model for human cancer, we performed a transcriptomal profiling of blood monocytes from RCC patients to investigate the contribution of these cells in cancer progression. As compared to monocytes from healthy donors (Mo), transcriptome analysis of monocytes from RCC patients (RCC-Mo) showed a distinct gene expression signature consisting of cytokines, chemokines and various inflammation-related genes. Validation of these genes by qPCR as well as other functional assays indicated RCC-Mo possess an inflammatory and tumor promoting phenotype.

Gene expression in blood of children with autism spectrum disorder

Gene expression in blood of children with autism spectrum disorder (ASD) was studied. Transcriptional profiles were compared with age and gender matched, typically developing children from the general population (GP) or IQ matched children with mental retardation or developmental delay (MR/DD).
Keywords: autism analysis

Carboplatin-induced gene expression changes in vitro are prognostic of survival in epithelial ovarian cancer

We performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro, and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization.  Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level.  Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints.
Time-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure.  Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data.  Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors.

We aimed to analyze the effects of Wnt-1 overexpression on the mRNA expression profile of human melanoma in a mouse xenograft model and correlated the results with then presence or absence of lymphangiogenesis and metastasis. Affymetrix gene expression analysis revealed activation of canonical and non-canonical targets genes in response to Wnt-1 as compared with controls. In regard to lymphangiogenic factors, the amount of VEGF-C was the single best marker to correlate with the amount of lymph-angiogenesis.

Differentially expressed genes in brain tissue from HtrA2 knockout mice

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here we demonstrate that loss of HtrA2 results in transcriptional up-regulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinson’s disease patients’ brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases.

A Transcriptomic Analysis of NET1 (a RhoA GEF Exchange Factor) in AGS Gastric Cancer Cells

Stable knockdown of NET1, a RhoGEF, was achieved in AGS Gastric Cancer cells.  This gene is known to be overexpressed in the disease.
Knockdown was achieved using lentiviral shRNA particles.  Gene expression was compared between knockdown and scrambled shRNA treated control cells.
Cells were treated with and without LPA, a known activator of RhoA.

Loss of Syk in normal breast cells in vivo and in vitro:  gene expression and phenotypic switch to stem-cell like with induction of invadopodia

Morphological and gene microarray analysis following Syk knockdown in MCF10A reveals a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodia and stem/ progenitor cell marker expression.

These results support the role of Syk in limiting normal stem/progenitor proliferation and invasion during morphogenesis, and support the role of Syk as a critical tumor suppressor for breast cancer.

SIRT3 opposes metabolic reprogramming of cancer cells through HIF1a destabilization

Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates and redox potential required for the generation of biomass. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. SIRT3 loss promotes a metabolic profile consistent with high glycolysis required for anabolic processes in vivo and in vitro. Mechanistically, SIRT3 mediates metabolic reprogramming independently of mitochondrial oxidative metabolism and through HIF1a, a transcription factor that controls expression of key glycolytic enzymes. SIRT3 loss increases reactive oxygen species production, resulting in enhanced HIF1a stabilization. Strikingly, SIRT3 is deleted in 40% of human breast cancers, and its loss correlates with the upregulation of HIF1a target genes. Finally, we find that SIRT3 overexpression directly represses the Warburg effect in breast cancer cells. In sum, we identify SIRT3 as a regulator of HIF1a and a suppressor of the Warburg effect.

Identification of p130Cas/ErbB2 dependent invasive signatures in transformed mammary epithelial cells.

Understanding transcriptional changes during cancer progression is of crucial importance to develop new and more efficacious diagnostic and therapeutic approaches. It is well known that ErbB2 is over-expressed in about 25% of human invasive breast cancers. We have previously demonstrated that p130Cas over-expression synergizes with ErbB2 in mammary cell transformation and promotes ErbB2-dependent invasion in three-dimensional (3D) cultures of human mammary epithelial cells.  Here, by comparing coding and non-coding gene expression profiling, we define the invasive signatures associated with concomitant p130Cas over-expression and ErbB2 activation in 3D cultures of mammary epithelial cells. Specifically, we have found that genes involved in amminoacids synthesis (CBS and PHGDH), cell motility, migration (ITPKA, PRDM1), and angiogenesis (HEY1) are up-regulated while genes involved in the inflammatory response (SAA1, S100A7) are down-regulated. In parallel, we have shown that the expression of specific miRNAs is altered. Among these, mir-200b, miR-222, miR-221and miR-424 are up-regulated while miR-27a, miR-27b and miR-23b are down-regulated. Overall this study present, for the first time, gene expression changes underlying the invasive behaviour following p130Cas over-expression in an ErbB2 transformed mammary cell model.

Regulation of gene expression in IECs by HDAC3 under germ-free conditions

The development and severity of inflammatory bowel diseases (IBD) and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria.  However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that intestinal epithelial cells (IECs) isolated from IBD patients exhibit decreased expression of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3). Further, genome-wide analyses of murine IECs that lack HDAC3 (HDAC3ΔIEC) revealed that HDAC3 deficiency resulted in dysregulated gene expression coupled with alterations in histone acetylation.  Critically, conventionally-housed HDAC3ΔIEC mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria.  In addition, HDAC3ΔIEC mice exhibited significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 plays a central role in maintaining intestinal homeostasis.  Strikingly, rederivation of HDAC3ΔIEC mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis, and intestinal barrier function were largely restored in the absence of commensal bacteria.  Collectively, these data indicate that the HDAC3 is a critical factor that integrates commensal bacteria-derived signals to calibrate epithelial cell responses required to establish normal host-commensal relationships and maintain intestinal homeostasis.

Male 130 day old rat adipose tissue from control and obese rats

Transcriptional profiling of liver samples from Lmna Gly609Gly knock-in mice

Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a point mutation in the LMNA gene that activates a cryptic donor splice site and yields a truncated form of prelamin A called progerin. Small amounts of progerin are also produced during normal aging. Studies with mouse models of HGPS have allowed the recent development of the first therapeutic approaches for this disease. However, none of these earlier works have addressed the aberrant and pathogenic LMNA splicing observed in HGPS patients because of the lack of an appropriate mouse model. We report herein a genetically modified mouse strain that carries the HGPS mutation. These mice accumulate progerin, present histological and transcriptional alterations characteristic of progeroid models, and phenocopy the main clinical manifestations of human HGPS, including shortened life span and bone and cardiovascular aberrations. By using this animal model, we have developed an antisense morpholino–based therapy that prevents the pathogenic Lmna splicing, dramatically reducing the accumulation of progerin and its associated nuclear defects. Treatment of mutant mice with these morpholinos led to a marked amelioration of their progeroid phenotype and substantially extended their life span, supporting the effectiveness of antisense oligonucleotide–based therapies for treating human diseases of accelerated aging.

Effect of maternal exposure to dioxin on the pituitary and hypothalamic expression of genes in PND70 male pups

Our previous studies have revealed that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 μg/kg) at gestational day (GD) 15 reduces the pituitary synthesis of luteinizing hormone (LH) during late fetal and early postnatal period, leading to imprinting of defects in sexual behaviors at adulthood. However, it remains obscure how the attenuation of pituitary LH links to sexual immaturity. To address this issue, we firstly performed a DNA microarray analysis to identify the gene(s) responsible for dioxin-induced sexual immaturity, using the pituitary and hypothalamus of male pups, at the age of postnatal day (PND)70, born from TCDD-treated dams. Among the reduced genes, we focused on gonadotropin-releasing hormone (GnRH) in the hypothalamus, because of its role in sexual behaviors suggested so far. The present study strongly suggests that maternal exposure to TCDD fixes the status of the lowered expression of GnRH in the offspring by reducing steroidogenesis at perinatal stage, and this is the mechanism for the imprinting of defects in sexual behaviors at adulthood.

Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia

Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. The molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that analysis on the changes in gene expression could help to delineate such mechanisms. In addition, the differences that can be anticipated between CCH and CIH could be potentially dissected.  Current study used CCH and CIH mouse models combined with cDNA microarrays to determine the changes of gene expression in CCH or CIH mice heart.
Keywords = heart

Equivalence of biologic responses induced in the mouse uterus by E2, GEN, and DES

Three independent replicates of 4 groups of immature (19/20 days of age) Alpk:APfCD-1 mice were treated with arachis oil (AO) vehicle, 2.5ug/kg 17beta-estradiol (E2), 2ug/kg diethylstilbestrol (DES), or 50mg/kg genistein (GEN), via 3 daily subcutaneous injection, and sacrificed 72hr after initial dose.

STING molecule has been reported to be important adaptor molecule for cytosolic DNA sensing. We investigated gene expression by cytosolic DNA stimulation using bone marrow derived dendritic cells. We comparared gene expression profile between WT and STING knock out BMDCs after cytosolic DNA stimulation.

Effect of PGE receptor EP3 subtype agonist on mouse asthma model

We examined the effect of PGE receptor EP3 subtype on mouse OVA-induced asthma model. We treat EP3 agonist 3h after OVA challenge.  After 24 h of 3rd OVA challenge, whole lung was isolated and the gene expression change was determined.

Low-density lipoprotein receptor-5 contributes to whole body metabolism by regulating fatty acid metabolism in the osteoblast

Wnt signaling is critical for normal skeletal development as well as whole-body metabolic function.  In this study, we described a previously unrecognized function for Wnt-Lrp5 signaling in the osteoblast that allows bone to acquire the resources necessary to fuel bone formation.  Mice lacking the Lrp5 co-receptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass and also develop peripheral adiposity with corresponding reductions in energy expenditure.  Conversely, mice expressing a high-bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels.  In this context, Wnt-initiated signals downstream of Lrp5, but not Lrp6, regulate the expression of key enzymes required for fatty acid β-oxidation.  These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate-specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism

Glaucoma is a neurodegenerative disease in which vision is lost as a result of the apoptosis of retinal ganglion cells. When the trabecular meshwork cells,  that regulate aqueous humor outflow, are stressed as they are in some forms of glaucoma, they considerably up-regulated secretion of a protein called myocilin into the aqueous. The function of this protein is unclear; but since some aqueous humor flows to the posterior of the eye, the effects of myocilin will also be felt by the retinal cells. We have a transgenic mouse that secretes large amounts of myocilin into aqueous humor. In these mice, there is a deposition of myocilin on membranes of certain cells suggesting myocilin might have some signaling functions. This signaling function could explain why stresses that increase intraocular pressures can increase the likelihood for glaucoma, cause the myocilin to be secreted at very high levels by the trabecular meshwork. Myocilin may be important in treating glaucoma.
This experiment will determine if myocilin is altering gene expression in the retina. The results should show variations in expression levels and will give us some indication of the pathways that are important to preserve retinal ganglion cells after a diagnosis of glaucoma.
Our hypothesis is that myocilin is acting like a signaling protein in the eye. It acts not only in the anterior segment but also in retina as a result of the flow of some aqueous to the back of the eye. This signaling function is protective to certain ocular cells. This function of myocilin may influence cells in the retina and may counter the apoptotic signals in the retinal ganglion cells that occur during glaucoma.
Retinas from transgenic mice and from mice of the same strain that do not have the transgene will be dissected. The mice will be three weeks of age. Because of the small size of the retina, samples will be pooled to obtain the RNA necessary to run the microarray. Three control and three transgenic samples will be run and compared with each other. We have data indicating that myocilin is at high levels in the aqueous humor of the transgenic animals.

Sox3 has been shown to be expressed within neural progenitors of the developing mouse central nervous system. However, identification of Sox3 targets within neural progenitors has remained elusive.

Using microarrays we compared populations of in vitro derived mouse neural progenitor cells, with or without Sox3, to identify putative Sox3 neural progenitor targets.

Pre-B cell leukemia homeobox protein Pbx1 is associated with lupus susceptibility in mice and humans

Sle1a.1 is part of the Sle1a lupus susceptibility locus which results in the production of activated and autoreactive CD4+ T cells as well as a reduction in the peripheral regulatory T cell (Treg) pool.  Sle1a.1 CD4+ T cells showed a defective response to retinoic acid (RA) expansion of TGFβ-induced Tregs.  At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d over-expression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells, and to decrease their apoptotic response to RA.  PBX1-d is expressed more frequently in lupus patients than in healthy controls, and its presence correlates with an increased memory T cell population.  These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

Expression data from Mayo Clinic Pancreatic Tumor and Normal samples

We used microarrays to identify the expression differences of FKBP5 gene between the pancreatic tumor and normal samples.On average normal samples had more FKBP5 expression compared to tumor samples

A microRNA network regulates expression and biosynthesis of CFTR and CFTR-ΔF508.

Production of functional proteins requires multiple steps including gene transcription and post-translational processing.  MicroRNAs (miRNA) can regulate individual stages of these processes.  Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain.  We discovered that microRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A.  Treating airway epithelia with a miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl- permeability independently of elevated mRNA levels.  A miR-138 anti-miR had the opposite effects.  Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis.  The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl- transport to cystic fibrosis airway epithelia.  This novel miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process broader than previously recognized.  This discovery also provides new therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Olfactory epithelium of SR-A mutant mice following bilateral olfactory bulbectomy

Sham and bilateral OBX were performed on wild-type and SR-A mutant mice.
Tissue collected 2, 8, 16, and 48 hours following OBX. Expression profiles were determined using Affynetrix MG U74Av2 chips.

DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours, compared to control cells.

Ozone is a very common environmental pollutant and has been associated with the exacerbation of cardiopulmonary diseases like asthma. In this study the molecular mechanisms underlying the effect of ozone on airways hyperpermability were investigated. Two strains of mice, HeJ (Tlr4 mutant) and OuJ (wildtype) were exposed continuously to air or 0.3ppm of ozone. Lungs were removed and RNA was collected to generate expression profiles.

Gene Profiling of Endometrium Reveals Progesterone Resistance and Candidate Genetic Loci in Women with Endometriosis

The transition of regularly cycling endometrium from the proliferative or Estrogen-dominant phase of the menstrual cycle to the Progesterone-dominant Early and Mid Secretory phases requires wide-spread changes in gene expression that shift the endometrium from a proliferative capacity to a differentiated 'decidual' phenotype in preparation for implantation.  This process appears delayed in women with severe endometriosis, suggestive of a progesterone resistant endometrium in this disease.

Regulatory consequences of TARBP2 over-expression in MDA-231 cell-line

Transient transfection of TARBP2 expression vectors (along with GFP controls) followed by transcriptomic profiling revealed that over-expression of TARBP2 resulted in a significant enrichment of sRSE-carrying transcripts among the down-regulated transcripts.

E12.5 CD9+ Mouse Placental Trophoblast Microarray, Wild-type vs c-Met KO

The placenta serves as the structural interface for nutrient and waste exchange for proper fetal development. Although defects in placental function result in various placental disorders, molecular mechanisms orchestrating placental development and function are poorly understood. Gene targeting studies have shown that Hgf or c-Met KO embryos exhibit growth retardation and markedly smaller size of the placenta, and die by E14.5. Stem/progenitor cells in various tissues express c-Met and they participate in morphogenesis and tissue repair. Thus, we hypothesized that the HGF/c-Met signaling pathway is essential for the emergence, proliferation, and/or differentiation of putative stem/precursor cells of labyrinth trophoblasts at the midgestation stage.
To examine the downstream mechanisms of HGF/c-Met signaling pathway that regulate placental labyrinth development, we performed microarray analysis and compared the transcriptional profiles of wild-type and c-Met g-KO placentas. The highly enriched gene ontology categories among the transcripts that were down-regulated in the mutant placentas were related to cell cycle, transcription, and placenta development. Surprisingly, the most highly enriched GO category among the up-regulated genes was immune response. Furthermore, genes classified as “unsaturated fatty acid metabolic process” were also significantly enriched among the up-regulated genes. This expression data suggested that HGF/c-Met signaling pathway positively regulates progression of cell cycle and transcription of placenta specific genes, and negatively regulates inflammatory reaction and fatty acids synthesis in the trophoblasts, thereby coordinating many critical cellular processes in the placenta.

FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis

Notch is normally activated by cleavage and nuclear translocation of its intracellular domain (ICN1), which turns on downstream target genes. Human T cell acute lymphoblastic leukemia (T-ALL), an aggressive immature T cell malignancy, is associated with Notch 1 gain-of-function mutations in more than 50% of the cases. Efforts to date to identify direct Notch1 targets have been confounded by the lack of a method to turn Notch1 on in a controlled fashion in T-ALL cells that are poised to respond to Notch signals. Of note, because Notch signaling activates transcriptional repressors that feedback to dampen the expression of many target genes (a process referred to as incoherent logic), it is likely that many direct targets are missed in Notch off analyses, which are further complicated by an inability to identify direct targets in a clear-cut fashion. We have overcome this limitation by developing a GSI washout method that results in the rapid translocation of activated Notch1 to the nucleus. We intend to use this method to study the assembly and loading of transcriptional complexes onto downstream targets, the kinetics of target activation. To date, our efforts have been devoted to comparing the gene expression signature of Notch-on and Notch-off in the human T-ALL cell line CUTLL. In addition to previously identified Notch1 target genes, we have also identified a series of novel genes upregulated by GSI washout in the presence of cycloheximide, suggesting that they are likely to be direct targets.
Additional controls included transduction of cells with dominant negative MAML1, a specific antagonist of canonical Notch1 signaling, prior to Notch1 reactivation, and a mock GSI  washout to control for cycloheximide effects.

Expression data from control and Ascorbic Acid (AA) stimulated Mc-3T3-E1 osteoblasts

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation.  Collagen synthesis and secretion is markedly upregulated upon Ascorbic Acid (AA) stimulation.  Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone secretion and deposition.  RabGTPases are major regulators on protein trafficking throughout the cell.  In this study, we identified the Rab GTPases that are upregulated during 5-day AA differentiation of OBs using microarray analysis, namely Rab1, Rab3d and Rab27b.
We used microarrays to detail the global programme of gene expression underlying procollagen production and trafficking and identified up-regulated genes during this process.

Gene expression in LPS stimulated IkappaB-beta knockout bone marrow derived macrophage (BMDM)

Inflammation is beneficial when it is part of the innate immune response, but harmful when it occurs in an unregulated, chronic manner. We now report that IkappaB-beta, a member of the classical IkappaB family, serves a dual role of both inhibiting and facilitating the inflammatory response. IkappaB-beta degradation releases NF-kappaB dimers which upregulate proinflammatory target genes such as TNF-alpha. Suprisingly absence of IkappaB-beta results in a dramatic reduction of TNF-alpha in response to LPS even though the activation of NF-kappaB is normal. The inhibition of TNF-alpha mRNA expression can be correlated to the absence of nuclear, hypophosphorylated-IkappaB-beta bound to p65:cRel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaB-beta acts through p65:cRel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaB-beta knockout mice are resistant to LPS induced septic shock and collagen-induced arthritis, and therefore blocking IkappaB-beta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha producting during the inflammatory response.

Transcriptional Adaptation to Clcn5 Knockout in Proximal Tubules of the Mouse Kidney

Dent disease has multiple defects attributed to proximal tubule malfunction including low molecular weight proteinuria, aminoaciduria, phosphaturia and glycosuria. In order to understand the changes in kidney function of the Clc5 transporter gene knockout mouse model of Dent disease, we examined gene expression profiles from proximal tubules of mouse kidneys.
Overall 720 genes are expressed differentially in the proximal tubules of the Dent Clcn5 knockout mouse model compared to those of control wild type mice. The fingerprint of these gene changes may help us to understand the phenotype of Dent disease.

mouse embryo, littermates, Tcof1, Treacher Collins sydnrome, comparative hybridisation

The object of this study was to identify genes transcriptionally upregulated and downregulated in response to Tcof1 haploin-sufficiency during mouse embryogensis
Keywords: mouse embryo, littermates, Tcof1, Treacher Collins sydnrome, comparative hybridisation

We generated mice with a transgenic BAC on a B6 background. The BAC contains Glo1, and the transgenic mice were found to overexpress Glo1.
We performed a microarray on whole-brain RNA of adult mice to identify differentially expressed genes resulting from Glo1 overexpression.

Expression data from PDE1C knockdown glioblastoma short-term cultures

In our lab we detected focal genomic amplification of PDE1C in 90% of short term GBM cultures. Knocking down of PDE1C was associated with compromised capacity opf these cultures to proliferate, migrate and invade. Therefore we carried out affymetric whole genome expression analysis to identify the down stream gene effectors of this function effects.

In order to characterize gene expression networks linked to AT1 angiotensin receptors in the kidney, we carried out genome-wide transcriptional analysis of  RNA from kidneys of wild-type (WT) and AT1A receptor-deficient mice (KOs) at baseline and after 2 days of angiotensin II infusion (1 ug/kg/min), using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. At baseline, 405 genes were differentially expressed (>1.5X) between WT and KO kidneys. Of these, more than 80% were up-regulated in the KO group including genes involved in inflammation, oxidative stress, and cell proliferation. After 2 days of angiotensin II infusion in WT mice, expression of ~805 genes was altered (18% up-regulated, 82% repressed). Genes in metabolism and ion transport pathways were up-regulated while there was attenuated expression of protective genes against oxidative stress including glutathione synthetase and mitochondrial SOD2. Angiotensin II infusion has little effect on blood pressure in KOs.  Nonetheless, expression of more than 250 genes was altered in kidneys from KO mice during angiotensin II infusion; 14% were up-regulated, while 86% were repressed including genes involved in immune responses, angiogenesis, and glutathione metabolism.  Between WT and KO kidneys during angiotensin II infusion, 728 genes were differentially expressed; 10% were increased and 90% were decreased in the WT group.  Differentially regulated pathways included those involved in ion transport, immune responses, metabolism, apoptosis, cell proliferation, and oxidative stress. This genome-wide assessment should facilitate identification of critical distal pathways linked to blood pressure regulation.

right lung gene expression of C57BL/6J, A/J and C3H/HeJ mice following bleomcyin exposure

Impact of curcumin on human monocytes (U937 cells) exposed to oxidative stress

Oxidative stress as a result of cigarette smoking is an important etiological factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. The activity of the transcriptional co-repressor Histone deacetylase-2 (HDAC2) is dramatically reduced in COPD and cells exposed to oxidative stress or cigarette smoke. Moreover, curcumin (diferuloylmethane), a dietary polyphenol, at concentrations upto 1uM specifically restores cigarette smoke extract (CSE)- or oxidative stress- impaired HDAC2 activity. The aim of this study was to therefore identify any links through those gene sets that are affected by oxidative stress and subsequent treatment with curcumin in order to determine whether or not this could explain the impact of curcumin on restoration of oxidant impaired HDAC2 transcriptional co-repressor activity.

Effects of a high-fat maternal diet and embryonic Cited2 genotype on gene expression and development in mouse

Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects, and partially penetrant left-right patterning defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2 deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume.
Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction.  We show that a high-fat diet reduces Pitx2c levels >2 fold in Cited2 deficient embryos and that Pitx2c deficiency results in cleft palate. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality and cleft palate.

Primart mouse embryonic fibroblasts (pass 3 to 4) were treated with tunicamycin (2 micrograms/ml) for specified times - genotypes were WILDTYPE and CHOP-/-. Each time point n=4

Vav proteins orchestrate a keratinocyte auto/paracrine program critical for skin tumorigenesis.

Cutaneous squamous tumors rely on autocrine/paracrine loops for proper fitness.  Targeting this Achilles’ heel is therefore considered a potential avenue for patient treatment.  However, the mechanisms that engage and sustain such programs during tumor ontogeny are poorly understood.  Here, we show that two Rho/Rac activators, the exchange factors Vav2 and Vav3, control the expression of an epithelial autocrine/paracrine program that regulates keratinocyte survival and proliferation as well as the creation of an inflammatory microenvironment.  Vav proteins are also critically involved in some of the subsequent autocrine signaling loops activated in keratinocytes.  The genetic inactivation of both Vav proteins reduces tumor multiplicity without hampering skin homeostasis, thus suggesting that pan-specific Vav therapies may be useful in skin tumor prevention and treatment.

DNA microarray analysis following gefinitib/luteolin administration to PC-3 cells

To further investigate the molecular mechanism of cell death induction by luteolin and/or gefitinib, we performed DNA microarray analysis, which may reveal the change of gene expression pattern following treatment with these chemicals. We examined PC-3 cells after 24h of treatment with 60μM luteolin and/or 60μM gefitinib because subG1 population has not appeared yet at this timing, which helps to avoid the secondary effects of apoptosis on the alteration of the gene expression pattern.

The Snf1-Related Kinase, Hunk, Is Essential for Mammary Tumor Metastasis

We previously identified a novel SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for Hunk in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer. Together, these findings establish a role for Hunk in metastasis and an in vivo function for this kinase.

Gene expression pattern of skin biopsies of epidermolysis bullosa simplex patients in comparison with control subjects

Tha altered biological pathways in Epidermolysis bulloda simplex, a rare monogenetic skin disease, have not been well characterized. Thus, the goal of this study is to characterize the expression profile of EBS patients compared with normal subjects using genomic expression analyses. Microarray analyses were performed with RNA isolated from skin biopsies. Robust multiarray analysis (RMA) normalization and Smyth’s moderated t test were used to select differentially expressed genes. Expression profiling comparisons show that 28 genes are differentially expressed in EBS patients compared to control subjects and 41 genes in EBS-DM compared to their matched controls. Nine genes involved in fatty acid metabolism and 2 genes in epidermal keratinisation are common altered expressed genes between the two subgroups. These two biological pathways contribute both to the formation of the cell envelope barrier and seem to be defective in the severe EBS phenotype. This study demonstrates, for the first time, the relevance of metabolic cluster, specifically fatty acid metabolism in EBS biology. Difference of expression for three (AWAT2, ELOVL , and SPRR4 ) of the five selected genes were validated using real-time reverse transcription–polymerase chain reaction. To our knowledge, the distinctive pattern of gene expression that characterizes EBS versus healthy skin tissue has never been reported.

Comparative transcriptome profiling of Amyloid Precursor Protein APP family members in the adult cortex

The β-amyloid precursor protein APP and the related APLPs, undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer disease (AD), the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase generated APP intracellular domain AICD, functions as a transciptional regulator in heterologous reporter assays, although its role for endogenous gene regulation has remained controversial.  To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators we performed a DNA microarray transcriptome profiling of the frontal cortex of adult wild type, APP-/-, APLP2-/- and APPsα knockin (KI) mice, APPα/α, expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included regulation of neurogenesis, regulation of transcription and regulation of neuron projection development. Comparative analysis of transcriptome changes and qPCR validation identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity related genes that were down-regulated in knock-out cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including Bace1, Kai1, Gsk3b, p53, Tip60 and Vglut2. Only Egfr was slightly up-regulated in APLP2-/- mice. Comparison of APP-/- and APPα/α with wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2-/- on different genetic backgrounds revealed that background related transcriptome changes may dominate over changes due to the knockout of a single gene.   Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.

Microarray analysis  of in vivo inhibition of mir-122 with an antisense oligonucleotide.

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRs is a powerful technique for their functionalization.  To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in vivo using a 2’-O-methoxyethyl phosphorothioate ASO.  This microrarray experiment was used to identify changes in mRNA levels due to the inhibition of miR-122 that accompanied phenotypic observations of reduced cholesterol and triglycerides, increased fatty acid oxidation, and decreased fatty acid and cholesterol synthesis rates observed in hepatocytes.
Keywords: single treatment vs. control

Gene expression profiles in white blood cells in response to exercise

White blood cells (WBCs) express tens of thousands of genes. Their expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes which may serve as surrogate markers for monitoring exercise and training load.
Five male probands performed an exhaustive treadmill test (ET) at 80% of their VO2max, and a moderate treadmill test (MT) at 60% VO2max for exactly the same time one to two week later. White blood cells (WBCs) were isolated by the erythrocyte lysis method. Gene expression profiles were measured using the Affymetrix GeneChip® technology. After scaling, normalisation, and filtering groupwise and pairwise comparisons of gene expression intensities were performed and validated by real-time PCR.
We found 450 genes up-regulated and 150 down-regulated (> 1.5-fold change; ANOVA with Benjamini-Hochberg correction for multiple testing, p<0.05) upon exhaustive exercise. Analysis of mean expression levels after MT showed that the extent of up- and down-regulation was workload dependent. The genes for the stress proteins HSPA1A, HSPH1 and the matrix metalloproteinase MMP-9 showed the most prominent increases whereas the mRNA concentrations of the YES1 oncogene (YES1), of CD160 (BY55), and of a member of the mitochondrial electron transport chain (ATP5s) were most strongly reduced. Remarkably, we could largely reproduce the data from a previous report by Connolly et al. (01) even though we used considerably different methodology. After acute exercise the genes with increased expression levels were highly significantly associated with the gene ontology terms heat-shock proteins, apoptosis and inflammation.
The results demonstrate that gene expression changes in WBCs can reflect intensity and duration of exercise. Further analysis is needed to confirm the applicability of expression fingerprints as useful tools for monitoring exercise and training loads in order to avoid training associated health risks.

Gene expression changes in the microdissected colon epithelium are similar to those of intact colon in the Il10-/- mouse model of IBD

Epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are involved in the initiation and maintenance of intestinal inflammation. LMD is a technique that allows the extraction of specific cell types, such as colonic epithelial cells, to analyse gene expression. LMD of colon epithelial cells followed by microarray analysis could be of more value than microarray analysis of intact colon for determining which pathways are active in the colon mucosa in the early and late stages of inflammation due to increased sensitivity to changes in specific cell populations. An experiment was performed using microarray analysis of intact colon samples and microdissected colon epithelial cell samples from Il10-/- and C57BL/6J mice at 6 and 12 weeks of age to study the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of colitis.
Results showed that intact colon and colon epithelial cell gene expression profiles were similar in terms of pathways between Il10-/- and C57BL/6J mice at 12 weeks of age and between Il10-/- mice at 12 and 6 weeks of age. More immune-related pathways were identified at 6 weeks of age in epithelial cells than intact colon. This suggests that LMD and targeting of specific cell types may be of particular use when studying the early stages of inflammation before the intestinal morphology is detectably altered, by increasing analysis sensitivity to mucosal gene expression changes.

bioinformatics;gene expression;memory;synaptic plasticity;myelin;inflamation;aging;repeat sample

Male Fischer 344 rats aged 4 months (young, n=10), 14 months (mid-aged, n=10), and 24 months (aged, n=10) were trained sequentially on two tasks: Morris Spatial Water Maze (SWM) and Object Memory Task (OMT). The training/testing sequence lasted 7 d, and hippocampal tissue was collected 24 hr later. Training and testing occured on each day except for days 2 and 3 of the 7 d sequence.
(01/10/05: Series was updated to correct mislabeling of all sample signal values within the Young Treatment Group)

To investigate the role of HES1 during glucocorticoid signaling in the liver, we employed whole-genome microarray expressions in liver mRNA extracted from control and Hes1-liver knockout male mice (HESKOL) that had been adrenalectomized to remove endogenous glucocorticoids.

C1q expression increases significantly in the peripheral blood of patients with active tuberculosis compared to healthy controls and individuals with latent TB infection. The percentage of C1q-expressing CD14 positive cells is significantly increased in active TB patients. C1q expression in the peripheral blood correlates with sputum smear positivity in tuberculosis patients and is reduced after anti-tuberculosis chemotherapy. Notably, receiver operating characteristic analysis showed that C1qC mRNA levels in peripheral blood efficiently discriminate active from latent tuberculosis infection and healthy controls. Additionally, C1qC protein level in pleural effusion shows improved power in discriminating tuberculosis from non-tuberculosis pleurisy when compared to other inflammatory markers, such as IL-6 and TNF-α
C1q expression correlates with active disease in human tuberculosis. C1q could be a potential diagnostic marker to discriminate active tuberculosis from latent tuberculosis infection as well as tuberculosis pleurisy from non-tuberculosis pleurisy.

Substantial evidence implicates IGF-I signaling in the development and progression of breast cancer.  To identify transcriptional targets of IGF action in breast cancer cells, we performed gene expression profiling (>22,000 RNA transcripts) of IGF-I-stimulated MCF-7 cells, a well characterized breast cancer cell line that is highly responsive to IGFs. We defined an IGF-I gene signature pattern of hundreds of genes either up-regulated or down-regulated at both 3 and 24 hrs in vitro.  After removing genes considered generic to cell proliferation, the signature was examined in four different public profile datasets of clinical breast tumors (representing close to 1000 patients), as well as in profile datasets of experimental models for various oncogenic signaling pathways. Genes with early and sustained regulation by IGF-I were highly enriched for transcriptional targets of the estrogen, Ras, and PI3K/Akt/mTOR pathways.  The IGF-I signature appeared activated in most estrogen receptor-negative (ER-) clinical breast tumors and in a substantial subset (~25%) of ER+ breast tumors.  Patients with tumors showing activation of the IGF-I signature tended to have a shorter time to disease recurrence (including patients not receiving adjuvant therapy), both when considering all patients and the subset of ER+ patients. We found evidence for cross-talk and common transcriptional endpoints between the IGF-I and estrogen systems.  Our results support the idea that the IGF-I pathway is one mechanism by which breast tumors may acquire hormone independence and a more aggressive phenotype.
Keywords: two group comparison

Affymetrix gene expression of Transitional Type-1 B-cells from Wild-type (CBA), Xid and Btk KO mice

Splenic Transitional Type-1 B-cells from CBA wild-type mice, X-linked immunodeficiency mice and Bruton's tyrosine kinase knock-out mice. Two replicates where run on Affymetrix 420 2.0 arrays for CBA wild-type,  Xid samples and the Btk KO samples.
Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional type 2 (T2) cells are also located in distinct anatomic locations. Based on a limited number of markers it has previously been reported that the earliest phenotypic sign of Btk deficiency is manifested at the T2 stage in mice. Here, we report on distinct genome-wide transcriptomic signature differences found in T1 B-lymphocytes from Btk-defective compared to normal mice and demonstrate that Btk deficiency is visible already at this stage.

Gene expression signature of EGR3 silencing in M12 human prostate cancer cells

EGR3 expression is upregulated in human prostate cancer compared to normal prostate tissue and is associated with absence of relapse, while low EGR3 expression in tumors is predicitive of disease relapse (Pio et al., PLOS One 2013; 8(1):e54096). However the function of EGR3 in prostate cancer is unknown. Human prostate cancer cells M12 containing high levels of EGR3 were used for shRNA-mediated silencing of EGR3. Gene expression analysis of EGR3 knockdown cells reveals a role in inflammation and the existence of a crosstalk with the NFkB pathway.

Expression data from MALP-2-stimulated macrophages from wild-type, IRAK-2-/- and IRAK-1-/IRAK-2-/- mice

IL-1R-associated kinases (IRAKs) participate in Toll-like receptor (TLR) signal transduction. MALP-2 is a TLR2 ligand, and stimulation of macrophages with MALP-2 activates expression of various genes including proinflammatory cytokines.
We used microarrays to examine influence of IRAK-2 or IRAK-1/IRAK-2 deficiency in MALP-2-inducible gene expression.

Temporal profiling of gene expression in cochleae of wild type and alpha9 null mice

Efferent inhibition of cochlear outer hair cells is mediated by nicotinic cholinergic receptors containing alpha9 (a9)  and alpha10 subunits. Mice lacking a9 nicotinic subunits fail to exhibit classic olivocochlear responses and are characterized by abnormal synaptic morphology at the base of outer hair cells. To detail molecular changes induced upon the loss of a9 subunit, we sampled cochlear RNA from wild type and a9 null mice at postnatal (P) days spanning periods of  synapse formation and maturation (P3, P7, P13 and P60). Our findings point to a delay in cochlear maturation starting at the onset of hearing (P13), as well as an up-regulation of various GABA receptor subunits in adult mice lacking the a9 nicotinic subunit.

It has been shown that dexamethasone (Dex) impairs the normal lung septation that occurs in the early postnatal period. Treatment with retinoic acid (ATRA) abrogates the effects of Dex. To understand the molecular basis for the Dex indiced inhibition of the formation of the alveoli and the ability of ATRA to prevent the inhibition of septation, gene expression was analyzed in 4-day old mice treated with diluent (control), Dex-treated and ATRA+Dex-treated.

Gene expression microarray profiles of cumulus cells in lean and overweight-obese polycystic ovary syndrome patients

Objective: The etiology of PCOS is mostly unknown. Existing data support both genetic and environmental factors in its pathogenesis.  Design: Prospective case - control study. Setting: University Hospital. Patients:  25 patients undergoing IVF-ICSI treatment.  Intervention: Genome-wide oligonucleotide microarray technology was used to study differential gene-expression patterns of cultured human cumulus cells from IVF patients divided into 4 groups according to disease state (PCOS vs. Control) and BMI (Obese vs. Lean).  Results: Two differential PCOS gene expression profiles were established: Lean-Type was formed by comparing PCOS lean (PL) vs. non-PCOS lean (NL) individuals; Obese-Type was formed by comparing PCOS obese (PO) vs. non-PCOS (NO) obese patients.   Conclusions:  Different molecular pathways are associated with PCOS in Lean and Obese individuals, as demonstrated by gene expression profiling of cumulus cells. Our findings provide insights into the molecular pathogenesis of PCOS.
We used microarrays to study the gene expression of human cultured cumulus cells. We  compared the genes expression of lean PCOS,  Obese PCOS, lean controls and obese controls. Different molecular pathways are associated with PCOS in Lean and Obese patients.

Signaling of Neisseria meningitidis MC58 mutants to primary human umbilical vein endothelial cells (HUVEC)

We examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of primary human umbilical vein endothelial cells (HUVEC) infected by piliated and adherent wild-type (WT), frpC/frpA-deficient mutant, or the non-adherent (ΔpilD) N. meningitidis MC58 bacteria defective in production of the type IV pilus, respectively. Surprisingly, no significant difference was found between the transcriptomes of HUVECs infected by bacteria producing, or not the RTX FrpC and FrpA proteins, thus failing to provide any hints on their biological activity. In contrast, pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response or adhesion. In particular, genes for signaling pathway proteins involved in early embryonic development, such as transforming growth factor-β (TGF-β)/Smad, Wnt/β-catenin, and Notch/Jagged were found to be upregulated upon adhesion of N. meningitidis together with genes for a number of transcription factors. This reveals that adhering piliated meningocci manipulate signaling pathways controlling human cell proliferation, survival and defense mechanisms, while establishing a commensal relationship with the host.
Keywords: time course, infection response, genetic modification

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their HLA genes. This has led to the proposed use of histocompatible, patient-specific stem cells, however the preparation of many different stem cell lines for clinical use is a daunting task.  Here we develop two distinct genetic engineering approaches that address this problem.  First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line.  A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population.  Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the B2M gene in ESCs.  Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies.  These B2M-/- ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes.  Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

Induction of the IL-9 gene by HTLV-I Tax stimulates the spontaneous proliferation of primary adult T-cell leukemia cells

The mechanism whereby IL-9 stimulated monocytes, promote proliferation of co-cultured ATL cells remains undefined. To obtain preliminary clues concerning this contribution, transcription profiles were obtained between monocytes derived from ATL patients and those derived from healthy donors as assessed by cDNA microarray.
Keywords: disease state design

Lung adenocarcinoma subtypes definable by lung development-related miRNA expression profiles in association with clinicopathologic features [mRNA analysis - Whole Human Genome 4 x 44K Microarray]

Accumulation of genetic and epigenetic changes alters regulation of a web of interconnected genes including miRNAs, which confer hallmark capabilities and characteristic cancer features. In this study, the miRNA and mRNA expression profiles of 126 non-small cell lung cancer specimens were analyzed, with special attention given to the diversity of lung adenocarcinomas. Of those, 76 adenocarcinomas were classified into two major subtypes, developing lung-like and adult lung-like, based on their distinctive miRNA expression profiles resembling those of either developing or adult lungs, respectively. A systems biology-based approach using a Bayesian network and nonparametric regression was employed to estimate the gene regulatory circuitry functioning in patient tumors in order to identify subnetworks enriched for genes with differential expression between the two major subtypes. miR-30d and miR-195, identified as hub genes in such subnetworks, had lower levels of expression in the developing lung-like subtype, while introduction of miR-30d or miR-195 into the lung cancer cell lines evoked shifts of mRNA expression profiles towards the adult lung-like subtype. Conversely, the influence of miR-30d and miR-195 was significantly different between the developing lung- and adult lung-like subtypes in our analysis of the patient dataset. In addition, RRM2, a child gene of the miR-30d-centered subnetwork, was found to be a direct target of miR-30d. Together, our findings reveal the existence of two miRNA expression profile-defined lung adenocarcinoma subtypes with distinctive clinicopathologic features and also suggest the usefulness of a systems biology-based approach to gain insight into the altered regulatory circuitry involved in cancer development.

Deciphering the Wnt-dependent gene signature in colorectal cancer cells

Microarray-based gene expression data were generated from RNA from Ls174T colorectal carcinoma cell lines in which Wnt-dependent transcriptional activity can be abrogated by inducible overexpression of a dominant-negative form of Tcf4 or siRNA against β-catenin.

Expression analysis of ZBP-89 deficient human primary erythroid progenitors

The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood.  Closely spaced binding sites for GATA, NF-E2/maf and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression.  We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner.  Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system.  We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation, and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci.  ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression.  ZBP-89 physically associates with the histone acetyltransferases (HATs) p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci.  Lentiviral shRNA knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation.  Addition of the HDAC inhibitor valproic acid partially reverses the reduced globin gene expression.  These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation.
Keywords: Human primary erythroid progenitors

Expression data from Patients with Bipolar (BP) Disorder and Matched Control Subjects

There are currently no biological tests that differentiate patients with bipolar disorder (BPD) from healthy controls. While there is evidence that peripheral gene expression differences between patients and controls can be utilized as biomarkers for psychiatric illness, it is unclear whether current use or residual effects of antipsychotic and mood stabilizer medication drives much of the differential transcription. We therefore tested whether expression changes in first-episode, never-medicated bipolar patients, can contribute to a biological classifier that is less influenced by medication and could potentially form a practicable biomarker assay for BPD.
We employed microarray technology to measure global leukocyte gene expression in first-episode (n=3) and currently medicated BPD patients (n=26), and matched healthy controls (n=25). Following an initial feature selection of the microarray data, we developed a cross-validated 10-gene model that was able to correctly predict the diagnostic group of the training sample (26 medicated patients and 12 controls), with 89% sensitivity and 75% specificity (p<0.001). The 10-gene predictor was further explored via testing on an independent test cohort consisting of three pairs of monozygotic twins discordant for BPD, plus the original enrichment sample cohort (the three never-medicated BPD patients and 13 matched control subjects), and a sample of experimental replicates (n=34). 83% of the independent test sample was correctly predicted, with a sensitivity of 67% and specificity of 100% (although this result did not reach statistical significance). Additionally, 88% of sample diagnostic classes were classified correctly for both the enrichment (p=0.015) and the replicate samples (p<0.001).

Selective enhancement and repression of glucocorticoid receptor signaling by coregulator Hic-5

The glucocorticoid receptor (GR) recruits many coregulators via the well characterized AF2 interaction surface in the GR ligand binding domain, but LIM domain coregulator Hic-5 binds to the relatively uncharacterized tau2 activation domain in the hinge region of GR. Requirement of Hic-5 for glucocorticoid-regulated gene expression in U2OS osteosarcoma cells was defined by Hic-5 depletion and global gene expression analysis. Hic-5 depletion had selective and dramatic effects, positive and negative, on both activation and repression of GR target genes. For some hormone-induced genes, Hic-5 facilitated recruitment of the Mediator complex and RNA polymerase II. In contrast, many genes were not regulated by hormone until Hic-5 was depleted. On these genes Hic-5 acted at a very early step of the regulatory process, preventing efficient GR binding on enhancers, chromatin remodeling, and thus preventing glucocorticoid-driven transcriptional regulation. Overall, Hic-5 has selective and diverse roles on GR target genes, functioning as coactivator on some genes and corepressor on others, and either facilitating or opposing the glucocorticoid-driven actions of GR. Hic-5 exhibits multiple mechanisms of action, either regulating GR binding to DNA and chromatin remodeling, or facilitating later steps in transcription complex assembly.
We investigate the relationship between GR and Hic5 and identify classes of genes that respond differently when cells are induced with hormone and when Hic5 is knocked down

Expression data from affected skin from psoriasis mouse models and normal skin from control mice

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments.   However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional profiling is used to compare gene expression patterns manifested by human psoriatic skin lesions with those that occur in five psoriasis mouse models (K5-Tie2, imiquimod, K14-AREG, K5-Stat3C and K5-TGFbeta1). While the cutaneous gene expression profiles associated with each mouse phenotype exhibited statistically significant similarity to the expression profile of psoriasis in humans, each model displayed distinctive sets of similarities and differences in comparison to human psoriasis. For all five models, correspondence to the human disease was strong with respect to genes involved in epidermal development and keratinization. Immune and inflammation-associated gene expression, in contrast, was more variable between models as compared to the human disease. These findings support the value of all five models as research tools, each with identifiable areas of convergence to and divergence from the human disease. Additionally, the approach used in this paper provides an objective and quantitative method for evaluation of proposed mouse models of psoriasis, which can be strategically applied in future studies to score strengths of mouse phenotypes relative to specific aspects of human psoriasis.
Global transcriptional profiling was utilized to evaluate the similarity between human psoriasis and the psoriasis-like phenotypes that develop in five mouse models (K5-Tie2, IMQ, K14-AREG, K5-Stat3C, K5-TGFbeta1)

Identification of Murine Uterine Genes Regulated in a Ligand-Dependent Manner by the Progesterone Receptor.

Progesterone (P4) acting through its cognate receptor, the progesterone receptor (PR), plays an important role in uterine physiology. The PR knockout (PRKO) mouse has demonstrated the importance of the P4-PR axis in the regulation of uterine function. To define the molecular pathways regulated by P4-PR in the mouse uterus, Affymetrix MG U74Av2 oligonucleotide arrays were used to identify alterations in gene expression after acute and chronic P4 treatments. In the analysis, retinoic acid metabolic genes, cytochrome P 450 26a1 (Cyp26a1), alcohol dehydrogenase 5, and aldehyde dehydrogenase 1a1 (Aldh1a1); kallikrein genes, Klk5 and Klk6; and specific transcription factors, GATA-2 and Cited2 [cAMP-corticosterone-binding protein/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail], were validated as regulated by the P4-PR axis. Identification and analysis of these responsive genes will help define the role of PR in regulating uterine biology.

Transcriptomic analysis of murine embryos lacking endogenous retinoic acid signaling

Retinoic acid (RA), an active derivative of the liposoluble vitamin A (retinol), acts as an important signaling molecule during embryonic development, regulating phenomenons as diverse as anterior-posterior axial patterning, forebrain and optic vesicle development, specification of hindbrain rhombomeres, pharyngeal arches and second heart field, somitogenesis, and differentiation of spinal cord neurons. This small molecule directly triggers gene activation by binding to nuclear receptors (RARs), switching them from potential repressors to transcriptional activators. The repertoire of RA-regulated genes in embryonic tissues is poorly characterized. We performed a comparative analysis of the transcriptomes of murine wild-type and Retinaldehyde Dehydrogenase 2 null-mutant (Raldh2-/-) embryos - unable to synthesize RA from maternally-derived retinol - using Affymetrix DNA microarrays. Transcriptomic changes were analyzed in two embryonic regions: anterior tissues including forebrain and optic vesicle, and posterior (trunk) tissues, at early stages preceding the appearance of overt phenotypic abnormalities. Several genes expected to be downregulated under RA deficiency appeared in the transcriptome data (e.g. Emx2, Foxg1 anteriorly, Cdx1, Hoxa1, Rarb posteriorly), whereas reverse-transcriptase-PCR and in situ hybridization performed for additional selected genes validated the changes identified through microarray analysis. Altogether, the affected genes belonged to numerous molecular pathways and cellular/organismal functions, demonstrating the pleiotropic nature of RA-dependent events. In both tissue samples, genes upregulated were more numerous than those downregulated, probably due to feedback regulatory loops. Bioinformatic clustering analysis allowed us to extract groups of genes displaying similar behaviors in mutant tissue samples. These data give an overview of the gene expression changes occurring under a state of embryonic RA deficiency, and provide new candidate genes and pathways for a better understanding of retinoid-dependent molecular events.

Gene expression profile of Dicer knockout in mouse DP thymocytes

The RNase III enzyme dicer is essential for the processing of microRNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability and translation in a wide range of organisms. To provide a model system to explore the role of dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional dicer allele. Deletion of dicer at an early stage of T cell development compromised the survival of αβ lineage cells, while the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of cytosine DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin) and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one-third of mammalian mRNAs are putative miRNA targets, dicer appears to be dispensable for CD4/8 lineage commitment, a process where epigenetic regulation of lineage choice has been well documented. Thus, although dicer appears critical for the development of the early embryo, it may have limited impact on the implementation of lineage-specific gene expression programs.

Expression changes in intestinal crypts upon deletion of beta-catenin

The Wnt signaling pathway is deregulated in over 90% of human colorectal cancers. β Catenin, the central signal transducer of the Wnt pathway, can directly modulate gene expression by interacting with transcription factors of the TCF/LEF-family. In the present study we investigate the role of Wnt signaling in the homeostasis of intestinal epithelium using tissue-specific, inducible beta-catenin gene ablation in adult mice. Block of Wnt/beta-catenin signaling resulted in rapid loss of transient-amplifying cells and crypt structures. Importantly, intestinal stem cells were induced to terminally differentiate upon deletion of beta-catenin resulting in a complete block of intestinal homeostasis and fatal loss of intestinal function. Transcriptional profiling of mutant crypt mRNA isolated by laser capture micro dissection confirmed those observations and allowed to identify genes potentially responsible for the functional preservation of intestinal stem cells.

A stably transfected inducible system for KLF4 was established in RKO cells as previously described (Chen et al., 2001; Chen et al., 2003).  This cell line, called EcR-RKO/KLF4, contains stably transfected receptors for the insect hormone, ecdysone (EcR), and a full-length mouse KLF4 cDNA driven by a promoter that responds to ecdysone and its analogue, ponasterone A.  Cells were treated with 5 µM ponasterone A (PA), for various time durations.  To control for the experiment, the vehicle, ethanol, was added for the same periods of time.
Keywords: Time course

Plasma Cells in Muscle in Inclusion Body Myositis and Polymyositis

Microarray data from muscle biopsy specimens from subjects with inclusion body myositis, polymyositis, and normals
Keywords: Research study

Comprehensive description of the transcriptome of hypothalamo-neurohypophyseal system in euhydrated and dehydrated rat

The hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei  are important integrative structures that regulate co-ordinated  responses to perturbations in cardiovascular homeostasis. Through  descending projections from parvocellular neurons to the brainstem,  the PVN acts as an autonomic 'premotor nucleus', regulating reflex  changes in sympathetic nerve activity that are involved in blood  pressure and blood volume regulation. Endocrine responses are  mediated through axonal projections from SON and PVN magnocellular  neurons (MCNs) to the capillaries of the posterior pituitary neural  lobe. In response to dehydration, a massive release of the  antidiuretic peptide hormone vasopressin (VP) into the circulation is  accompanied by a dramatic functional remodelling of the HNS. We have  used microarrays to comprehensively catalogue the genes expressed in  the PVN, the SON and the neurointermediate lobe (NIL) of the  pituitary gland. Further, we have identified transcripts that are  regulated as a consequence of dehydration, as well as RNAs that are  enriched in either the PVN or the SON. We suggest that these  differentially expressed genes represent candidate regulators and  effectors of HNS activity and remodelling.
Keywords: Dehydration

Expression data of EZH2-dependent genes in prostate cancer cell lines

EZH2 is frequently over-expressed in aggressive and metastatic solid tumors, including castration resistant prostate cancer (CRPC). We sought to determine EZH2-dependent gene expression programmes in prostate cancer progression, and found an intriguing functional switch of EZH2 from a repressor to an activator during CRPC development.
We used microarrays to detail the global profiling of gene expression that are differentially regulated upon EZH2 depletion in two different prostate cancer cell lines.

siRNA-mediated knockdown of Sp3 induces a pro-inflammatory pattern of cytokine production: relevance to MS

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system and is generally considered to be autoimmune in nature. We previously demonstrated that the transcription factor Sp3 is significantly down-regulated in immune cells from MS patients. The potential role of Sp3 down-regulation in MS pathogenesis is not well understood. The function of endogenous Sp3 was assessed in vitro after siRNA-mediated knockdown of its transcript in Jurkat cells. Sp3 protein levels were reduced an average of 70%. ELISA studies demonstrated decreased endogenous production of IL-10 and TGFβ1 and increased endogenous production of TNFα (p<0.05 in all assays).  Subsequent microarray analysis demonstrated significantly altered expression of 36 genes (p<0.001 for each gene) compared with control samples. Analysis showed differential expression (p<0.005) of 8 gene pathways.  Many of the genes and pathways that were regulated by Sp3 are involved in immune function, specifically with regard to apoptosis, cell-to-cell adhesion, integrin signaling, T-cell differentiation, and cytokine production. This study identifies mechanisms by which Sp3 may regulate immune function and suggests a basis for its potential contribution to MS disease pathogenesis.
Keywords: siRNA knockdown; Jurkat T-cells; Multiple Sclerosis

Expression data from mock- or LMO3 silenced differentiating adipose stem cells

In this study we aimed to gain further insight on the role of GCs in adipocyte differentiation. For the future drugability of candidate targets it is of utmost importance to find factors relevant to human biology. Thus, we analyzed the transcriptome of GC induced primary human adipose stem cells (hASC) to identify novel factors downstream of GC action
We used microarrays to detail the global programme of gene expression following glucocorticoid treatment and identified distinct classes of up- and downregulated genes during this process.

Gene expression profiling in BCR/ABL-expressing LSCs and BCR/ABL-expressing Hif1a-/-LSCs

Using a mouse model of chronic myelogenous leukemia (CML), here we report that HIF1α plays a crucial role in survival maintenance of leukemia stem cells (LSCs). Deletion of HIF1α impairs the propagation of CML through impairing cell cycle progression and inducing apoptosis of LSCs. Deletion of HIF1α results in elevated expression of p16Ink4a and p19Arf in LSCs, and knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. To further identify the pathways in which Hif1a regulates function of LSCs, we performed a comparative DNA microarray analysis using total RNA isolated from BCR-ABL-expressing wild type LSCs and BCR-ABL-expressing Hif1a-/- LSCs. The result was validated by quantitative real-time PCR analysis of non-BCR-ABL-expressing Lin-Sca-1+c-Kit+ cells, BCR-ABL-expressing wild type LSCs, and BCR-ABL-expressing Hif1a-/- LSCs.

The Differentiation and Stress Response Factor, XBP-1, Drives Multiple Myeloma Pathogenesis

Multiple myeloma (MM) evolves from highly prevalent premalignant condition termed Monoclonal Gammopathy of Undetermined Significance (MGUS).  We report an MGUS-MM phenotype arising in transgenic mice with Emu-directed expression of the unfolded protein/ER stress response and plasma cell development spliced isoform factor XBP-1s.  Emu-XBP-1s elicited elevated serum Ig and IL-6 levels, skin alterations and with advancing age, a significant proportion of Emu-xbp-1s transgenic mice develop features diagnostic of human MM including bone lytic lesions. Transcriptional profiles of Emu-xbp-1s B lymphoid and MM cells show aberrant expression of genes known to be dysregulated in human MM including Cyclin D1, MAF, MAFB, and APRIL. This genetic model coupled with documented frequent XBP-1s overexpression in human MM serve to implicate chronic XBP-1s dysregulation in the development of this common and lethal malignancy.
Keywords: XBP-1, MGUS, multiple myeloma, transgenic mouse

Overexpression of transcription factor to determine downstream targets.

Overexpression of USF1 in HEK293T cells in vitro to ascertain the genes downstream of USF1. Will identify direct targets as well as indirect targets of USF1.

A screen of 5 anti-inflammatory compounds for their effects in explanted, cultured rat spinal cord slices.  All injured (explanted) cords are cultured for 4 hrs.

Differential gene expression profiles between SUM149 cells transfected with control siRNA and SUM149 cells transfected with siRNA targeting tarzarotene-induced gene 1 (TIG1)

We identified tazarotene-induced gene 1 (TIG1) as a potential tumorigenic gene in IBC. To investigate the underlying mechanism by which TIG1 promotes tumor growth and invasiveness of IBC cells, we first sought to identify TIG1 functional partners by using DNA microarray analysis to compare gene expression profiles between SUM149 cells transfected with control siRNA and SUM149 cells transfected with siRNA targeting TIG1. We identified receptor tyrosine kinase Axl as a functional partner of TIG1.

Effect of histone deacetylase inhibitors on osteoblast gene expression

Background:
Osteoblast differentiation requires the coordinated stepwise expression of multiple genes.  Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure.  We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin).  Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation.

Results:
To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions.  The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays.  Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR.  Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced.  Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed.  We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.

Conclusions: This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1.  Also known as NHERF1, Slc9a3r1 is required for optimal bone density.  Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs.  These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

The pathogenesis of acne has been linked to multiple factors such as increased sebum production, inflammation, follicular hyperkeratinization, and the action of Propionibacterium acnes within the follicle. 13-cis Retinoic Acid (13-cis RA, isotretinoin) is the most potent agent in acne treatment.  Surprisingly, its mechanism of action in acne is still unknown.  Gene expression profiling of skin from 6 patients treated with isotretinoin was performed to gain insights into its mechanism of action. Skin biopsies were obtained from the patients at baseline and at one-week isotretinoin treatment. Gene array expression profiling was conducted using Affymetrix HG-U133A 2.0 arrays in order to examine changes in gene expression as a result of treatment. After treatment, 43 genes were significantly changed: 38 up-regulated and 5 down-regulated. A significant proportion of these genes are involved in pathways that regulate differentiation, tumor suppression, serine proteases, serine protease inhibitors and solute transfer. These studies are the first describing the initial changes in gene expression associated with isotretinoin therapy and are valuable in identifying potential therapeutic targets in acne.
Keywords: drug treatment effects

effects of 72 hour 13-cis RA treatment on cultured human sebocyte cells (SEB-1)

The pathogenesis of acne has been linked to multiple factors such as increased sebum production, inflammation, follicular hyperkeratinization, and the action of Propionibacterium acnes within the follicle. 13-cis Retinoic Acid (13-cis RA, isotretinoin) is the most potent agent in acne treatment.  Surprisingly, its mechanism of action in acne is still unknown.  Gene expression profiling of cultured human immortalized sebocytes (SEB-1)  treated with 13-cis RA  was performed to gain insights into its sebocyte-specific mechanism of action. SEB-1 sebocytes were cultured with 0.1 uM 13-cis RA for 72 hours or vehicle control. Gene array expression profiling was conducted using Affymetrix HG-U95Av2 arrays in order to examine changes in gene expression as a result of treatment. A total of 85 genes (78 different genes) were significantly influenced by 13-cis RA:  58 were upregulated and 27 were down-regulated.  There were changes in several genes involved in apoptosis and innate immunity.  These studies are the first describing the sebocyte- specific response in gene expression associated with isotretinoin therapy and are valuable in identifying potential therapeutic targets in acne.
Keywords: effects of 72 hour 13-cis RA treatment on cultured human sebocyte cells (SEB-1)

Effect of EGF and/or HER2 on the growth of MCF10A cells on extracellular matrix: time course

Mammary epithelial cells MCF10A  and HER2 overexpressing MCF10A  cells were grown on matrigel in the absence or presence of epidermal growth factor. Cells were lysed and RNA was collected  at 1.5,3,5,7,9 days.

gene expression profiling, paired samples, dichotomous conditions

Targets of pharmacological histone deacetylase inhibition in human umbilical vein endothelial cells

We and others have previously reported the paradoxical downregulation of endothelial nitric oxide synthase (eNOS, NOS3) steady-state mRNA by pharmacological histone deacetylase (HDAC) inhibitors. This microarray experiment was designed to identify novel targets of an HDAC inhibitor (trichostatin A (TSA), 500 nM, 24h, Sigma) in primary human endothelial cells (human umbilical vein endothelial cells, HUVEC).
Keywords: gene expression profiling, paired samples, dichotomous conditions

IL-1β-driven neutrophil biogenesis and survival preserve anti-bacterial defense in IKKβ deficiency

IKKβ-dependent NF-κB activation is associated with inflammation and tumorigenesis and is critical for innate immunity. Despite risk of immune suppression, pharmacological blockade of IKKβ/NF-κB has been considered as an attractive strategy for treatment of inflammatory diseases and cancer. Unexpectedly, treatment with IKKβ inhibitors caused neutrophilia, which also occurs in mice with inducible IKKβ deletion, termed IkkβΔ mice. IkkβΔ mice show hyperproliferation of granulocyte progenitors and increased neutrophil lifespan. Deletion of IL-1 receptor 1 in IkkβΔ mice restored normal blood cellularity and prevented neutrophil-driven inflammation. However, IkkβΔ/Il-1r1-/- mice, in contrast to IkkβΔ mice, are highly susceptible to bacterial infections indicating that either IKKβ/NF-κB or IL-1R signaling are sufficient for maintaining anti-microbial defenses, but inactivation of both pathways severely compromises innate immunity.

Cavin-3 is a tumor suppressor protein of unknown function. Using a combination of in vivo knockout and in vitro gain/loss of function approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae, a lipid-raft specialization that contains an ERK activation module, to the membrane skeleton of the plasma membrane. Loss of cavin-3 reduces the number of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation and resistance to apoptosis. The in vivo consequences of cavin-3 loss are increased lactate production and cachexia.

To gain insights into the genetic profile of every stage of the melanoma progression pathway, and to determine to what extent these profiles are similar or distinct, we performed whole-genome expression profiling of tissue specimens representing normal skin, benign and atypical nevi, and early and advanced-stage melanomas. The results of this study provide first-time evidence that significant molecular changes occur distinctly at the border of/transition from melanoma in situ to primary melanoma, and that genes involved in mitotic cell cycle regulation and cell proliferation constitute the two leading categories of genes associated with these changes.
Keywords: Stage comparative analysis

Synergy between PPARgamma ligands and platinum-based drugs in cancer

PPARγ is a member of the nuclear receptor family for which agonist ligands have anti-growth effects.  However, clinical studies using PPARγ ligands as a monotherapy failed to show a beneficial effect.  Here we have studied the effects of PPARγ activation with chemotherapeutic agents in current use for specific cancers.  We observed a striking synergy between rosiglitazone and platinum-based drugs in several different cancers both in vitro and using transplantable and chemically induced “spontaneous” tumor models.  The effect appears to be due in part to PPARγ-mediated downregulation of metallothioneins, proteins that have been shown to be involved in resistance to platinum-based therapy.  These data strongly suggest combining PPARγ agonists and platinum-based drugs for the treatment of certain human cancers
Keywords: Gene expression, change, synergy of interaction

Bipolar Disorder, Monozygotic Twins, Discordant, Trasnsformed Lymphocytes

Expression profiling in monozygotic twins discordant for bipolar disorder

To identify genes dysregulated in bipolar disorder (BD1) we carried out global gene expression profiling using whole-genome microarrays. To minimize genetic variation in gene expression levels between cases and controls we compared expression profiles in lymphoblastoid cell lines from monozygotic twin pairs discordant for the disease. We identified 82 genes that were differentially expressed by ≥ 1.3-fold in 3 BD1 cases compared to their co-twins, and which were statistically (p ≤ 0.05) differentially expressed between the groups of BD1 cases and controls. Using qRT-PCR we confirmed the differential expression of some of these genes, including: KCNK1, MAL, PFN2, TCF7, PGK1, and PI4KCB, in at least 2 of the twin pairs. In contrast to the findings of a previous study by Kakiuchi and colleagues with similar discordant BD1 twin design1 our data do not support the dysregulation of XBP1 and HSPA5. From pathway and gene ontology analysis we identified up-regulation of the WNT signalling pathway and the biological process of apoptosis. The differentially regulated genes and pathways identified in this study may provide insights into the biology of BD1.
Keywords: Bipolar Disorder, Monozygotic Twins, Discordant, Trasnsformed Lymphocytes

Endogenous expression of an oncogenic PI3K mutation leads to activated PI3K pathway signaling and an invasive phenotype

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a rational target for therapeutic intervention. In order to investigate the primary phenotype(s) mediated by mutant PIK3CA in a clean and highly patient-relevant context, we utilized a non-tumorigenic MCF10A parental and isogenic knock-in cell line that harbors a common activating PIK3CA kinase domain mutation (H1047R). We found that introduction of an endogenously mutated PIK3CA primarily results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells.  Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective and selective on reversing this phenotype compared to cell-proliferation, highlighting a potential new paradigm for studying PI3K-pathway targeted agents.

Mice were a mixed background between 129/SvEv and C57BL/6.
Rp1 knockout mice (Rp1-/-) and wildtype littermates examined.
At P7, P10, P14, P18 and P21, mice were sacrificed and retinas removed.
Each RNA sample included a pool of neural retinas from 3-4 mice.
Retinas were all collected at 1-2 pm.

Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on gene expression in the NCI-H460 human lung large cell carcinoma cell line

To understand the effect of nicotine on sensitivity of cancer cells to radiation or anti-cancer drugs, NCI-H460 human lung large cell carcinoma cells were treated with 6 Gy ionizing radiation or 1 uM cisplatin after exposure to 5 uM nicotine or cigarette smoke extract. Whole genome expression in total RNA extracted from the treated cells was quantified using the GeneChip™ Human Gene 1.0 ST microarray platform from Affymetrix® (Santa Clara, CA).

Gene expression Profiling of Peripheral Blood Leukocytes after Endotoxin Challenge in Humans

Peripheral blood mononuclear cells were isolated 0, 30 min, 6 h, 24 h, and 7 day after intravenous endotoxin challenge.

Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods.
To test our hypothesis we propose 3 specific aims:
1) To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice
2) To compare the pattern of gene expression in our mouse model with that in the hippocampus of rats prenatally exposed to alcohol (FAS model) and
3) To compare the pattern of gene expression in our mouse model with that shown in post-mortem tissues of patients with schizophrenia.
In our previous protocols we examined the pattern of gene expression in our HuD transgenic mice and in rats prenaltally exposed to alcohol. A report by another group (Hakak et al, 2001) showed that three of the HuD targets were upregulated in the prefrontal cortex of patients with schizophrenia. To evaluate whether other target of HuD may be affected in this illness, in the current protocol, we want to compare the pattern of expression in our transgenic mice with in tissue from patients with schizophrenia
Based on the behavioral and molecular properties of our HuD transgenic mice we hypothesize that these animals may be good models for the studying the basis of learning disabilities and of diseases that show deficits in PPI such as fetal alcohol syndrome and schizophrenia.
All 28 samples are derived from cerebellar tissues for patients with schizophrenia and matched controls.  The specimens were obtained from the Maryland Brain Collection according to NIH guidelines for confidentially and privacy. The protocol used in these studies was reviewed by our HRRC which found that our studies do not fall within the category of protocols monitored by the IRB (see attached letter form the HRRC). Specimens from 14 patients with a diagnosis of schizophrenia performed according to DSM-IV criteria and 14 sex-, age- and PMI-matched controls was included in this study. No differences were found between patients and control subjects in the average age (45±12 versus 43±10 years, p=0.86) or PMI (12±5 versus 16±6 hours, p=0.11). We will provide 28 samples containing 5 ug of RNA each in DEPC water (see validation of the quality of the RNA below). In addition, we include in our study animals treated with haloperidol as control for medication. These samples will be submitted in a separate protocol.

Comparison of neural stem cell lines overexpressing Nurr1 to clonal controls

The orphan nuclear receptor Nurr1 has been shown to be critical for the development of ventral midbrain dopaminergic neurons. Consequently, the development of ES cells overexpressing Nurr1 has raised hope for the development of cell replacement therapies for Parkinson's Disease to replace degenerated dopaminergic neurons.  However, the molecular consequences of Nurr1 on gene expression in these cells remain unknown.  To address this, stable, clonal, c17.2 neural stem cell lines were established that overexpressed the orphan nuclear receptor Nurr1 (clone 42 & clone 48) or parental control cell line (puroB & puroD, respectively).

Gene expression profiles of striatum and cerebellum from knock-in mouse model of Huntington's disease

Huntington’s disease (HD) involves marked early neurodegeneration in the striatum whereas the cerebellum is relatively spared despite the ubiquitous expression of full-length mutant huntingtin, implying that inherent tissue-specific differences determine susceptibility to the HD CAG mutation. To understand this tissue specificity, we compared early mutant huntingtin-induced gene expression changes in striatum to those in cerebellum in young Hdh CAG knock-in mice, prior to onset of evident pathological alterations. Endogenous levels of full-length mutant huntingtin caused qualitatively similar, but quantitatively different gene expression changes in the two brain regions. Importantly, the quantitatively different responses in striatum and cerebellum in mutant mice were well accounted for by the intrinsic molecular differences in gene expression between striatum and cerebellum in wild-type animals. Tissue-specific gene expression changes in response to the HD mutation, therefore, appear to reflect the different inherent capacities of these tissues to buffer qualitatively similar effects of mutant huntingtin. These findings highlight a role for intrinsic quantitative tissue differences in contributing to HD pathogenesis, and likely to other neurodegenerative disorders exhibiting tissue-specificity, thereby guiding the search for effective therapeutic interventions.

Gene expression profile after TRPV4 knockdown in 3T3-F442A adipocytes

Comparing gene expression profile in 3T3-F442A adipocytes with shRNA against TRPV4 or GFP. TRPV4 is an ion channel expressed in adipocytes. Results provided information that how loss-of-function of TRPV4 affects gene expression in adipocytes.

Drosha knockdown in human cells reduces viability but does not affect snRNA or rRNA synthesis or processing

We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor.  Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels.  Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors.  In fungi, RNase III homologs process rRNA precursors and certain mRNAs.  Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation.  Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs
Keywords: siRNA knockdown

To identify mediators of obesity-linked reductions in PGC-1, we tested the effects of cellular nutrients in C2C12 myotubes.  While overnight exposure to high insulin, glucose, glucosamine, or amino acids had no effect, saturated fatty acids (FA) potently reduced PGC-1a and b mRNA expression.
Keywords: Nutrient Effect

Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma

Gene expression profiles of 75 tissue samples were analyzed representing the stepwise carcinogenic process from pre-neoplastic lesions (cirrhosis and dysplasia) to HCC, including four neoplastic stages (very early HCC to metastatic tumors) from patients with HCV infection. Gene signatures that accurately reflect the pathological progression of disease at each stage were identified and potential molecular markers for early diagnosis uncovered. Pathway analysis revealed dysregulation of the Notch and Toll-like receptor pathways in cirrhosis, followed by deregulation of several components of the Jak/STAT pathway in early carcinogenesis, then up-regulation of genes involved in DNA replication and repair and cell cycle in late cancerous stages.
Keywords: 8 pathological stages of HCV-induced HCC

genetic modification, strain comparison, cell stimulation, time course, anti-bacterial response, innate immune response, inflammatory response

response to LPS of WT and IRAK4 kinase dead mouse bone marrow macrophages

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Though regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. In order to investigate the role of IRAK-4 kinase function in vivo, ‘knock-in’ mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase deficient IRAK-4 protein (IRAK-4 KD). Analysis of bone marrow macrophages obtained from WT and IRAK-4 KD mice with a number of experimental techniques demonstrated that the IRAK-4 KD cells greatly lack responsiveness to stimulation with the Toll-like receptor 4 (TLR4) agonist LPS. One of the techniques used, microarray analysis, identified IRAK-4 kinase-dependent LPS response genes and revealed that the induction of LPS-responsive mRNAs was largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in TLR4-mediated induction of inflammatory responses.
Keywords: genetic modification, strain comparison, cell stimulation, time course, anti-bacterial response, innate immune response, inflammatory response

Expression data from CD19-positive splenic B cells isolated from 1-month old ID4+/-TCL1-tg and ID4+/+TCL1-tg mice

The function of ID4 in CLL development was studied in vivo using TCL1 transgenic mouse model that develop leukemia similar to human CLL. TCL1 mice with ID4 single knockout gene have accelerated CLL progression.
Results from the animal study suggest ID4 as a tumor suppressor gene that might regulate cell proliferation and apoptosis in B lymphocytes.

Development of heart valves requires Gata4 expression in endothelial-derived cells

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in formation of portions of the atrial and ventricular septae, and generation of thin, pliable valves. The transcription factor Gata4 is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.

We analyzed gene expression profiles of unstimulated Herpesvirus Saimiri (HVS) transformed T cells (CD4+) of patients harboring a homozygous R335W mutation in the IL-2 inducible T cell kinase (ITK) compared to healthy control HVS cells.
We identified a set of 1927 Affymetrix probe sets showing significant misregulation in the ITK deficient cells (Welch's T-test, p<0.05).

rat heart ventricles, LAD - left anterior descending coronary artery, IR - ischemia-reperfusion

Rats underwent surgery for LAD ligation for 30 min followed by reperfusion. Heart ventricles were collected 2d or 7d after reperfusion.
Keywords: rat heart ventricles, LAD - left anterior descending coronary artery, IR - ischemia-reperfusion

Background: Causative genes for autosomal dominantly inherited familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) have been well characterized. There is, however, another 10-15 % early onset colorectal cancer (CRC) whose genetic components are currently unknown.  In this study, we used DNA chip technology to systematically search for genes differentially expressed in early onset CRC.

Mice lacking topoisomerase IIβ (Top IIβ) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild type and top2β knockout mouse embryos were generated. Surprisingly, only a small number (1-4%) of genes were affected in top2β knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated.
By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIβ is not required for early differentiation programming, but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIβ and HDAC2, a known TopIIβ-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIβ binding to the 5’ region of a number of TopIIβ-sensitive genes. Further
studies of a TopIIβ-sensitive gene, Kcnd2, revealed the presence of TopIIβ in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIβ in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.

To identify HGF/Met regulated genes, we performed expression microarray analysis after inducible activation of Met receptor in primary cultures of hepatocytes established from wild-type control (Alb-Cre +/-) and Met conditional knockout mice (Alb-Cre +/-; Met Fl/Fl).
Keywords: time series design

Transcription array by profiling in WT and SRC-2 null mouse liver

The molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway while fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.

Expression data from chemically-induced skin papillomas (benign tumours)

Cancer stem cells (CSCs) have been reported in various cancers including skin squamous cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here, we found that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells (SCs), was the most upregulated transcription factor in CSCs of squamous skin tumours. Sox2 is absent in normal epidermis and begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which Sox2 is frequently genetically amplified, the expression of Sox2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis dramatically decreases skin tumour formation following chemical induced carcinogenesis. Using Sox2-GFP knockin mice, we showed that Sox2 expressing cells in invasive SCC are greatly enriched in tumour propagating cells (TPCs) that further increase upon serial transplantations.  Lineage ablation of Sox2 expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of Sox2 expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to their regression and decreases their ability to be propagated upon transplantation into immunodeficient mice, supporting the essential role of Sox2 in regulating CSC functions. Transcriptional profiling of Sox2-GFP expressing CSC and upon Sox2 deletion uncovered a gene network regulated by Sox2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct Sox2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion, and paraneoplastic syndrome. Altogether, our study demonstrates that Sox2, by marking and regulating the functions of skin tumour initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.
We used microarrays to profile tumour epithelial cells upon Sox2 deletion to uncover a gene network regulated by Sox2 in primary tumour cells in vivo.

Gene expression data from SOX2 knock-out 2102Ep and NTera-2 human embryonal carcinoma cell lines

SOX2 is an oncogene and a core pluripotency transcription factor. SOX2 has multiple roles in various malignancies, in the maintainance of pluripotency and during various stages of embryonic development. Human embryonal carcinoma cells express SOX2 and the loss of this results in their differentiation.
We silenced SOX2 in two human embryonal carcinoma cell lines and measured whole-genome mRNA expression. Many genes related to embryogenesis and tissue morphogensis were upregulated. Other upregulated genes were markers of mesodermal development and epithelial-to-mesenchymal transition.

Comparison of post-mortem tissue from Brodman Brain BA22 region between schizophrenic and control patients

Transcriptional analysis of the superior temporal cortex (BA22) in schizophrenia: Pathway insight into disease pathology and drug development
Schizophrenia is a highly debilitating psychiatric disorder which is known to have heritable genetic and environmental components. To gain some insight into the mechanisms underpinning both positive and negative symptoms of the disease, we determined the genome wide expression of mRNA transcripts in post-mortem tissue from the superior temporal cortex (Brodmann Area 22, BA22) in schizophrenic and control patients. The BA22 region is known to mediate the positive pathophysiology of schizophrenia; we compared this to the anterior prefrontal cortex (BA10) from the same subjects, which is known to mediate negative symptoms. Following adjustments for confounding clinical, sample and experimental sources of variation, we carried out gene set enrichment analysis in each region using pathway data. We identified an over-representation of genes involved in cytoskeletal remodelling, neurodevelopment, cell adhesion, cellular signalling, neurotransmission and autophagy. Collectively our analysis indicates a disruption of processes underpinning synaptic plasticity in both regions. Region-specific changes support the dysregulation of distinct pathways in the BA10 and BA22 regions. This may highlight new therapeutic opportunities to treat both negative and positive symptoms of the disease.

Glucocorticoid-induced reversal of IL1b stumulated gene expression in OE-E6/7 cells

Studies indicate that high-grade serous ovarian carcinoma (HGSOC), the most common epithelial ovarian carcinoma histotype, originates from the fallopian tube epithelium (FTE).  Risk factors for this cancer include reproductive parameters associated with lifetime ovulatory events.  Ovulation is an acute inflammatory process during which the FTE is exposed to follicular fluid containing both pro- and anti-inflammatory molecules, such as interleukin-1 (IL1), tumor necrosis factor (TNF), and cortisol.  Repeated exposure to inflammatory cytokines may contribute to transforming events in the FTE, with glucocorticoids exerting a protective effect.  The global response of FTE cells to inflammatory cytokines or glucocorticoids has not been investigated.  To examine the response of FTE cells and the ability of glucocorticoids to   oppose this response, an immortalized human FTE cell line, OE-E6/E7, were treated with IL1β, dexamethasone (DEX), IL1β and DEX, or vehicle and genome-wide gene expression profiling was performed.  IL1β altered the expression of 47 genes of which 17 were reversed by DEX.  DEX treatment alone altered the expression of 590 genes, whereas combined DEX and IL1β treatment altered the expression of 784 genes.  Quantitative real time RT-PCR studies validated the gene array data for IL8, IL23A, TACC2 and PI3. Consistent with the array data, Western blot analysis showed increased levels of PTGS2 protein induced by IL1β that was blocked by DEX. These findings support the hypothesis that pro-inflammatory signaling is induced in FTE cells by inflammatory mediators and raises the possibility that dysregulation of glucocorticoid signaling could contribute to increased risk for HGSOC.

Effect of SOCS3 on the transcriptional response of bone marrow-derived macrophages to IL-6

Effects of SOCS3 on the transcriptional response of bone marrow-derived macrophages to IL-6.

Fetal liver cells from SOCS3+/+ or SOCS3-/- embryos were used to reconstitute recipient mice. Donor derived bone marrow from these mice was differentiated to macrophages.  Macrophages were either unstimulated, or stimulated for 100 or 400 minutes with 10 ng/ml IL-6.

We hypothesized that the estrogen-related receptor a (ERRa), which recruits PGC-1a to metabolic target genes in heart, exerts protective effects in the context of stressors known to cause heart failure. ERRa-/- mice subjected to left ventricular (LV) pressure overload developed signatures of heart failure including chamber dilatation and reduced LV fractional shortening. 31P-NMR studies revealed abnormal phosphocreatine depletion in ERRa-/- hearts subjected to hemodynamic stress, indicative of a defect in ATP reserve. Mitochondrial respiration studies demonstrated reduced maximal ATP synthesis rates in ERRa-/- hearts. Cardiac ERRa target genes involved in energy substrate oxidation, ATP synthesis, and phosphate transfer were downregulated in ERRa-/- mice at baseline or with pressure overload. These results demonstrate that ERRa, a potential therapeutic target, is indispensable for the adaptive bioenergetic response to hemodynamic stressors known to cause heart failure.
Keywords: Genetic modification, stress response

Expression data of Sox9+ and Ngn3+ mouse pancreas cells at different stages of development

Genes specific to Sox9+ pancreatic progenitors were identified by comparing the gene expression in embryonic and adult Sox9+ cells.
We used microarray analysis to detail the global changes in gene expression as Sox9 positive embryonic pancreatic progenitors differentiatiate into adult ductal cells or the endocrine lineage.

Comparison of gene expression profiles in naïve WT and Asc-null bone marrow derived dendritic cells

The adaptor protein ASC contributes to innate immunity through the assembly of caspase-1-activating inflammasome complexes. We demonstrate that ASC plays an inflammasome-independent cell-intrinsic role in adaptive immune cells. Asc-/- mice displayed defective antigen presentation by dendritic cells and lymphocyte migration due to impaired Rac-mediated actin polymerization. Genome-wide analysis showed that ASC, but not Nlrp3 or caspase-1, controls mRNA stability and expression of DOCK2, a guanine nucleotide exchange factor that mediates Rac-dependent signaling in immune cells. DOCK2-deficient dendritic cells showed similar defective antigen uptake as Asc-/- cells. Ectopic expression of DOCK2 in ASC-deficient cells restored Rac-mediated actin polymerization, antigen uptake and chemotaxis. Thus, ASC shapes adaptive immunity independently of inflammasomes by modulating DOCK2-dependent Rac activation and F-actin polymerization in dendritic cells and lymphocytes.

Interleukin-17 mediated paracrine network promote tumor resistance to anti-angiogenic therapy

While angiogenesis inhibitors have provided significant clinical benefit as cancer therapeutics, the mechanisms of anti-VEGF resistance remain incompletely understood. We uncovered an interleukin-17 mediated paracrine network of signaling between the adaptive and innate immune system associated with resistance to anti-VEGF treatment in multiple tumor models.

Comparative gene expression profile of PPARg and RARa ligand treated human dendritic cells

Our data indicated that activation of the PPARg nuclear receptor induces a retinoid response in human dendritic cells. In order to assess the contribution of retinoid signaling to the PPARg response we decided to use a combination of pharmacological activators and inhibitors of these pathways. Cells were treated with the synthetic PPARg ligand rosiglitazone (RSG), or with RSG along with the RARa antagonist (AGN193109) to block RARa mediated gene expression, or the RARa specific agonists (AM580) alone.  This design allows one to determine if retinoid signaling is a downstream event of PPARg activation and what portion of PPARg regulated genes are regulated via induced retinoid signaling.
Keywords: ligand response

Gene Expression Profiling of 17 beta-Estradiol and Genistein Effects on Mouse Thymus

Compared the effects of Estrogen and its analog Genistein on mouse thymus. Endogenous estrogen were minimized by removal of ovaries. Samples were collected at 4 time points: 0 days, 2 days, 3 days and 6 days from the start of the treatment. Two biological replicates at each time point.
Keywords: comparison of hormone and its analog; Time course

Expression analysis data from large T antigen-immortalized murine embryonic fibroblasts (MEFs)

Microarrays were used to examine the genome-wide expression in FIH null, VHL null and VHL/FIH double null MEFs.
We used these data to analyze how deletion of FIH or VHL alone affects gene expression and if VHL and FIH have synergistic effects and differential selectivity on regulating gene expression.

Epilepsy is a major neurological disorder that affects approximately 1% of the population. The processes that lead to the development of epilepsy (epileptogenesis) are largely unknown. Levetiracetam is a novel antiepileptic drug (AED) that in the kindling model inhibits epileptogenesis in addition to being effective in controlling established epilepsy. The mechanisms of action of levetiracetam as an AED and an antiepileptogenic drug are unknown. By identifying the effect of chronic levetiracetam therapy on gene expression in the brain we hope to be able to identify genes that are involved in epileptogenesis. By comparing the gene expression profiles of levetiracetam and phenytoin treatments, we hope to be able to distinguish between genes that are important for the antiepileptic (anti-seizure) effect and genes that are important for the antiepileptogenic effect of levetiracetam. Phenytoin is a well-established AED; its mechanism of action involves inhibition of sodium channels. In contrast to levetiracetam, available data suggest that phenytoin in certain situations may enhance rather than inhibit the development of epilepsy.
We will identify changes in gene expression in the hippocampus, frontal cortex, and brain stem caused by chronic treatment (3 months) with levetiracetam or phenytoin. We will search for differences in the gene expression profiles after chronic treatment with the two drugs, to explain the different clinical effects of the two drugs.
We hypothesize that chronic treatment with levetiracetam leads to changes in the expression of genes that are involved in both the antiepileptogenetic and antiepileptic effect of the drug, whereas chronic treatment with phenytoin affects genes that may be related to pro-epileptogenetic as well as antiepileptic effects of that drug. We further hypothesize that i) regional differences in the effects of levetiracetam and phenytoin may reflect important functional differences in the actions of these two drugs, and ii) there may be different mechanisms involved in their anti-seizure effect.
Male Wistar rats receive levetiracetam (150 mg/kg x 2), phenytoin (75 mg/kg x 2) or control solution (N=7 in each group) through a gastric tube twice daily for 90 days. Experimental animals and controls are housed together in the same cages in groups of three, facilitating paired comparisons among the three groups in animals that have experienced the same environment throughout the 3 month experimental period. Four hrs after the last dose animals are anesthetized, decapitated, and total RNA is extracted from brain stem, frontal cortex, and hippocampus. RNA will be sent to the TGEN facility for hybridization to the rat U230A chips. The number of chips needed is (2 drugs + 1 control) * (3 brain regions) * (7 animals per group) = 63 chips. We will initially select those genes for analysis that are called “present” by the Affymetrix software in 4 or more animals for at least one of the 6 experimental groups. Then t-tests with Benjamini & Hochberg correction for protection against multiple comparisons will be used to identify genes that are differentially expressed at a false discovery rate of .05 among any of the six groups. We will be primarily interested in the following expression profiles, analyzed as paired comparisons by the Affymetrix software:

1. phenytoin vs control in hippocampus
2. phenytoin vs control in brain stem
3. levetiracetam vs control in hippocampus
4. levetiracetam vs control in brain stem

We will then compare the profiles of both drugs in each brain region (e.g., phenytoin vs levetiracetam in hippocampus) to select genes that are drug-specific. We will also take a Venn diagram approach to identify genes that are drug-specific across both brain regions tested.

Effect of ZFX shRNA mediated knockdown in the human AML cell line NOMO-1

Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) maintain the undifferentiated phenotype and proliferative capacity of their respective cells of origin, hematopoietic stem/progenitor cells and immature thymocytes. The mechanisms that maintain these progenitor-like characteristics are poorly understood. We report that the transcription factor Zfx is required for the development and propagation of experimental AML caused by MLL-AF9 fusion, and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx activated progenitor-associated gene expression programs and prevented differentiation. Key Zfx target genes included mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescued the propagation of Zfx-deficient AML. These studies identify a common mechanism that controls the cell-of-origin characteristics of acute leukemias derived from disparate lineages and transformation mechanisms.

NANOG-OCT4-SOX2 Regulatory Module in Human Embryonic Stem Cells (dataset 4)

The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined.  Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs.  Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner.  OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs.  Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs.

A newly identified spliced isoform of metadherin differently regulates the global transcriptomic profile in MCF-7 breast cancer cells

The current study analyzed the metadherin (MTDH)-mediated altered gene expression profiles in ER positive MCF-7 cells. Some of these altered gene expressions were further inter connected to various pathways which may eventually be recognized as drug targets or biomarkers in those breast cancers where MTDH plays a role in cancer progression/metastasis.

A newly identified spliced isoform of metadherin differently regulates the global transcriptomic profile in MDA-MB-231 breast cancer cells

The current study analyzed the metadherin (MTDH)-mediated altered gene expression profiles in ER negative MDA-MB-231 cells. Some of these altered gene expressions were further inter connected to various pathways which may eventually be recognized as drug targets or biomarkers in those breast cancers where MTDH plays a role in cancer progression/metastasis.

protasome inhibitor, time course, MCF7 cells, genome wide  gene expression response, siRNA

Argyrin A is a p27 stabilizing drug with potent antiproliferative activity in vivo

Reduction in the cellular levels of the cyclin kinase inhibitor p27kip1 are frequently found in many human cancers and correlate directly with patient prognosis. Specifically ubiquitin dependent proteasomal turnover has been shown to cause reduced p27 expression in many human cancers. We recently demonstated that expression of a stabilized version of p27kip1 (p27kip1T187A) in a genetically modified mouse significantly reduced the number of intestinal adenomatous polyps which progressed to invasive carcinomas. Based on this work we set out to identify compounds which lead to a re-expression of p27 in cancer tissues. In this work we identify Argyrin A a compound derived from myxobacterium archangium gephyra as a potent inducer of p27kip1 expression. Argyrin A induces apoptosis in human colon cancer xenografts and tumor vasculature in vivo leading to a profound reduction in tumor size at well tolerated levels. Argyrin A functions are strictly dependent on the expression of p27kip1 as neither tumor cells nor endothelial cells which do not express p27kip1 respond to this compound. Surprisingly the molecular mechanism by which Argyrin A exerts its p27 dependent biological function is through a potent inhibition of the 20S proteasome.
Keywords: protasome inhibitor, time course, MCF7 cells, genome wide  gene expression response, siRNA

STEAP4 is a plasma membrane metallo-reductase involved in the transport of iron and copper. Recently, STEAP4 was implicated in promoting insulin sensitivity by acting in white adipose tissue (WAT) to control the production of inflammatory cytokines such as IL-6. Indeed, the loss of STEAP4 expression in mice leads to increased production of inflammatory cytokines in visceral WAT and systemic insulin resistance. In this report, we demonstrate that in mouse liver STEAP4 is produced at significant levels and that STEAP4 transcription is induced by IL-6. We further demonstrate that the STEAP4 gene is a direct target of phosphorylated STAT3 in mouse liver. In addition, hepatic STEAP4 expression is regulated by feeding and fasting, and obesity leads to the induction of STEAP4 expression in the liver. Interestingly, the regulation of STEAP4 in both feeding and fasting and the obese state appears to require the transcription factor C/EBPalpha that may act in concert with STAT3 as they both bind to the proximal STEAP4 promoter in vivo. Taken together these data suggest the transcriptional regulation of hepatic STEAP4 may play a critical role in the response to nutritional and inflammatory stress and contribute to the protective effect of STEAP4 in vivo.

Investigation of the role of FOXP3 in CD4+ T effector cells. FOXP3 is transiently upregulated in T effector cells under activation. This temporary expression in Teff cells is insufficient to suppress expression of reported targets of FOXP3 repressor activity. The role of FOXP3 in T effector cells remains unclear.
We used microarray analysis to detail the differentially expressed genes between FOXP3 wild type and 2T>C(mut) clones and identified classes of up-regulated or down-regulated genes based upon FOXP3 expression.

Succinate semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive disorder effecting approximately 350 people around the world. Patients suffering from SSADH deficiency experience language acquisition failure, memory deficiencies, autism, increased aggressive behaviors, and seizures. There is a chemical buildup of both gamma-aminobutyric acid (GABA) and gamma-hydroxybutyric acid (GHB) in the neurological system of these patients. The Aldh5a1-/- knock out mouse model of SSADH deficiency shows the same chemical imbalances as the human disease, with additional fatal tonic-clonic seizures at three weeks of age.  The elucidation of seizure causing pathways will facilitate treatment of seizure phenotypes in diseases with related epilepsy.
Gene expression patterns within the hippocampus, cerebellum, and cortex of SSADH deficient mice (Aldh5a1-/- mice) will be compared to wild type mice at a time point immediately prior to fatal seizures.
We hypothesis that the SSADH deficient mice experience a dysfunction of glutamate/GABA/ glutamine neurotransmitter cycle linked to astroglial metabolism and/or uptake of neuronally-released glutamate. The increased levels of GHB and GABA lead to down regulation of GABA-B-Receptor leading to seizures. The SSADH deficient phenotype may also be caused by ongoing oxidative damage and the pathological role of succinic semialdehyde.
SSADH deficient mice (Aldh5a1-/- knock out) exhibit fatal seizures around three weeks of age. Mutant and wild type mice will be sacrificed between 17 and 19 days of life, and brain sections will be extracted and frozen (using a standard protocol). Hippocampus, cerebellum, and cortex from three mutant mice and three wild type mice will individually be expression profiled on the Affymetrix platform, giving a total of eighteen arrays. Comparative analysis will then be carried out, evaluating the transcript differences between mutant and wild type mice in each brain region.

A number of human neurodegenerative diseases result from the expansion of a glutamine repeat within the disease-causing protein. Spinocerebellar ataxia type1 is one such disease, caused by expansion of a polyglutamine tract in the novel protein ataxin-1. To faithfully model SCA1 in the mouse, we generated  knock-in mice carrying 154 CAG repeats in the mouse Sca1 locus. These mice reproduced many aspects of the human disease. Despite ubiquitous expression of the mutant protein, they developed slowly progressive selective neurodegeneration , which is most distinct in Purkinje cells and spinal cord. Alterations in gene expression have been proposed to be involved in the pathogenesis of polyglutamine disease including SCA1, but the changes of gene expression in an authentic disease model have not been characterized. We believe that knowledge of these genes will give insight into the pathophysiology of SCA1 and may ultimately be relevant to the treatment of SCA1.
We will determine gene expression patterns in the cerebellum and forebrain at three different time points.
We propose that the genes whose expression is affected  by mutant ataxin-1 expression are effectors for neuronal dysfunction and neuronal degeneration in the knock-in mice. We also hypothesize the difference in the expression levels of these genes accounts for selective vulnerability of neurons.
We will examine three time points: 4 weeks, 11 weeks, and 20 weeks of age. We have shown that the mice developed motor incoordination as revealed  by rotating rod test as early as 5 weeks of age but it was not until 10 weeks that they start showing clear  neurological  phenotype such as clasping.  At each point, we will compare the pattern between the mutant and wild-type littermate. Animals will be prepared and sacrificed by a standard procedure. Cerebellum and spinal cord are the most affected areas whereas forebrain shows less neurodegeneration. Tissue will be rapidly dissected from cerebellum and forebrain, frozen in liquid nitrogen, and stored at ?80 C until analysis. Tissues will be sent to the centers (as opposed to RNA). We will be providing 9 tissue samples from three litters for each time point to mitigate any expression differences resulting from subtle differences of procedure or environment where the mice grow.

C-JUN promotes BCR-ABL induced lymphoid leukemia by inhibiting methylation of the 5´ region of Cdk6

The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, since lack of c-JUN impairs proliferation of p185BCR-ABL transformed cells without affecting viability. The decreased proliferation of c-JunD/D cells is associated with the loss of cyclin dependent kinase 6 (CDK6) expression. In c-JunD/D cells CDK6 expression becomes down-regulated upon BCR-ABL induced transformation which correlates with CpG island methylation within the 5´ region of Cdk6. We verified the impact of Cdk6 deficiency by using Cdk6-/- mice that developed BCR-ABL induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition we show that re-expression of CDK6 in BCR-ABL transformed c-JunD/D cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the AP-1 transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1 regulated DNA methylation upon BCR-ABL induced transformation, thereby accelerating leukemogenesis.

Transcriptome from circulating cells suggests dysregulated pathways associated with long-term recurrent events following first-time myocardial infarction.

Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for cardiovascular disease. However, the utility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome.  Whole-genome microarray and targeted cytokine expression profiling on blood samples from normal cardiac function controls and first-time AMI patients within 48-hours post-MI revealed expected differential gene expression profiles enriched for inflammation and immune-response pathways in AMI patients. To determine molecular signatures at the time of AMI that could prognosticate long-term outcomes, transcriptional profiles from sub-groups of AMI patients with (n=5) or without (n=22) any recurrent events over an 18-month follow-up were compared. This analysis identified 559 differentially expressed genes. Bioinformatic analysis of this differential gene set for associated pathways revealed 1) increasing disease severity in AMI patients is associated with a decreased expression of the developmental epithelial-to-mesenchymal transition, and 2) modulation of cholesterol transport genes that include ABCA1, CETP, APOA1, and LDLR is associated with clinical outcome. In conclusion, differentially regulated genes and modulated pathways were identified that predicted recurrent cardiovascular outcomes in first-time AMI patients. This cell-based approach for risk stratification in AMI warrants a larger study to determine the role of metabolic remodeling and regenerative processes required for optimal outcomes. A validated transcriptome assay could represent a novel, non-invasive platform to anticipate modifiable pathways and therapeutic targets to optimize long-term outcome for AMI patients.

Glycerol kinase (Gyk) knockout mouse; mouse model of human Glycerol Kinase Deficiency; Gyk KO versus WT liver expression analysis; Affymetrix mus 430 2.0 GeneChip

Targeted disruption of glycerol kinase gene in mice: expression analysis in liver

Glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologic crises. Liver shows the highest level of glycerol kinase (GK) activity in humans and mice.  Absence of genotype-phenotype correlations in patients with GKD indicate the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal glycerol kinase (Gyk) knockout (KO) and wild type (WT) mice. Unsupervised learning revealed the overall gene expression profile of the KO mice was different from that of WT. Real time PCR confirmed differences for selected genes.  Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories.  Pathway Assist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors SREBP-1c, ChREBP, HNF-4alpha, and PPAR-alpha, had increased activity in the Gyk KO mice compared with WT mice; while SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in gene expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse knockout model of a human disease.
Keywords: Glycerol kinase (Gyk) knockout mouse; mouse model of human Glycerol Kinase Deficiency; Gyk KO versus WT liver expression analysis; Affymetrix mus 430 2.0 GeneChip

Whole brain gene expression in X-monosomic and normal female mice

Whole brain gene expression was examined in the following strains of mice:
1. P0 maternal monosomic 39,Xm females, C57BL/6J x C3H/Paf
2. P0 paternal monosomic 39, Xp females, In(X)/C3H x C57BL/6J
3. P0 normal 40,XX females, In(X)/C3H x C57BL/6J
4. P0 normal 40,XX females, C57BL/6J x C3H/Paf

Expression profiling of tumor cells from MYCN-driven neuroblastoma upon BRD4 or AURKA inhibition

Amplification of MYCN is the most prominent genetic marker of high-stage neuroblastoma, a childhood tumor originating from the neural crest. We generated a cell line (mNB-A1) from tumors developed in transgenic mouse and treated these cells with DMSO (n=6), the BRD4-inhibitor JQ1 (n=3) or the AURKA-inhibitor MLN8237 (n=3) for 24 h.

The aim of this study was to measure the influence of beverages on blood gene expression. We wanted to explore the underlying mechanisms of the cardioprotective effects of red wine.
Keywords: Treatment effect

Identification of Sox10 target genes by comparative expression profiling

RT4D6 Schwannoma cells were treated with siRNA specific for Sox10 and a mutant siRNA. Total RNA from the cells were used to screen for differentially expressed genes.

Bleomycin induces molecular changes directly relevant to idiopathic pulmonary fibrosis:  A model for “active” disease

Genomic profiling of RNA from cultured human fibroblasts of donor samples in the 10-14th passage was carried out to determine expression changes in the fibroblasts of individual with different degrees of pulmonary fibrosis. Donors consisted of individuals with rapid progressing pulmonary fibrosis, slow progressing pulmonary fibrosis, or no fibrosis.

Loss of histone demethylase KDM6B enhances aggressiveness of pancreatic cancer through downregulation of C/EBPα

Genetic mutations in pancreatic ductal adenocarcinoma (PDAC) with critical roles have been well examined. The recent discovery of alterations in genes encoding histone modifiers suggests their possible roles in the complexity of cancer development. We previously reported loss of heterozygosity of the KDM6B gene, which encodes a histone demethylase for trimethylated histone H3 lysine 27 (H3K27me3), a repressive chromatin mark, in PDAC cells. In this study, we demonstrated that loss of KDM6B enhanced aggressiveness of PDAC cells. KDM6B has been regarded as a tumor suppressor that mediates oncogenic KRAS-induced senescence. Consistently, KDM6B was highly expressed in pancreatic precancerous lesions (pancreatic intraepithelial neoplasms); then, the expression decreased as the malignant grade progressed. We found that knockdown of KDM6B in PDAC cells promoted tumor sphere formation and increased peritoneal dissemination and liver metastasis in vivo. Microarray and chromatin immunoprecipitation analysis implicated CEBPA for aggressiveness induced by KDM6B knockdown. CEBPA knockdown recapitulated the phenotypic change of PDAC cells after KDM6B knockdown, which was reversed by forced expression of C/EBPα. Moreover, similar protein expression patterns of KDM6B and C/EBPα in human PDAC emphasized their functional correlation. Notably, pharmacological inhibition of the H3K27 methylase EZH2 in PDAC cells inhibited tumor sphere formation along with the upregulation of CEBPA expression, and this effect was impaired in KDM6B knockdown cells, highlighting the role for KDM6B in the activation of CEBPA. Together, our results propose a significant role for the KDM6B-C/EBPα axis in the PDAC phenotype.

The tumor stroma is believed to contribute to some of the most malignant characteristics of epithelial tumors.  However, signaling between stromal and tumor cells is complex and remains poorly understood.  Here we show that genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumors.
Global gene expression profiling in mammary stromal cells identified a Pten-specific signature associated with massive extra-cellular matrix (ECM) remodeling, innate immune cell infiltraion and increased angiogenesis.  Execution of this transcriptional program was mediated, in part, by the induction, phosphorylation and recruitment of Ets2 to target promoters.  Remarkably, Ets2 inactivation in Pten stroma-deleted tumors was sufficient to decrease tumor growth and progression.  These findings identify the Pten-Ets2 axis as a critical stroma-specific signaling pathway that suppresses mammary epithelial tumors.

NIH3T3 cells, myc, ras, IRF-1, proliferation, transformation, G1/S cell cycle ceckpoint

Tumor suppression by Interferon regulatory factor-1 relies on down-regulation of cyclin D1

Interferons have been ascribed to mediate antitumor effects. IRF-1 is a major target gene of interferons. It inhibits cell proliferation and oncogenic transformation. Here we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of non-transformed cells by IRF-1. These include cell cycle regulating genes. Activation of IRF-1 decreases cyclin D1 expression and CDK4 kinase activity concomitant with dephosphorylation of pRb. These effects of IRF-1 are mediated by inhibition of the MEK-ERK pathway and a transcriptional repression of cyclin D1. IRF-1 mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Ablation of cyclin D1 by RNA interference experiments prevents transformation and tumor growth in nude mice. The data demonstrate that cyclin D1 is a key target for IRF-1 mediated tumor suppressive effects.
Keywords: NIH3T3 cells, myc, ras, IRF-1, proliferation, transformation, G1/S cell cycle ceckpoint

Nuclear receptor-mediated alleviation of alcoholic fatty liver by polyphenols contained in alcoholic beverages

To elucidate the effect of the polyphenols contained in alcoholic beverages on the metabolic stress induced by ethanol consumption, four groups of mice were fed for five weeks on Lieber's diet with or without ethanol, with ethanol plus ellagic acid, and with ethanol plus trans-resveratrol. Alcoholic fatty liver was observed in the group fed the ethanol diet but not in those fed the ethanol plus polyphenol diets. Liver transcriptome analysis revealed that the addition of the polyphenols suppressed the expression of the genes related to cell stress that were up-regulated by ethanol alone. Conversely, the polyphenols up-regulated the genes involved in bile acid synthesis, unsaturated fatty acid elongation, and tetrahydrofolate synthesis that were down-regulated by ethanol alone. Because parts of these genes were known to be regulated by the constitutive androstane receptor (CAR), we performed the same experiment in the CAR-deficient mice. As a result, fatty liver was observed not only in the ethanol group but also with the ethanol plus polyphenol groups. In addition, there was no segregation of the gene expression profiles among these groups. These results provide a molecular basis for the prevention of alcohol-induced stress by the polyphenols in alcoholic beverages.

We carried out a global survey of age-related changes in mRNA levels in the C57BL/6NIA mouse hippocampus and found a difference in the hippocampal gene expression profile between 2-month-old young mice and 15-month-old middle-aged mice correlated with an age-related cognitive deficit in hippocampal-based explicit memory formation. Middle-aged mice displayed a mild but specific deficit in spatial memory in the Morris water maze.
Keywords: age comparison

Mutant thyroid hormone receptors (TRs) isolated from distinct cancer types display distinct target gene specificities: a unique regulatory repertoire associated with renal clear cell  carcinomas.

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that regulate a diverse array of biological activities, including metabolism, homeostasis, and development.  TRs also serve as tumor suppressors, and aberrant TR function (via mutation, deletion, or altered expression) is associated with a spectrum of both neoplastic and endocrine diseases.  A particularly high frequency of TR mutations has been reported in renal clear cell carcinoma (RCCC) and in hepatocellular carcinoma (HCC). We have shown that HCC-TR mutants regulate only a fraction of the genes targeted by wild-type TRs, but have gained the ability to regulate other, unique, targets.  We have suggested that this altered gene recognition may contribute to the neoplastic phenotype.  Here, to determine the generality of this phenomenon, we examined a distinct set of TR mutants associated with RCCCs. We report that two different TR mutants, isolated from independent RCCC tumors, possess greatly expanded target gene specificities that extensively overlap one another, but only minimally overlap that of the WT-TRs, or those of two HCC-TR mutants.  Many of the genes targeted by either or both RCCC-TR mutants have been previously implicated in RCCC, and include a series of metallothioneins, solute carriers, and genes involved in glycolysis and energy metabolism.  We propose that TR mutations from RCCC and HCC are likely to play tissue-specific roles in carcinogenesis, and that the divergent target gene recognition patterns of TR mutants isolated from the two different types of tumors arises from different selective pressures during development of RCCC versus HCC.

Identification of genes modulated by Nurr1 overexpression in HB-1

Transcriptome comparison of murine wild-type and dumbo retinas at P15

Hmx1 is a transcription factor expressed in the developing eye and ear and in some other parts of the nervous system. Dumbo mice are carrying the Hmx1 p.Q64X loss-of-function mutation (Munroe et al., 2009. BMC Developmental Biology). Transcriptomic analyses of this mouse model allows to decipher biological pathways under the control of Hmx1. In our study, we used it to better understand the role of Hmx1 in the retina and to identify several of its target genes.

exposure to dna damaging agent, comparison of phenotypic response to dna damaging agent

Supplementary information #3. Z-normalized 4 0ver 4 hr ratios of the genes differently expressed when sensitive and insensitive persons were compared. On these figures the cluster of Figure 1 has been based

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual’s susceptibility to the major sporadic cancer types, including colon, lung and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with a low mutagen sensitivity were compared with seven with a high score. RNA was isolated from cells exposed to bleomycin (4 hr) and unexposed cells. Micro-array analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all fourteen cell lines, included genes involved in multiple processes, most prominent in cell proliferation and DNA repair. When comparing the insensitive and sensitive individuals other differentially expressed genes were found, that were involved in proliferation (e.g. BUB1) and signal transduction (e.g. DUSP4). This difference in expression profiles between mutagen sensitive and insensitive individuals justifies further studies aiming to elucidate the genes responsible for the development of sporadic cancers.
Keywords: exposure to dna damaging agent, comparison of phenotypic response to dna damaging agent

Identification of genes that are regulated by TAK1 in human cutaneous T cell lymphoma HuT-102 cells

We previously reported that human T cell lymphotropic virus 1 (HTLV-1) Tax oncoprotein constitutively activates TAK1. Here, we established Tax-positive HuT-102 cells stably downregulated TAK1 expression by short-hairpin RNA (HuT-shTAK1 cells), and investigated the physiological function of TAK1. Microarray analysis demonstrated that several interferon (IFN)-inducible genes including chemokines such as CXCL10 and CCL5 were significantly downregulated in HuT-shTAK1 cells. In contrast, Tax-mediated constitutive activation of NF-kB was intact in HuT-shTAK1 cells. IRF3, a critical transcription factor in innate immunity to viral infection, was constitutively activated in a Tax-dependent manner. Activation of IRF3 and IRF3-dependent gene expression were dependent on TAK1 and TBK1. On the other hand, IRF4, another IRF family of transcription factor overexpressed in a Tax-independent manner, negatively regulated the TAK1-dependent IRF3 transcriptional activity. Together, HTLV-1 manipulates IFN signaling by regulating both positive and negative IRFs.

ZXDC1 augments the expression of various markers of monocyte/macrophage differentiation when over-expressed in the U937 cell line treated with the phorbol ester PMA.  Likewise, knockdown of ZXDC1 restricts the induced expression of these markers.  We sought to identify specfic gene targets of ZXDC1 during the process of monocyte/macrophage differentiation in U937 by performing gene expression profiling in cells exhibiting reduced expression of ZXDC1 compared to controls.
We used microarray to identify genes regulated by ZXDC during PMA-induced differentiation of U937 monoblasts towards a monocyte/macrophage phenotype

Skeletal muscles of untreated children with juvenile dermatomyositis

Background :To evaluate the impact of the duration of chronic inflammation on gene expression in skeletal muscle biopsies (MBx) from untreated children with juvenile dermatomyositis (JDM) and identify genes and biological processes associated with the disease progression, expression profiling data from 16 girls with active symptoms of JDM greater or equal to 2 months were compared with 3 girls with active symptoms less than 2 months.
Results: Seventy-nine genes were differentially expressed between the groups with long or short duration of untreated disease.  Genes involved in immune responses and vasculature remodeling were expressed at a higher level in muscle biopsies from children with greater or equal to 2 months of symptoms, while genes involved in stress responses and protein turnover were expressed at a lower level.  Among the 79 genes, expression of 9 genes showed a significant linear regression relationship with the duration of untreated disease.  Five differentially expressed genes--HLA-DQA1, smooth muscle myosin heavy chain, clustering, plexin D1 and tenomodulin--were verified by quantitative RT-PCR.  The chronic inflammation of longer disease duration was also associated with increased DC-LAMP+ and BDCA2+ mature dendritic cells, identified by immunohistochemistry.
Conclusions: We conclude that chronic inflammation alters the gene expression patterns in muscle of untreated children with JDM.  Symptoms lasting greater or equal to 2 months were associated with dendritic cell maturation and anti-angiogenic vascular remodelling, directly contributing to disease pathophysiology.

Liver and quadricpes muscle gene expression, WT vs. PGC1beta mutant

Hypomorphic Mutation in PGC1beta causes mitochondrial dysfunction and liver insulin resistance

PGC1beta is a transcriptional coactivator that potently stimulates mitochondrial biogenesis and respiration of cells. Here, we have generated mice lacking exons 3 to 4 of the Pgc1beta gene (PGC1beta E3,4-/E3,4- mice). These mice express a mutant protein that has reduced coactivation activity on a subset of transcription factors, including ERRalpha, a major target of PGC1beta in the induction of mitochondrial gene expression. The mutant mice have reduced expression of OXPHOS genes and mitochondrial dysfunction in liver and skeletal muscle as well as elevated liver triglycerides. Euglycemic-hyperinsulinemic clamp and insulin signaling studies show that PGC1beta mutant mice have normal skeletal muscle response to insulin, but have hepatic insulin resistance. These results demonstrate that PGC1beta is required for normal expression of OXPHOS genes and mitochondrial function in liver and skeletal muscle. Importantly, these abnormalities do not cause insulin resistance in skeletal muscle but cause substantially reduced insulin action in the liver.
Keywords: Liver and quadricpes muscle gene expression, WT vs. PGC1beta mutant

To identify MED1 target genes involved in prostate tumorigenesis.

The major type of protein arginine methyltransferase is PRMT1. Since the growth of embryos from Prmt1−/− mice was arrested shortly after implantation, PRMT1 must play a critical role in early mouse development.
We used microarray to investigate the effect of PRMT1 in early embryonic development.

To unravel the molecular mechanisms mediating the effects of androgens on spermatogenesis, testicular gene expression was compared in mice with a Sertoli cell-selective androgen receptor knockout (SCARKO) and littermate controls on postnatal d 10. At this age testicular cell composition is still comparable in SCARKOs and controls. Microarray analysis identified 692 genes with significant differences in expression. A more than 2-fold up- or downregulation by androgen action in Sertoli cells was observed for 28 and 6 genes respectively. The biological relevance of the “strongly” upregulated genes was supported by the finding that several of them were previously described to be androgen-regulated or essential for spermatogenesis. Serine protease inhibitors were overrepresented in the same subgroup suggesting a role for androgens in cell junction dynamics and tissue restructuring events during spermatogenesis. A time course experiment (d8-d20), followed by cluster analysis allowed the identification of typical expression patterns of differentially expressed testicular genes during initiation of spermatogenesis. Three genes with a pattern closely resembling that of Pem, a prototypal androgen-regulated gene in Sertoli cells, were selected for confirmation by RT-PCR and further analysis. The data confirm that the SCARKO model allows identification of novel androgen-regulated genes in the testis. This particular series represents  all data from  d 10. The additional expression data from the time course (d8-d20) is represented by series GSE2259 ("Testicular gene expression in SCARKO mice during prepuberty").

We compared expression of genes in brains of SIRT1 brain-specific knockouts (BSKO) to those of wild-type littermate controls (WT).

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic, long term exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, as well as exposures to military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cells lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation.  Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, providing targets for focused in vitro studies.

Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons

Gene expression data in estrogen receptor alpha positive breast tumors with and without PIK3CA mutations.

PI3K/AKT pathway plays one of pivotal roles in breast cancer development and maintenance. PIK3CA, coding PIK3 catalytic subunit, is the oncogene which shows the high frequency of gain-of-function mutations leading to the PI3K/AKT pathway activation in breast cancer. In particular in the ERα-positive breast tumors PIK3CA mutations have been observed in 30% to 40%. However, genes expressed in connection to the pathway activation in breast tumorigenesis remain largely unknown.
To identify downstream relevant target genes (and signaling pathways) turned on by the aberrant PI3K/AKT signal in breast tumors, we analyzed gene expression by pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations.

Global analyses of gene expression in early experimental knee osteoarthritis

OBJECTIVE: To analyze genome-wide changes in chondrocyte gene expression in a surgically induced model of early osteoarthritis (OA) in rats, to assess the similarity of this model to human OA, and to identify genes and mechanisms leading to OA pathogenesis. METHODS: OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression. Expression patterns of selected dysregulated genes, including Col2a1, Mmp13, Adamts5, Ctsc, Ptges, and Cxcr4, were validated by real-time polymerase chain reaction, immunofluorescence, or immunohistochemistry 2, 4, and 8 weeks after surgery. RESULTS: After normalization, comparison of OA and sham-operated samples showed 1,619 differentially expressed probe sets with changes in their levels of expression >/=1.5-fold, 722 with changes >/=2-fold, 135 with changes >/=4-fold, and 20 with changes of 8-fold. Dysregulated genes known to be involved in human OA included Mmp13, Adamts5, and Ptgs2, among others. Several dysregulated genes (e.g., Reln, Phex, and Ltbp2) had been identified in our earlier microarray study of hypertrophic chondrocyte differentiation. Other genes involved in cytokine and chemokine signaling, including Cxcr4 and Ccl2, were identified. Changes in gene expression were also observed in the contralateral knee, validating the sham operation as the appropriate control. CONCLUSION: Our results demonstrate that the animal model mimics gene expression changes seen in human OA, supporting the relevance of newly identified genes and pathways to early human OA. We propose new avenues for OA pathogenesis research and potential targets for novel OA treatments, including cathepsins and cytokine, chemokine, and growth factor signaling pathways, in addition to factors controlling the progression of chondrocyte differentiation.

K-ras is one of the most frequently mutated human oncogenes.  However, activation of K-ras can lead to either senescence or proliferation in primary cells.  The precise mechanism governing these distinct outcomes remains unclear.  Here we describe a novel loss-of-function screen to assess the role of specific genes identified as potential key regulators of K-ras driven oncogenesis.  Using this approach, we identify the transcription factor Wt1 as an inhibitor of senescence in primary cells expressing oncogenic K-ras.  Deletion or suppression of Wt1 expression leads to senescence of primary cells expressing oncogenic K-ras under the control of the native promotor and at physiological levels, but had no effect on cells expressing wild-type K-ras.  Furthermore, loss of Wt1 in lung cancer cell lines that harbor mutant K-ras leads to apoptosis.  Taken together, these observations reveal a novel role for Wt1 as a key regulator of the complex genetic network required for the oncogenic effect of the small GTPase K-ras.

We compare the expression profiles of K-ras mutant mouse cell line (LKR13) upon shRNA knockdown of K-ras itself or Wt1. The study provides insights into the transcriptional role of Wt1 in the context of oncogenic K-ras.

Expression data from mouse embryonic stem cells upon TRIM71 KD and parental ctrl cells

We identify mammalian TRIM71 as repressor of mRNAs that inhibits translation and affects mRNA stability. In this data set we compare the expression profile of mouse ES upon Trim71 KD versus that of the parental cells.

Primary human erythroid progenitor cells HDAC1 and HDAC2 shRNA knockdown samples

Gene expression profiling was performed on primary human erythroid progenitor cells expressing a control shRNA (luciferase), two different HDAC1 shRNAs, and two different HDAC2 shRNAs.
The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a novel bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

K-ras is one of the most frequently mutated human oncogenes.  Activation of K-ras can lead to either senescence or proliferation in primary cells. The precise mechanism governing these distinct outcomes remains unclear. Here we utilized a loss-of-function screen to assess the role of specific genes identified as potential key regulators of K-ras driven oncogenesis. Using this approach, we identify the transcription factor Wt1 as an inhibitor of senescence in primary cells expressing oncogenic K-ras.  Deletion or suppression of Wt1 expression leads to senescence of primary cells expressing oncogenic K-ras under the control of the native promotor at physiological levels, but has no effect on cells expressing wild-type K-ras.  Wt1 contributes to K-ras driven lung tumorigenesis in vivo and loss of Wt1 is specifically deleterious to human lung cancer cell lines that are dependent on oncogenic K-ras.  Taken together, these observations reveal a novel role for Wt1 as a key regulator of the complex genetic network required for the oncogenic effect of the small GTPase K-ras.

We compare the expression profiles of Wild type, K-ras mutant, Wt1-null, and double K-ras mutant/Wt1-null mouse embryonic fibroblasts (MEFs). The study provides insights into the transcriptional role of Wt1 in the context of oncogenic K-ras.

TOX3 is Expressed in Mammary ER Positive Epithelial Cells and Regulates a Subset of ER Target Genes in a Ligand-Independent Manner in Luminal Breast Cancer

To assess how the TOX3 nuclear protein can modulate gene expression in luminal epithelial cells, MCF7 cells were transfected with a TOX3 expression vector or vector control. In both instances, GFP was coexpressed, allowing isolation of transfected cells by flow cytometry before transcriptome analysis. Experiments were carried out under estrogen depleted conditions, and cells isolated 48 hours after transfection.

Gene expression time-course in the human skin during elicitation of allergic contact dermatitis

Genes involved in the inflammatory response resulting in allergic contact dermatitis (ACD) are only partly known. In this study, we introduce the use of high density oligonucleotide arrays for gene expression profiling in human skin during the elicitation of ACD. Skin biopsies from normal and nickel-exposed skin were obtained from 7 nickel-allergic patients and 5 non-allergic controls at four different time points during elicitation of eczema: 0h, 7h, 48h and 96h. Each gene expression profile was analysed by hybridization to high density oligonucletide arrays.
Cluster analysis of 74 genes found to be differentially expressed in the patients over time revealed that the patient samples may be categorised into two groups: An early time point group (0h and 7h) and a late time point group (48h and 96h). Compared to the early time points, late time point skin samples were characterised by the up-regulation of inflammatory molecules including genes involved in the class I antigen presenting pathway and genes involved in lymphocyte adhesion and motility
Correspondence analysis including both patients and controls revealed three distinct groups: i) the control group, ii) the early time point patient group (0h and 7h) and iii) the late time point patient group (48h and 96h).

A Novel Statistical Algorithm for Gene Expression Analysis Differentiates PXR-Dependent and Independent Mechanisms of Toxicity

The objective of the present study is to examine the potential role of the pregnane x receptor (PXR) in mice treated with an small molecule inhibitor for beta-secretase (CMP013)

TMEM88 knockdown during human embryonic stem cell cardiac differentiation

TMEM88 is indispensable for heart development and acts in the pre-cardiac mesoderm to specify lineage commitment of the cardiovascular progenitor cell through inhibition of Wnt signaling.

Klf5 controls bone marrow homing of stem cells and progenitors through Rab5-mediated membrane Beta1/Beta2-integrin expression

The Kruppel-like factor 5 (Klf5) regulates pluripotent stem cell self-renewal but its role in somatic stem cells is unknown. Klf5 deficient hematopoietic stem cells and progenitors (HSC/P) fail to engraft after transplantation. This HSC/P defect was associated with impaired bone marrow (BM) homing and lodging and decreased retention in BM. The Klf5Δ/Δ HSC/P homing defect associated with decreased adhesion to fibronectin and expression of membrane-bound β1/β2-integrins. In vivo inducible gain-of-function of Klf5 in HSC translated into increased HSC/P adhesion. The expression of Rab5 family members, mediators of β1/β2-integrin recycling in the early endosome, was decreased in Klf5Δ/Δ HSC/P. Klf5 binds directly to the promoter of Rab5a/b and overexpression of Rab5b rescued the expression of activated β1/β2-integrins, adhesion and BM homing of Klf5Δ/Δ HSC/P. Altogether, these data indicate that Klf5 is indispensable for adhesion, homing, lodging and retention of HSC/P in the BM through Rab5-dependent post-translational regulation of Beta1/Beta2 integrins.

Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells

We report a Jak2V617F knock-in mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a selective competitive advantage over wild type HSCs. In contrast, myeloid progenitor populations are expanded and skewed towards the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F positive MPN.

The effect of trypsin-2 on human tongue squamous cell carcinoma cell line gene expression

The level of trypsin-2 has been shown to correlate with the malignancy and metastatic potential of many cancer.
We used microarrays to detail how tryspin-2 overexpression changes the gene profile of human tongue squamous cell carcinoma cell line (HSC-3).

Zymosan stimulation of MyD88-deficient bone marrow-derived macrophages

Innate immune pattern recognition receptors play critical roles in pathogen detection and initiation of antimicrobial responses. We and others have previously demonstrated the importance of the beta-glucan receptor Dectin-1 in the recognition of pathogenic fungi by macrophages and dendritic cells, and have elucidated some of the mechanisms by which Dectin-1 signals to coordinate the antifungal response. While Dectin-1 signals alone are sufficient to trigger phagocytosis and Src-Syk-mediated induction of antimicrobial reactive oxygen species, collaboration with Toll-like receptor (TLR)2 signaling enhances NF-kB activation and regulates cytokine production. In this study we demonstrate that Dectin-1 signaling can also directly modulate gene expression via activation of nuclear transcription of activated T cells (NFAT) transcription factors. Dectin-1 ligation by zymosan particles or live Candida albicans yeast triggers NFAT activation in macrophages and dendritic cells. Dectin-1-triggered NFAT activation plays a role in the induction of Egr2 and Egr3 transcription factors, and cyclooxygenase 2 (Cox-2). Furthermore, we show that NFAT activation regulates IL-2, IL-10 and IL-12 p70 production by zymosan-stimulated dendritic cells. These data establish NFAT activation in myeloid cells as a novel mechanism of regulation of the innate antimicrobial response.
Keywords: stress response

The long-term goal of this project is to establish whether and how chronic nicotine exposure affects nervous system function.  The biological targets of nicotine action are diverse members of the superfamily of neurotransmitter-gated ion channels called nicotinic acetylcholine receptors (nAChR).  nAChR play multiple, critical roles in chemical signaling throughout the brain and body.  They also must be involved in nicotine dependence, which drives tobacco product use responsible for tremendous economic and personal costs.
To define changes in gene expression induced by nicotine exposure in a model neuronal cell lines expressing at least two nicotinic receptor subtypes.
Nicotine exposure exerst at least some of its effects on nervous system function by altering gene expression.
Cells of the SH-SY5Y human neuroblastoma will be exposed to an efficacious dose of nicotine or to control medium for two different periods.

To investigate the specific gene expression program by which mutant-p53 and Pin1 control invasion and metastasis in breast cancer cells, we compared the transcriptomic profile of control,  mutant-p53 depleted or Pin1 depleted MDA-MB-231 cells.

Adenoid cystic carcinoma (ACC) is an uncommon malignant neoplasm (incidence of 0.35 per 100,000) that occurs in different body sites. It most often arises in salivary glands and other secretory glands in the head and neck region. To find the gene expression signatures of ACC, we performed microarray experiments to compare ACC xenograft tumor models and normal tissue samples. We used microarrays to detail the global program of gene expression underlying adenoid cystic carcinoma and identified distinct classes of up- and down-regulated genes compared to non-neoplastic salivary tissue.

Expression data from murine irf4-/- and irf4+/+ mature B cells purified by magnetic cell separation

Gene expression profiling of murine irf4-/- and irf4+/+ splenic B cells identifies genes regulated by the transcription factor IRF4 in quiescent mature B cells.

Gene expression profiles of Cbfb-deficient and control Treg cells were compared.

Abstract: Naturally arising regulatory T (Treg) cells express the transcription factor FoxP3, which critically controls the development and function of Treg cells. FoxP3 interacts with another transcription factor Runx1 (also known as AML1). Here we showed that Treg cell-specific deficiency of Cbfβ, a cofactor for all Runx proteins, or that of Runx1, but not Runx3, induced lymphoproliferation, autoimmune disease, and hyper-production of IgE. Cbfb-deleted Treg cells exhibited impaired suppressive function in vitro and in vivo, with altered gene expression profiles including attenuated expression of FoxP3 and high expression of interleukin-4. The Runx complex bound to more than 3000 gene loci in Treg cells, including the Foxp3 regulatory regions and the Il4 silencer. In addition, knockdown of RUNX1 showed that RUNX1 is required for the optimal regulation of FoxP3 expression in human T cells. Taken together, our results indicate that the Runx1-Cbfβ heterodimer is indispensable for in vivo Treg cell function, in particular, suppressive activity and optimal expression of FoxP3.

Expression data from bone marrow CD34+ cells of MDS patients and healthy controls

In order to gain insight into the molecular pathogenesis of the myelodysplastic syndromes (MDS), we performed global gene expression profiling and pathway analysis on the hematopoietic stem cells (HSC) of 183 MDS patients as compared with the HSC of 17 healthy controls. The most significantly deregulated pathways in MDS include interferon signaling, thrombopoietin signaling and the Wnt pathway. Among the most significantly deregulated gene pathways in early MDS are immunodeficiency, apoptosis and chemokine signaling, whereas advanced MDS is characterized by deregulation of DNA damage response and checkpoint pathways. We have identified distinct gene expression profiles and deregulated gene pathways in patients with del(5q), trisomy 8 or –7/del(7q). Patients with trisomy 8 are characterized by deregulation of pathways involved in the immune response, patients with –7/del(7q) by pathways involved in cell survival, whilst patients with del(5q) show deregulation of integrin signaling and cell cycle regulation pathways. This is the first study to determine deregulated gene pathways and ontology groups in the HSC of a large group of MDS patients. The deregulated pathways identified are likely to be critical to the MDS HSC phenotype and give new insights into the molecular pathogenesis of this disorder thereby providing new targets for therapeutic intervention.

TEC (B6 vs Aire KO). Thymic epithelial cells from B6 strain and AIRE KO are compared.

Table 6: Cryptosporidium parvum-infected HCT-8 cells, 72 hours post-infection (average of 3 replicates)

• The origin of biological samples (In vitro infection of HCT-8 cells with Cryptosporidium parvum)

Monolayers of the HCT-8 cells (ATCC CCL-244, American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and additional nutritional supplements. Cells in log phase were plated at 2×106 cells per 150 mm tissue culture dish and infected with sterilised Cryptosporidium parvum oocysts (Iowa strain). Purified oocysts were suspended in phosphate buffered saline, sterilised in 33% bleach for 7 min on ice, washed in Hank's buffered saline solution, and added to HCT-8 cultures at a ratio of one oocyst per cell. Following a 2 h excystation period at 37 °C, cells were washed with warm Hank's buffered saline solution, and cells were incubated in fresh supplemented media. Mock-infected cultures were treated identically, with the exception that oocysts were not added to the cultures.

Three independent mock- and C. parvum-infected cultures were prepared for analysis.

•  The origin of mRNA samples

At 6, 12, 24, 48, and 72h post-inoculation, cell culture media were removed and cultures were immediately lysed by the addition of 3.5 ml of Trizol® reagent (GIBCO BRL Life Technologies) directly to the culture plate. Total RNA was prepared as described by the manufacturer, and poly(A) mRNA was isolated using oligo-dT cellulose columns (Amersham Pharmacia Biotech). The qualities of total RNA and poly(A) mRNA preparations were assessed by Northern blot.

•  Protocols for conducting microarray hybridization

Poly(A) mRNA was converted into ‘target’ suitable for hybridisation to Affymetrix microarray chips according to protocols provided by Affymetrix Inc. (Santa Clara, CA). Briefly, 2.0  g of poly(A) mRNA was reverse-transcribed to prepare double-stranded cDNA using T7-(dT)24 primer (GENSET Corp., La Jolla, CA) and Superscript II reverse transcriptase (GIBCO). Approximately 1  g of cDNA was used for in vitro transcription in the presence of biotinylated UTP and CTP using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Inc). This labelled cRNA was fragmented in 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94 °C for 35 min. The integrity of cRNA and the efficiency of fragmentation were monitored by Northern blot analysis (not shown).
Fragmented biotinylated cRNA targets were hybridised to HG-U95Av2 chips (Affymetrix) that contain probe sets for 12,600 human gene/transcripts. Fifteen micrograms of fragmented cRNA and appropriate controls were hybridised to the chips at 45 °C for 16 h with constant rotation at 60 rev./min. The chips were subsequently washed and stained with streptavidin–phycoerythrin conjugate using the GeneChip Fluidics station protocol EukGE_WS2 (Affymetrix). Following washing and staining, microarray chips were scanned twice at 3  m resolution using a Hewlett-Packard confocal scanner and hybridisation intensities for each of the genes/transcripts were collected from scanned images.

•  Analysis of microarray data

Gene expression data were initially analysed with the GeneChip® expression analysis software (Affymetrix Microarray Suite, version 5.0). The fluorescence intensity of the genes/transcripts was measured for each probe array and, to minimise discrepancies due to non-biological variations, normalised by global scaling to 1000.

Further data analyses were performed using GeneSpring software (version 6.0; Silicon Genetics, Redwood City, CA). Per gene normalisation was applied in which the expression signal of each gene in C. parvum-infected cells (raw data) was normalised to the median of its measurements in the mock-infected samples (control); and the ratio of expression levels between mock and infected samples was calculated as the mean value of normalised signal vs. control signal among three replicates. To identify genes with significantly altered expression, a series of statistical analyses (filtering) were performed: cut-off values for ratio of expression levels 1.80 and 0.55 were used to filter genes with expression level fold changes greater than ±1.8 in all three independent samples. Genes with fold change variations >1.5 across the three samples were excluded. Furthermore, a ‘statistical group comparison’ using Student's t-test/ANOVA was conducted to compare mean expression levels between mock-infected and infected samples, and the genes with significant differential expression (P<0.05) were selected. Identified differentially expressed genes were then annotated using GeneSpring's ‘Build Simplified Ontology’ constructor which hierarchically groups genes into meaningful biological categories (gene lists) based on the Gene Ontology Consortium Classifications. Various lists of regulated genes were created by cross-referencing annotated gene lists and applying assorted statistical and visualisation methods.

Arid3a modulates the first cell fate decision by direct regulation of both embryonic and extraembryonic gene expression (microarray)

Arid3a, a transcription factor known for its requirement in B-lymphocyte development, has been recently identified as a member of ES cell pluripotency network. Arid3a is moderately expressed in ES cells, and its expression is gradually increased during differentiation. Since Arid3a shows the highest expression in placenta, we hypothesized that Arid3a may play important roles in TE development. We report that Arid3a is a central regulator of both TE-specific and pluripotency-associated gene expression during ES cell differentiation. While dispensable for self-renewal, we observed that knockdown of Arid3a delays differentiation of ES cells. Induction of Arid3a leads ES cells to promote differentiation, specifically towards TE lineage. Moreover, these Arid3a-overexpressing cells maintained in TE culture media are sufficient to generate functional trophoblast stem-like cells, suggesting roles of Arid3a in TE differentiation. By integrative analyses using the chromosomal targets of Arid3a with expression profiling, we revealed the dual roles of Arid3a, as a direct activator of TE-specific genes and a repressor of pluripotency-associated genes. We further revealed the repressive roles of Arid3a are mediated by histone deacetylases (HDACs). Taken together, our results demonstrate that Arid3a is a critical novel regulator in TE lineage specification.

Expression changes after loss of Dot1l in murine MLL-AF9 leukemia cells

MLL-fusions may induce leukemogenic gene expression programs by recruiting the histone H3K79 methyltransferase to MLL-target promoters. We evaluated gene expression changes after cre-mediated loss of Dot1l in leukemia cells obtained from mice injected with MLL-9 transformed lineage negative bone marrow cells.

To unravel the function of VAMP7 in the male sexual differentiation, we carried out in vitro studies of VAMP7 knockdown using siRNA, in the human embryonal carcinoma NTERA2/D1 cells.

We used Affymetrix HG U133 Plus 2.0 GeneChips to compare the transcriptome of MDA-MB-231 cells transfected with SDC1-siRNA against negative control siRNA-transfected cells.

Expression data of cystic fibrosis and non-cystic fibrosis airway cell lines under oxidative stress

CF's physiopathology is poorly explained by the mutation alone. The oxydative stress could be a major factor of this illness . Study its impact on transcriptome's CF cell line could be ameliorate our understanding of the evolution of cystic fibrosis.
we used microarray technology to evaluate  under oxydative stress,  the transcriptional state  of an epithelial lung cell issued from a human with cystic fibrosis and to identify a set of modulated genes associated to survival cell processes.

Comparison of MBT/Pas and BALB/cByJ MEFs response after infection with Rift Valley Fever virus

The Rift Valley Fever (RVF) is an arthropod-borne disease present in several countries of Africa and Middle East. It is caused by RVF virus which can infect both humans and animals. In humans, it leads to various manifestations including hepatitis, encephalitis and death, while in domestic animals it usually causes miscarriage in pregnant females and it is often fatal for the newborn. Not all people or animal infected by the virus present the same disease. Some patients exhibit unapparent or moderate febrile reactions, while others develop severe symptoms. This observation suggests that host genetic factors play a role in controlling the outcome of infection. In this work, we compare the response of two different inbred strains of mice, MBT/Pas and BALB/cByJ, to infection with RVF virus. These strains exhibit different profiles of susceptibility to RVF virus infection. Indeed, MBT/Pas mice rapidly develop high viraemia and die soon after infection, while BALB/cByJ mice have a lower viraemia and die later. Interestingly, mouse embryonic fibroblasts (MEFs) obtained from MBT/Pas foetuses allows higher viral production than BALB/cByJ MEFs.

Identification of Sp1 targets involved in proliferation and cancer

Sp1 is a transcription factor able to regulate many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, with a special emphasis to those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA (siSp1) to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1 siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis.  Underexpressed genes were selected since they represent putative genes activated by Sp1. These underexpressed genes were classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipidic metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the MatchTM software. After literature mining, 11 genes were selected for further validation of their expression levels using RT-real time PCR. Underexpression was confirmed for the 11 genes plus Sp1 in HeLa cells after siSp1 treatment. Additionally, EMSA and chromatin immunoprecipitation assays were performed to test for binding between Sp1 and the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Finally, the mRNA levels of RAB20, FGF21 and IHPK2, three genes related with proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their relationship with Sp1.

Gene expression profiles induced by shRNA-mediated knockdown of PDEF in MCF7 mammary epithelial cell line

Microarray gene expression analysis was performed in MCF7 cells transduced with a non-specific shRNA or PDEF-targeting shRNA, and both subjected to hormone depletion for 48 hours. Analyses of differentially expressed genes combined with gene ontology revealed a downregulation of cell cycle related-genes and an upregulation of apoptosis-related genes in PDEF knockdown cells. These target genes constitute potential effectors of the pro-survival role of PDEF.

Activation of beta-catenin in hepatocellular carcinoma (mouse Ctnnb1 knockout)

CTNNB1 is the most frequently mutated gene in hepatocellular carcinoma (HCC). However, its clinical relevance remains controversial. We determined an evolutionarily conserved β-catenin signature by comparative analysis of gene expression data from human HCC (GSE43619) and a mouse model.

Transcriptional profiling of the human frontal cortex from individuals ranging from 26 to 106 years of age. The postmortem brain tissue samples used in this study were neuropathologically normal for age, and were derived from non-demented individuals.

Primary tumor growth induces host tissue responses that are believed to support and promote tumor progression. Identification of the molecular characteristics of the tumor microenvironment and elucidation of its crosstalk with tumor cells may therefore be crucial for improving our understanding of the processes implicated in cancer progression, identifying potential therapeutic targets, and uncovering stromal gene expression signatures that may predict clinical outcome. A key issue to resolve, therefore, is whether the stromal response to tumor growth is largely a generic phenomenon, irrespective of the tumor type, or whether the response reflects tumor-specific properties. To address similarity or distinction of stromal gene expression changes during cancer progression, oligonucleotide-based Affymetrix microarray technology was used to compare the transcriptomes of laser-microdissected stromal cells derived from invasive human breast and prostate carcinoma. Invasive breast and prostate cancer-associated stroma was observed to display distinct transcriptomes, with a limited number of shared genes. Interestingly, both breast and prostate tumor-specific dysregulated stromal genes were observed to cluster breast and prostate cancer patients, respectively, into two distinct groups with statistically different clinical outcomes. By contrast, a gene signature that was common to the reactive stroma of both tumor types did not have survival predictive value. Univariate Cox analysis identified genes whose expression level was most strongly associated with patient survival. Taken together, these observations suggest that the tumor microenvironment displays distinct features according to the tumor type that provides survival-predictive value.

Human lung cancer (A549) cells were treated 50uM of the metal cation-containing chemotherapeutic drug motexafin gadolinium (MGd) for 4, 12, and 24 hrs and expression compared to control cells (treated with 5% mannitol for the same length of time)

Global expression analysis of DLBCL cell lines with LITAF over-expression or silencing

The multifunctional protein lipopolysaccharide-induced TNFalpha factor (LITAF) induces the secretion of inflammatory cytokines in monocytes and regulates protein degradation in neural cells. In B-cell lymphomas, LITAF is frequently inactivated by epigenetic mechanisms, but beyond these data little is known about its regulation and function. Immunohistochemical and gene expression profiling analyses of normal and malignant B-cells revealed that LITAF and BCL6 exhibited opposite expression patterns. Accordingly, chromatin immunoprecipitation and luciferase experiments showed that LITAF is transcriptionally repressed by BCL6 in germinal center (GC) lymphocytes and in B-cell lymphoma cells. Gain- and-loss-of-function assays demonstrated that LITAF does not exert any of its previous roles. Conversely, LITAF co-localized with autophagosomes in B-cells whereby activated autophagic responses, which were abrogated upon LITAF silencing. Therefore, BCL6-mediated transcriptional repression of LITAF may contribute to an appropriate GC reaction by suppressing autophagy in GC lymphocytes, whereas constitutive repression of autophagic responses may promote B-cell lymphoma development.

Transcription factor TFAP2C regulates major programs required for murine fetal germ cell maintenance and haploinsufficiency predisposes to teratomas in male mice

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor Tcfap2c / TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tcfap2c in primordial germ cell-like cells. We show that loss of Tcfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation maturation markers and induction of markers indicative of somatic differentiation, cell cycle, epigenetic remodeling, and pluripotency associated genes. Chromatin-immunoprecipitation analyses demonstrated binding of Tcfap2c to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of Tcfap2c in primordial germ cells. Since Tcfap2c deficient primordial germ cell like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tcfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tcfap2c develop germ cell cancer with high incidence. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate, that mice with a heterozygous deletion of the Tcfap2c target gene Nanos3 are also prone to develop teratoma. These data highlight Tcfap2c as a critical and dose-sensitive regulator of germ cell fate.

Differential gene expression in the proximal neck of human abdominal aortic aneurysm

The aim of this study was to assess the gene expression profile of biopsies obtained from the neck of human AAAs.

We used microarrays to analyze the global gene expression and identified differentially expressed gene list between wild-type anterior prostates and Pb-Cre4;PtenLoxP/LoxP anterior prostates, Pb-Cre4;PtenLoxP/LoxP;LrfLoxP/LoxP anterior prostates at 12 weeks of age.

Expression data from human MSI colorectal cancer and normal colonic mucosa

Microsatellite instability (MSI), caused by defective mismatch repair, is observed in a subset of colorectal cancers (CRCs). We evaluated somatic mutations in microsatellite repeats of genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat.

Overexpression of Gfi1b in a pro-B Abelson murine leukemia virus transformed cell line

We have conducted a screen for factors that downregulate expression of the genes encoding the V(D)J  recombinase (RAG1 and RAG2) during B cell development.  We have identified the transcription factor Gfi1B as being one of the proteins capable of decreasing RAG transcription when overexpressed in Ableson transormed ProB cell lines.  We have yet to determine whether the overexpression of Gfi1B downregulates the RAGs directly, or whether it initiates a signalling programme that results in RAG downregulation.  We hypothesize that by comparing global gene expression patterns in cells that overexpress Gfi1B and those that do not, we can distinguish between these possibilities and additionally gain insight into the broader genetic program that may be influenced by Gfi1B during hematopoiesis.

Gene expression analysis in control and diabetic rats. Diabetes-induced erectile dysfunction in rat model of DM. 10 weeks of streptozotocin induced diabetes. F344 Rats.

Temporal control of gene expression by ERK MAP kinase during cell cycle progression from G0/G1 to S phase

The ERK family of MAP kinase plays a critical role in growth factor-stimulated cell cycle progression from G0/G1 to S phase. But, how sustained activation of ERK promotes G1 progression has remained unclear. Here, our systematic analysis on the temporal program of ERK-dependent gene expression shows that sustained activation of ERK is required for induction and maintenance of the decreased expression levels of a set of genes. Moreover, our cell biological analysis reveals that these ERK-dependent downregulated genes have the ability to block S phase entry. Cessation of ERK activation at mid or late G1 leads to a rapid increase of these anti-proliferative genes and results in the inhibition of S phase entry. These findings uncover an important mechanism by which the duration of ERK activation regulates cell cycle progression through dynamic changes in gene expression, and identify novel ERK target genes crucial for the regulation of cell cycle progression.

Rationale: Immortalized cells may exhibit important differences relative to their primary cell counterparts. Microarrays were used compare primary human umbilical vein endothelial cells (HUVECs) and the immortalized HUVEC cell line EA.hy926, in their response to inhibition of the mevalonate pathway by a HMG-CoA reductase inhibitor (atorvastatin). The effects of atorvastatin were reversed by the addition of mevalonate, to subtract non-specific changes in gene expression.
Methods: Confluent cell cultures of HUVECs and EA.hy926 cells (two independent experiments in each cell type) were incubated with 1) statin-free media; 2) 10 microM atorvastatin; 3) 500 microM mevalonate; or 4) a combination of 10 microM atorvastatin and 500 microM mevalonate. All cells were harvested at 24 h. Total RNA was isolated using Trizol reagent (Invitrogen). cDNA was prepared from 10 microgram total RNA using a double-stranded cDNA synthesis kit (Life Technologies) with a T7-dT24 primer for first-strand synthesis. cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Twenty microgram of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer, containing 40 mM Tris-acetate, pH 8.1, 125 mM KOAc, 30 mM MgOAc. Fifteen micrgram of fragmented cRNA, together with control cRNAs and grid alignment oligonucleotides, were hybridized overnight to GeneChip Human Genome U133A 2.0 arrays (Affymetrix) at 45°C under constant rotation. Arrays were washed and incubated with an anti-streptavidin antibody. Fluorescent signals were measured using an Agilent Gene Array Laser Scanner and analyzed with MicroArray Suite 5.0 (Affymetrix). Microarrays were scaled using default settings.

Analysis of the gene expression profile between MYC positive and MYC negative germinal center B-cells.

mononuclear cells;gene microarray;pulmonary arterial hypertension;Herpesvirus;biomarker;other

Microarray analysis of peripheral blood mononuclear (PBMC) cells in pulmonary arterial hypertension.
Keywords = mononuclear cells

Expression profiling of hearts from FVB males subjected to cardiac pressure overload by transverse aortic constriction (TAC). TAC performed on 8-10 weeks month old males and females. Hearts examined  30 weeks after surgery.

Summary:
HEK293 cells were transfected with control plasmid (pcDNA1/Neo; Invitrogen) or with the plasmid encoding HCaRG by a standard calcium phosphate co-precipitation method. The clones expressing the highest levels of HCaRG, HCaRG clone 8 and 9 were used in this experiment, while clone transfected with vector alone, Neo clone, served as control.
Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen).

Energy Substrate Uptake and Metabolism are Preserved in Hypertrophic Caveolin-3 Knockout Hearts
Keywords: gene knockout

Hearts Lacking Caveolin-1 Develop Hypertrophy with Normal Cardiac Substrate Metabolism

Comparison of gene expression profiles among Nrf1 liver-specific conditional knockout, Nrf2 knockout and Keap1 knockdown mice.

To identify Nrf1-dependent and Nrf2-dependent genes in the liver, we examined the gene expression profiles of Nrf1 Alb-CKO, Nrf2 knockout and Keap1 knockdown mouse livers by microarray analyses.

The role of Raf-1 kinase inhibitor protein in the regulation of pancreatic beta cell proliferation in mice

AIMS/HYPOTHESIS: Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function.
METHODS: Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment.
RESULTS: Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice.
CONCLUSIONS/INTERPRETATION: These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Transcriptional profiling of IRF4 -/- vs IRF4 +/- T-cells under Th17 polarizing conditions

Transcriptional profiling of T-cells isolated from spleen of IRF4 -/- mice and cultured under Th17 polarizing conditions for 42 hrs compared to cells similarly isolated and cultured from spleen of IRF4 +/- mice. The aim of the study was to identify global misexpression of genes in IRF4 -/- cells and hence identify key pathways regulated by IRF4 during Th17 differentiation.

Profiling of immortalized human lung epithelial cells following oncogenic KRAS expression and TBK1 suppression

The purpose of the dataset is to analyze expression of genes induced by KRAS and regulated by TBK1
The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. An alternative strategy for targeting KRAS is to identify gene products that, when suppressed or inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkB kinase, TBK1, was selectively essential in cells that harbor mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF- B anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 as a potential therapeutic target in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer.

Grhl2 regulated genes in the mouse lung epithelial MLE15 cell line

The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies, we identified the NK2 homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays, and by confocal microscopy we showed that these two transcription factors form a positive feed-back loop in vivo and in cell lines, and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration and cell-cell interactions.

Early effects of Serum Response Factor (SRF) gene deletion on the vascular gene expression

The objective of this study is to assess the early effects of the Serum Response Factor deletion on the vascular gene expression program.
To generate smooth muscle specific SRF knockout, 4 month-old [SRF-flex2neo -SM22CreERT2ki/+] mice (SRFSMKO mice) were injected i.p. with 1mg of tamoxifen for 3 days. All control (SRF-flex2neo littermates) and SRFSMKO mice were systematically injected with tamoxifen to normalize for any effects due to tamoxifen.

Gene Expression Preferentially Regulated by Tamoxifen in Breast Cancer Cells

The beneficial effect of the selective
estrogen receptor modulator (SERM) tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like
agonist effects.  Here we report that in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of more than 60 genes in estrogen receptor alpha
(ERa)-positive MCF-7 human breast cancer cells which are minimally regulated by estradiol or raloxifene.  This gene regulation by tamoxifen is mediated by ERa and is reversed by estradiol or ICI 182,780.  We find that the introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes, suggesting that ERbeta is capable of repressing certain actions of tamoxifen. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, the expression level of these genes was examined in breast tumors of women who had received treatment with tamoxifen.  High expression of two of the tamoxifen stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive tumors, and hence appear to be markers of a poor prognosis.
Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen-preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy.

We observed robust overexpression of type I interferon (IFN)–inducible genes and genomic signatures that indicate T cell and dendritic cell infiltration in lesional skin. Up-regulation of mRNAs for IFN-a subtypes was observed in lesional skin compared with nonlesional skin. Enrichment of mature dendritic cells and 2 type I IFN–inducible proteins, STAT1 and ISG15, were observed in the majority of lesional skin biopsies. Concordant overexpression of IFN-c and TNF-a–inducible gene signatures occurred at the same disease sites.

Systems biology approach to identify transcriptional reprogramming and microRNA targets during the progression of Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation throughout the kidney parenchyma. It is caused by mutations in either of two genes, PKD1 and PKD2. Mice that lack functional Pkd1 (Pkd1null/null), develop rapidly progressive cystic disease during embryogenesis, and serve as a model to study human ADPKD.  We examined the molecular pathways that modulate renal cyst growth in the Pkd1null/null model by performing global gene-expression proﬁling in embryonic kidneys at day 14 and 17. Gene Ontology and gene set enrichment analysis were used to identify overrepresented signaling pathways in Pkd1null/null kidneys. We found dysregulation of developmental, metabolic, and signaling pathways (e.g. Wnt, calcium, TGF-b and MAPK) in Pkd1null/null kidneys.

Sirtuins are a family of protein deacetylases, deacylases, and ADP-ribosyltransferases that regulate life span, control the onset of  numerous age-associated diseases, and mediate metabolic homeostasis. We have uncovered a novel role for the mitochondrial  sirtuin SIRT4 in the regulation of hepatic lipid metabolism during changes in nutrient availability. We show that SIRT4 levels  decrease in the liver during fasting and that SIRT4 null mice display increased expression of hepatic peroxisome proliferator activated  receptor   (PPAR ) target genes associated with fatty acid catabolism. Accordingly, primary hepatocytes from SIRT4  knockout (KO) mice exhibit higher rates of fatty acid oxidation than wild-type hepatocytes, and SIRT4 overexpression decreases  fatty acid oxidation rates. The enhanced fatty acid oxidation observed in SIRT4 KO hepatocytes requires functional SIRT1, demonstrating a clear cross talk between mitochondrial and nuclear sirtuins. Thus, SIRT4 is a new component of mitochondrial signaling in the liver and functions as an important regulator of lipid metabolism.

Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional neurological, lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1-/- mice (Npc1nih)relative to Npc1+/- at an asymptomatic stage, as well as early- and late-symptomatic stages.  Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates for the neurological disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher’s disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1-/- as well as Balb/c Npc1nmf164 mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1-/- mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry.
We used microarrays on the diseased organs, brain, liver and spleen of the Npc1-/- mice  to unserstand the molecular changes occur during the progression of NPC diseases. From the data, we have identified 12 potential genes which can be potentially developed as blood-based biomarker. We have also discovered up regulation of innate iimunity genes in all three organs of Npc1-/- mice and functionally validated them in liver and spleen.

TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells using the Cre-loxP strategy. While spermatogenesis is completed in Taf7l mutant mice, the weight of Taf7l mutant testis is decreased and the amount of sperm in the epididymis is sharply reduced. Mutant epididymal sperm exhibit abnormal morphology including folded tails. Sperm motility is significantly reduced, and Taf7l mutant males are fertile with reduced litter size. Microarray profiling reveals that the abundance of six gene transcripts (including Fscn1) in Taf7l mutant testis decreases by > 2-fold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ-cell differentiation. Our mouse studies suggest that mutations in human TAF7L gene might be implicated in X-linked oligozoospermia in men.

Effects of diethylstilbestrol (DES) on the anterior pituitary gland of the ACI, Copenhagen and Brown Norway Rat.

Anterior pituitary glands were isolated from 21 week old male rats either untreated or treated for 12 weeks with the synthetic estrogen diethylstilbestrol (DES). Three biological replicates were prepared for untreated, control animals and four biological replicates were prepared for DES-treated animals. This was done for each of 3 inbred rat strains: ACI, Copenhagen, and Brown Norway. Expression profiles were determined using Affymetrix Rat Genome 230 v. 2.0 arrays. Comparisons of untreated vs. treated animals within a strain will allow identification of estrogen responsive genes. Comparisons between strains, either treated or untreated, will identify strain (i.e., genetic) differences in expression.
Keywords: Estrogen Response

Basonuclin, which is a zinc-finger protein found in abundance only in the keratinocytes of the stratified epithelium, male germ cells and oocytes, qualifies as a maternal-effect gene because the source of pre-implantation embryonic basonuclin is maternal.  Using a transgenic-RNAi approach, we knocked-down basonuclin specifically in mouse oocytes, which led to female sub-fertility.  Basonuclin deficiency in oocytes perturbed both RNA polymerase I- and II-mediated transcription and oocyte morphology was affected as evidenced by cytoplasmic and cell surface abnormalities.  The affected oocytes, however, could still mature to and arrest at metaphase II and be ovulated, suggesting the impaired pathways were not essential for oocyte development and maturation.  Nevertheless, an early embryonic failure in pre-implantation development was identified and likely accounted for the sub-fertility phenotype.  These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.
Keywords: RNAi knockdown

Gene Expression Changes in the Rat Nasal Epithelium Following Formaldehyde Exposure

Formaldehyde, an important industrial chemical, is used for multiple commercial purposes throughout the industrialized world.  This simple, one carbon aldehyde is a natural metabolite formed in cells throughput the body.  However, it is also a rodent nasal carcinogen, when inhaled by rats every day for two-years at irritant concentrations.  High tumor incidences occur at concentration of 10 ppm and above; no tumors are observed at concentrations below 6.0 ppm.  The US Environmental Protection Agency (US EPA) is now (2007) conducting a risk assessment to try to evaluate possible cancer risks for much lower levels of human exposure.  Sensitive methods are needed to evaluate tissue responses below those concentrations that are clearly irritant or carcinogenic.  This microarray study was undertaken to evaluate the mode of action for nasal responses to inhaled formaldehyde in Fisher 344 rats over a range of exposure concentrations.  The range of concentrations used spanned those at which virtually no tissue responses were observed (0.7 ppm) to those that represent the highest concentration in the cancer studies (15 ppm) that produced nasal tumors in half the exposed group of rats.  The study identified doses at which there were no statistically significant changes in gene expression; intermediate doses with changes in a small number of genes not easily grouped by function; and then concentrations where changes were consistent with irritation and cell stress responses.
Keywords: time course, dose response, gene expression

Hematopoietic progenitor cells were isolated from 13.5 day mouse fetal livers by lineage depletion and expanded for three days. Fetal livers were isolated from both wild type and Gata-1 knock embryos. Gata-1 knock embryos contain a deletion of the Gata-1 promoter sequence that results in undetectable levels of Gata-1 protein specifically in the megakaryocyte lineage. Following progenitor outgrowth  megakaryocytes were enriched in a differentiation media for three days and isolated on a discontinuous BSA gradient. The resulting megakaryocytes were >90% pure as determined by acetylcholinesterase staining. These cells were lysed in Trizol and the resulting RNA was used for hybridization.
Keywords = Gata-1

Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. Originally, NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. We have now systematically analyzed the molecular mechanism of variable NMD efficiency and used different HeLa cell strains as a model system. The results of this analysis show that NMD efficiency can be remarkably variable and represents a stable characteristic of these strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the HeLa strain analyzed. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. We conclude that cellular concentrations of RNPS1 modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that controls NMD.
Keywords: NMD UPF1 knock down

Comparative Analysis of MMTV-neu tumors, preneoplastic MMTV-neu mammary gland, and Wild-type controls

To identify early events of erbB2-induced mammary tumorigenesis, we compared datasets from 14 genechip experiments including MMTV-neu tumors, preneoplastic neu mammary gland (adjacent neu), and age-matched, wild-type control mammary glands

The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein and organelle clearance. In skeletal muscle, both systems’ excessive activation induces severe muscle loss. Although altered proteasomal function has been observed in various myopathies, the specific role of proteasomal activity in skeletal muscle has not been determined by loss-of-function approaches. Here, we report that muscle-specific deletion of a crucial proteasomal gene, Rpt3, resulted in profound muscle atrophy and decrease in force. Rpt3 null muscles showed reduced proteasomal activity in early age, accumulation of basophilic structure, disorganization of sarcomere, and formation of vacuoles and concentric membranous structures in electronmicroscope. We also observed accumulation of ubiquitin, p62, LC3, TDP43, FUS and VCP proteins. Proteasomal activity is important to preserve muscle mass and to maintain myofiber integrity. Our results suggest that inhibition/alteration of proteasomal activity can contribute to myofiber degeneration and weakness in muscle disorders, such as inclusion body myositis, characterized by accumulation of abnormal inclusions.

We used microarrays to detail the global programme of gene expression underlying cardiac development by HDAC2 and identified distinct classes of up-regulated and down-regulated genes during this process.
Keywords: Genetic Modification

diabetes, mouse models, diabetic nephropathy, streptozotocin db/db

Persons with Down syndrome (DS) exhibit low muscle strength that significantly impairs their physical functioning. The Ts65Dn mouse model of DS also exhibits muscle weakness in vivo and may serve as a useful model to examine potential factors responsible for DS-associated muscle dysfunction. Therefore, the purpose of this experiment was to directly assess skeletal muscle function in the Ts65Dn mouse and to reveal potential mechanisms of DS-associated muscle weakness. Soleus muscles were harvested from anesthetized male Ts65Dn and wild-type (WT) colony controls.  In vitro muscle contractile experiments revealed normal force generation of unfatigued Ts65Dn soleus, but a 12% reduction in force was observed in Ts65Dn muscle during recovery following fatiguing contractions compared to WT muscle (p<0.05). Oxidative stress may contribute to DS-related pathologies, including muscle weakness, which may be the result of overexpression of chromosome 21 genes (e.g., copper-zinc superoxide dismutase (SOD1)). SOD1 expression was 25% higher (p<0.05) in Ts65Dn soleus compared to WT muscle but levels of other antioxidant proteins were unchanged. Lipid peroxidation (4-hydroxynoneal) was unaltered in Ts65Dn muscle although protein carbonyls were 20% greater compared to muscle of WT animals (p<0.05). Cytochrome c oxidase expression was reduced 22% in Ts65Dn muscle, suggesting a limitation in mitochondrial function may contribute to post-fatigue muscle weakness. Microarray analysis of Ts65Dn soleus revealed alteration of numerous cellular pathways including: proteolysis, glucose and fat metabolism, neuromuscular transmission, and ATP biosynthesis. In summary, the Ts65Dn mouse displays evidence of muscle dysfunction, and the potential role of mitochondria and oxidative stress warrants further investigation.

Thousands of enhancers are characterized in the human genome, yet few have been shown important in cancer. Inhibiting oncokinases, such as EGFR, ALK, HER2, and BRAF, is a mainstay of current cancer therapy but is hindered by innate drug resistance mediated by upregulation of the HGF receptor, MET. The mechanisms mediating such genomic responses to targeted therapy are unknown. Here, we identify lineage-specific MET enhancers for multiple common tumor types, including a melanoma lineage-specific MET enhancer that displays inducible chromatin looping and MET gene induction upon BRAF inhibition. Epigenomic analysis demonstrated that the melanocyte-specific transcription factor, MITF, mediates this enhancer function. Targeted genomic deletion (<7bp) of the MITF motif within the MET enhancer suppressed inducible chromatin looping and innate drug resistance, while maintaining MITF-dependent, inhibitor-induced melanoma cell differentiation. Epigenomic analysis can thus guide functional disruption of regulatory DNA to decouple pro- and anti-oncogenic functions of tumor lineage-enriched transcription factors mediating innate resistance to oncokinase therapy.

Microarray analysis of genes activated by mouse CAR and human CAR in vivo.

This dataset is a four-ligand x three-genotype Affymetrix microarray analysis of the regulation of liver genes in the mouse by the constitutive androstane receptor (CAR).

UTX gene is localized on the X chromosome, identified as a demethylase on histone H3 lysine 27.

Microarray data of differentiating embryonic stem cells overexpressing the transcription factor Msgn1

During mammalian gastrulation, pluripotent epiblast stem cells migrate through the primitive streak to form the multipotent progenitors of the mesoderm and endoderm germ layers. Msgn1 is a bHLH transcription factor and is a direct target gene of the Wnt/bcatenin signaling pathway. Msgn1 is expressed in the mesodermal compartment of the primitive streak and is necessary for the proper development of the mesoderm. Msgn1 mutants show defects in somitogenesis leading to a lack of trunk skeletal muscles, vertebra and ribs.
To study the molecular and cellular function of Msgn1 in Embryonic Stem Cells (ESC), we have generated doxycycline inducible gain-of-function ESC to overexpress Msgn1 in ESC. In order to identify Msgn1 targets, we performed transcriptional profiling of Msgn1 expressing ES cells and found that upon induction of Msgn1, multiple genes in the Notch pathway were differentially expressed compared to the uninduced cells. Moreover, Whole Mount Insitu Hybridization analysis in Msgn1 null mutants revealed that these Notch pathway genes required Msgn1 for their proper expression in vivo. Our studies demonstrate that Msgn1 is a critical effector of the Wnt pathway during mammalian somitogenesis, mediating crosstalk between the Wnt and Notch pathways.

Transcriptomic approach to renal toxicity for cisplatin treatment in Nrf2 WT/KO

NF-E2-related factor 2 (Nrf2), a transcription factor activated by oxidative stress, induces phase II conjugating or antioxidant enzymes. In the present study, we examined the effect of Nrf2 knockout (Nrf2 KO) on the kidney injury induced by cisplatin using cDNA microarray analyses. A transcriptomic approach enabled us to extract gene clusters which show significantly different mRNA expression patterns between wild type (WT) and Nrf2 KO mice. In the experimental set, WT vehicle (WT-Veh) and WT cisplatin (WT-Cis) groups were included in our previous GEO database (GSE35257) because the two groups were simultaneously shared with Nrf2 KO vehicle (Nrf2 KO-Veh) and Nrf2 KO cisplatin (Nrf2 KO-Cis) groups in the current report.   Copyright (c) 2012 by Korea Food & Drug Administration.

Multi-tissue, selective PPARγ modulation of insulin sensitivity and metabolic pathways in obese rats

We characterized the insulin sensitivity and multi-tissue gene expression profiles of lean and insulin resistant, obese Zucker rats untreated or treated with one of four PPARγ ligands (pioglitazone, rosiglitazone, troglitazone, and AG035029). We analyzed the transcriptional profiles of adipose tissue, skeletal muscle, and liver from the rats and determined whether ligand insulin-sensitizing potency was related to ligand-induced alteration of functional pathways.  Ligand treatments improved insulin sensitivity in obese rats, albeit to varying degrees.
Male Zucker fatty (fa/fa) and lean (fa/+) rats (Charles River, Wilmington, MA) were received at 6 weeks of age. Fatty rats were weight-matched upon arrival and randomly divided into one of five experimental groups.  The fatty rat groups varied by the type of chow they were fed - normal chow alone or with a PPARγ ligand admixture:  normal chow (fatty control, FC), rosiglitazone-treated (Rosi), pioglitazone-treated (Pio), troglitazone-treated (Tro), or AG035029-treated (AG). Lean control (LC) rats were all fed normal chow. Rats groups were maintained on the diets for 21 days.

Overexpression of Dmrt5 in in vitro differentiated neural progenitor cells regulates gene expression

To test the regulatory effects of Dmrt5 on gene expression, we designed tetracycline inducible lines of Dmrt5 transgenic mouse ESCs. Overexpression of Dmrt5 was induced upon addition of Doxycycline (Dox). To evaluate the effects of Dmrt5 on gene expression in different stages of in vitro differentiated NPC derived from mouse embryonic stem cells (ESC), we analyzed gene expression profiles at differentiation day 7 and day 9 with or without Dox. The data revealed that overexpression of Dmrt5 in in vitro differentiated neural progenitor cells (NPC) regulates gene expression. Addition of Dox to the medium of the control cell line rtTA did not significantly alter gene expression profile, demonstrating that the observed effects were through induction of Dmrt5, but not simply through Dox.

Integrative Genomic and Proteomic Analysis of Prostate Cancer Reveals Signatures of Metastatic Progression

An integrative analysis of this compendium of proteomic alterations and transcriptomic data was performed revealing only 48-64% concordance between protein and transcript levels. Importantly, differential proteomic alterations between metastatic and clinically localized prostate cancer that mapped concordantly to gene transcripts served as predictors of clinical outcome in prostate cancer as  well as other solid tumors.
Keywords: prostate cancer progression

Gene expression profile of small airway epithelium of normal non-smokers and normal smokers

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A array) in phenotypically normal smokers (n=6, 24 ± 4 pack-yr) compared to matched non-smokers (n=5). Compared to samples from the large (2nd to 3rd order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells. The small, but not large, airway samples included Clara cells, a cell found only in the small airway epithelium, and the small, but not the large, airway epithelium expressed genes for the surfactant apoproteins. Despite the fact that the smokers were phenotypically normal, analysis of the small airway epithelium of the smokers compared to the non-smokers demonstrated up- and -down-regulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, pro-fibrosis, mucin, responses to oxidants and xenobiotics, antiproteases and general cellular processes. In the context that COPD starts in the small airways, these changes in gene expression in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.
Keywords: response to cigarette smoking

Changes in human bladder epithelial cell gene expression associated with interstitial cystitis or antiproliferative factor treatment.

Explanted bladder epithelial cells from patients with interstitial cystitis (IC) have been shown to differ from explanted control cells in several ways, including production of an antiproliferative factor (APF), altered production of certain epithelial growth factors, and rate of proliferation. To better understand the role of the APF in abnormal bladder epithelial cell proliferation in IC, we studied gene expression patterns in normal bladder epithelial cells treated with APF vs. mock APF and compared them to expression patterns in IC vs. normal cells using microarray analysis. Oligo-dT-primed total cellular RNA was labeled with [33P]dCTP and hybridized to GeneFilter GF211 microarray membranes (Research Genetics) containing cDNA for 3,964 human genes. Thirteen genes that function in epithelial cell proliferation or differentiation were consistently differentially expressed in both IC (compared with control) and APF-treated (compared with mock APF-treated) normal bladder epithelial cells. The general pattern of gene expression in IC and APF-treated cells suggested a less proliferative phenotype, with increased expression of E-cadherin, phosphoribosylpyrophosphate synthetase-associated protein 39, and SWI/SNF complex 170-kDa subunit, and decreased expression of vimentin, {alpha}2-integrin, {alpha}1-catenin, cyclin D1, and jun N-terminal kinase 1; these findings were confirmed for the structural gene products (E-cadherin, vimentin, {alpha}2-integrin, and {alpha}-catenin) by immunohistochemistry. These results are compatible with the previously noted decreased proliferation rate of IC and APF-treated normal cells, and indicate that the mechanism whereby APF inhibits cell proliferation may involve both downregulation of genes that stimulate cell proliferation along with upregulation of genes that inhibit cell growth.

Modulation of pro- and antiapoptotic molecules in double-positive (CD4+CD8+) thymocytes following dexamethasone treatmen

Glucocorticoids play a role in regulation of T lymphocytes homeostasis and development. In particular, glucocorticoid treatment induces massive apoptosis of CD4+CD8+ double positive (DP) thymocytes. This effect is due to many mechanisms, mainly driven by modulation of gene transcription. To find out which genes are modulated, we analyzed DP thymocytes treated for 3 hours with dexamethasone or medium alone, by global gene expression profiling using the Affymetrix technology (MGU74Av2 GeneChip). The data were analyzed with MAS 5.0  imposing a cut off of 1.7 fold in up regulation and 1.35 fold in down regulation. To further filter results we also used the statistical software, SAM (d 0.88 see figure 1s in supplementary data of the above cited manuscript). Results indicate modulation of 156 genes, also confirmed by either RNAse protection assay or Real Time PCR. For data mining we also used Go Miner to explore the Gene Ontology data bank (see tables 1-3 of the above cited manuscript). The overall results demonstrated that dexamethasone caused the down-regulation of genes promoting survival of DP thymocytes (e.g. Notch1, Socs1 and Id3) or the modulation of genes activating cell death through the ceramide pathway (Ugcg, Sgpp1, Degs1 and Gpr65) or through the mithocondrial machinery. Among the latter, there are Bcl-2 family members (Bim, Bfl-1, Bcl-xL and Bcl-xbeta), genes involved in the control of redox status (thioredoxin reductase, TXNIP and idh2) and genes belonging to Tis11 family which are involved in mRNA stability. Our study suggests that dexamethasone treatment of DP thymocytes modulates several genes belonging to apoptosis-related systems that can contribute to their apoptosis.
Keywords: Dexamethasone/PBS treatment comparison

In the present investigation, we have exploited the opportunity provided by neoadjuvant treatment of a group of postmenopausal women with large operable or locally advanced breast cancer (in which therapy is given with the primary tumour remaining within the breast) to take sequential biopsies of the same cancers before and after 10-14 days treatment with letrozole.  RNA extracted from the biopsies has been subjected to Affymetrix microarray analysis and the data from paired biopsies interrogated to discover genes whose expression is most influenced by oestrogen deprivation.

The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map

The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a protomap in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The intermediate map in SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 → Eomes → Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.

Alternate protein kinase A activity identifies a unique population of stromal cells in adult bone

We investigated the Prkar1a+/- mice when bred with Prkaca+/- genetic backgrounds. Prkar1a+/-Prkaca+/- mice not only continued to develop bone lesions but also demonstrated a significant increase in both the number and the severity of the lesions, as well as a reduction in the age of first appearance of any bone abnormality.  Histological analysis of bone from Prkar1a+/-Prkaca+/- mice showed that lesions had similarity to tumors from humans with CNC and some resemblance (but also differences) from humans and mice with fibrous dysplasia (FD). Prkar1a+/- and Prkar1a+/-Prkaca+/- mouse lesions always involved a particular sub-population of cells that could be identified as belonging to the osteoblastic lineage, that were initially located on the endosteal surface of the periosteum and nearby trabecular bone proximal to the growth plate of the long bones and vertebrae, and resembled bone stromal cells (BSCs). The bone lesions expressed high level of mesenchymal proteins like n-cadherin, vimentin, snail1, twist1, mmp2 and mmp9 when compared to WT bone. On top of this, bone lesions from  Prkar1a+/-Prkaca+/- mice also showed an increased expression of keratin related genes, which are involved in hair follicle and epithelial differentiation, when compared to lesions from Prkar1a+/- mice.

Gene expression profiles in synovial biopsies from patients with arthritis

Rheumatoid arthritis (RA) is an inflammatory joint disorder that results in progressive joint damage when insufficiently treated. In order to prevent joint destruction and functional disability in RA, early diagnosis and initiation of appropriate treatment with Disease-Modifying Antirheumatic Drugs (DMARDs) is needed. However, in daily clinical practice, patients may initially display symptoms of arthritis that do not fulfil the classification criteria for a definite diagnosis of RA, or any other joint disease, a situation called “Undifferentiated Arthritis” (UA). Out of the patients with UA, 30 to 50% usually develop RA, and early identification of these remains a challenge.

At the present time, although several risk factors associated with the development of RA have been identified (6-9), a model that reliably predicts the probability of evolution of UA into RA in individual patients is lacking. In order to better identify early RA patients, an American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) collaboration recently developed new classification criteria. Although these criteria are more sensitive, the risk of over-diagnosis is an important issue to consider, especially in very early disease. In this context, the present study explores the feasibility of a molecular diagnosis of arthritis, based on the identification of disease-specific transcriptomic profiles in synovial biopsies from patients with arthritis according to the underlying condition.

In a previous study, we performed global analyses of gene expression in synovial biopsies from patients with RA, Systemic Lupus Erythematosus (SLE) and Osteoarthritis (OA), using high-density oligonucleotide spotted microarrays. We found that the gene expression profiles are strikingly different according to the underlying condition. Thus, the majority of the genes up-regulated in SLE are type I Interferon-inducible genes, as compared with the up-regulation genes involved in T cell and B cell activation in RA, and in extracellular matrix homeostasis in OA. Based on these results, similar analyses were performed in synovial biopsies from patients with seronegative arthritis (SA) and microcrystalline arthritis (MIC), in order to identify disease-specific molecular signatures.

Hippocampus; LTP; Microarray analysis; Avoidance learning; Aging; PPAR; Synaptic hyperpolarization; T2DM

Long-term pioglitazone improves learning and attenuates pathological markers in a mouse model of AD

Thiazolidinediones (TZDs) are agonists at peroxisome proliferator-activated gamma-type (PPAR-y) receptors and are used clinically for the treatment of type 2 diabetes where they have been shown to reestablish insulin sensitivity, improve lipids profile, and reduce inflammation. Recent work also suggests that TZDs may be beneficial in Alzheimer's disease (AD), ameliorating cognitive decline early in the disease process. However, there have been only a few studies identifying mechanisms through which cognitive benefits may be exerted. Starting at 10 months of age, the triple transgenic mouse model of AD (3xTg-AD) with accelerated amyloid-B (AB) deposition and tau pathology was treated with the TZD pioglitazone (PIO- Actos) at 18 mg/Kg body weight/day. After four months, PIO-treated animals showed multiple beneficial effects, including improved learning on the active avoidance task, reduced serum cholesterol, decreased hippocampal AB deposits, and enhanced short- and long-term plasticity. Baseline electrophysiological membrane properties and blood glucose levels were unchanged by PIO treatment. Gene microarray analyses of hippocampal tissue identified predicted transcriptional responses following TZD treatment as well as potentially novel targets of TZDs, including facilitation of estrogenic processes, and decreases in glutamatergic and ketone metabolic/ cholesterol dependent processes. Taken together, these results confirm prior animal studies showing that TZDs can ameliorate cognitive deficits associated with AD-related pathology, but also extend these findings by pointing to novel molecular targets in the brain.
Keywords : Hippocampus; LTP; Microarray analysis; Avoidance learning; Aging; PPAR; Synaptic hyperpolarization; T2DM

This study was undertaken in order to characterize the functions of Rex-1 and identify potential Rex-1 target genes.Both alleles of the Rex-1 gene were disrupted in J1 mouse embryonic stem cells. Gene expression levels in one of the resulting Rex-1 knockout cell lines was compared to that of J1 wild type cells.
Keywords: cell type comparison

C2C12 mouse myoblasts (American Type Culture Collection, Manassas, VA) were maintained in growth medium (DMEM supplemented with 10% Fetal Bovine serum, Invitrogen Corporation, Carlsbad, CA) in 5% CO2. Cells were plated at a density of 40-50% in 100mm dishes. When cells reached ~90% confluency 24 hours later, they were switched to differentiation media (DMEM containing 2% horse serum; GIBCO, Invitrogen Corporation, Carlsbad, CA) which was subsequently changed every 48 hours. All cells were allowed to differentiate for 4 days with feeding every two days (day 0, 2, and 4). Beginning at day 4, control cells were re-fed every 48 hours whereas experimental cells were not re-fed. After an additional 4 days (8 days post differentiation), cells were harvested and total RNA was isolated from control and "starved" (non re-fed cells)

Expression data for 2-month-old wild type and Tfam-SCKO (MPZ-Tfam KO) sciatic nerves

Gene expression analysis of 2-month-old Ctrl and Tfam-SCKO mice. At this age mitochondrial function is disrupted in the Schwann cells of Tfam-SCKO mice ,but their nerves display only very limited pathology.
Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells, SCs) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response through actions of heme-regulated inhibitor kinase (HRI), and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines, an intermediate of fatty acid b-oxidation. Importantly, we show that acylcarnitines are released from SCs and induce axonal degeneration. A maladaptive integrated stress response as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies.

Expression analysis in order to elucidatethe role of hormone-sensitive lipase in soleus muscle.

anesthesia, amygdala, LTM, Rampil, Isoflurane, fear, memory;other

Fear conditioning in rats leads to long term memory (LTM) formation. A central neural substrate for LTM is the basolateral amygdala. We sought the expression changes specific to LTM at 6h following anesthesia with isoflurane (a general anesthetic and an effective amnestic agent), following pain (a component of conditioning), and following conditioning in presence and absence of isoflurane.
Keywords = anesthesia, amygdala, LTM, Rampil, Isoflurane, fear, memory

RB pathway deregulation promotes invasion and disease progression in a mouse model of MYC-overexpressing mammary tumorigenesis

Breast cancer is a highly heterogeneous disease that is categorized into distinct tumor subtypes based on specific molecular attributes, which ultimately influence therapeutic options. Unlike ER+ and/or HER2+ cancers that are subject to specific targeted therapies, triple negative breast cancers (TNBCs) do not express these receptors, which leaves patients with limited treatment options. Thus, significant focus has been placed on identifying molecular attributes of basal-like disease that could be used to develop and/or direct novel treatment regimens. Activation of MYC signaling and inactivation of the RB-pathway are frequent events in many types of human cancers. These pathways influence many biological processes, such as cell proliferation, that contribute to the aggressiveness and therapeutic response of tumors. The current study examines the interaction of the MYC and RB pathways in mammary epithelial cell tumorigenesis.

The effect of imatinib therapy on tumor associated macrophages (TAMs) in murine gastrointestinal stromal tumor (GIST)

The gene expression profile of TAMs sorted from vehicle control tumors in GIST mice (Sommer et al, PNAS 2003) was compared to TAMs sorted from mice after 2 weeks of imatinib therapy
The Affymetrix Mouse Genome 430A 2.0 platform was used

Transcriptional Alterations in Skeletal Muscle Following Desmin Deletion

Desmin is a cytoskeletal protein in muscle involved in integrating cellular space and transmitting forces.  In this study we sought to determine the effects of desmin deletion on skeletal muscle at the transcriptional level across many pathways of muscle physiology.

Atxn1L is a novel regulator of Hematopoietic Stem Cell Quiescence

We compared gene expression differences in Atxn1L knockout vs wildtype HSCs
KO allele described in Pub Med ID: 22014525

We used microarrays to investigate a global change in gene expression by conditional depletion of Rybp in mouse ES cells.

Control and Liver Insulin Receptor KO mice (LIRKO) were sacrificed in the non-fasted state. RNA was prepared from liver samples and subjected to expression microarray analysis
Each array was hybridized with sample derived from 2-3 mice of the same genotype.

Oct4 and jhdm1b infected MEF with or without vitamin C treatment

Investigate the effect of jhdm1b on Oct4 mediated reprogramming
Investigate the effect of vitamin C on the function of jhdm1b

Transcriptome analysis of Ts1Cje (mouse model of Down syndrome) and euploids murine cerebellum during postnatal development
Keywords = Down syndrome

Co-regulated gene expression by estrogen receptor-α and liver receptor homolog-1 is a feature of the estrogen response in breast cancer cells

Estrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, is ERα-regulated in breast cancer cells. Further, LRH-1 stimulates proliferation and promotes motility and invasion of breast cancer cells. To determine the mechanisms of LRH-1 action in breast cancer cells, we carried out gene expression microarray analysis following siRNA-mediated LRH-1 knockdown. Interestingly, gene ontology (GO) category enrichment analysis of the genes differentially regulated in the presence or absence of LRH-1 identified estrogen responsive genes as the most highly enriched GO categories. To further define LRH-1 target genes, we performed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1. Remarkably, ChIP-seq showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα-regulated genes by LRH-1 through binding to estrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 over-expression stimulated ERα recruitment, whilst LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at estrogen response elements controls the expression of estrogen-responsive genes.

disease state comparison; Marfan syndrome; cultured skin fibroblasts

A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

Background:  Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms.  Results:  We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease.  Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations.  Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusions:  Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 x 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status).   An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.
Keywords: disease state comparison; Marfan syndrome; cultured skin fibroblasts

Four weeks expression data of the antipsychotics Clozapine and Haloperidol in the mouse brain (Affymetrix, GCRMA).

Atypical antipsychotic Clozapine has a superior antipsychotic and antimanic effects compared to other antisphicotics. Its widespread use was limited by the side effects of agranulocytosis, cardiomyopathy and metabolic anomalies such as weight gain, and diabetes. Very little is known about mechanisms by which Clozapine works. The aim of this experiment is to compare the chronic gene expression profile (i.e four weeks) of the atypical antipsychotic Clozapine to the typical antipsychotic drug Haloperidol using gene expression Microarray in order to understand the intercellular mechanism behind the therapeutic and the toxic effects of Clozapine.

Expression profiling of pancreatic beta-cells harboring a pancreatic-specific deletion of PPAR-beta

Peroxisome proliferator-activated receptor beta/delta protects against obesity by reducing dyslipidemia and insulin resistance via effects in various organs, including muscle, adipose tissue, liver, and heart.  However, nothing is known about the function of PPAR-beta in pancreas, a prime organ in the control of glucose metabolism. To gain insight into so far hypothetical functions of this PPAR isotype in insulin production, we specifically ablated Ppar-beta in pancreas.  The mutated mice developed a chronic hyperinsulinemia, due to an increase in both beta-cell mass and insulin secretion. Gene expression profiling indicated a broad repressive function of PPAR-beta impacting the vesicular compartment, actin cytoskeleton, and metabolism of glucose and fatty acids. Analyses of insulin release from the islets revealed an increased second-phase glucose-stimulated insulin secretion. Higher levels of PKD, PKC-delta and diacyglycerol in mutated animals lead to an enhanced formation of trans-Golgi network (TGN)-to-plasma-membrane transport carriers in concert with F-actin disassembly, which resulted in increased insulin secretion and its associated systemic effects. Taken together, these results provide evidence for PPAR-beta playing a repressive role on beta-cell growth and insulin exocytosis, which shed new light on its anti-obesity action.

PKCz-mediated Gaq stimulation of the ERK5 pathway is involved in cardiac hypertrophy

Background: Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gaq-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gaq acts as an adaptor protein that facilitates PKCz-mediated activation of ERK5 in epithelial cells.  Since the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in Gq-dependent  signaling in cardiac cells.
Methodology/Principal Findings: We have explored the mechanisms involved in Gq-coupled GPCR-mediated stimulation of the ERK5 pathway and its functional consequences in cardiac hypertrophy using both cultured cardiac cells and an animal model of angiotensin- dependent induction of cardiac hypertrophy in wild-type and PKCz knockout mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in cardiomyocytes and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCz in primary cultures of cardiac cells and in cardiomyocytes isolated from PKCz-deficient mice. Moreover, these mice do not develop cardiac hypertrophy upon chronic challenge with angiotensin II, as assessed by morphological, biomarker, electrocardiographic and global gene expression pattern analysis.
Conclusion/Significance: Our data put forward that PKCζ is essential for Gq- dependent ERK5 activation in cardiac cells and indicate a key cardiac physiological role for this recently described Gaq/PKCz/MEK5 signaling axis.

Harmane Alters the Expression of a Subset of Genes with Diverse Functions in Primary Human Astrocytes

Essential tremor (ET) is the most common movement disorder in adults, but little is known about the molecular mechanisms underlying ET pathogenesis.  Harmane is a member of a group of tremorogenic chemicals.  In humans, increased blood harmane concentration is associated with increased risk of ET.  Astrocytes are essential for brain function, and astrocyte dysfuctions are associated with many neurodegenerative diseases.  Therefore, we identified the molecular targets of harmane in primary human astrocytes by using microarray gene expression profiling and computational analysis algorithms. We found that harmane altered the expression of a limited number of genes encoding diverse functions.  Notably, the transcript levels of two GABA receptors and a GABA transporter were altered by harmane, consistent with previous evidence suggesting that the GABAergic neurotransmission system may be disrupted in ET.  Also, we found that the transcript levels of two prominent proinflammatory enzymes, the inducible nitric oxide synthase NOS2A and the cyclooxygenase COX2, and 10 other targets of the proinflammatory IFN-gamma signaling pathway were up-regulated by harmane.  These results together raise the possibility that perturbation of the expression of functions involved in neurotransmission and inflammatory activation of astrocytes might be important mechanisms underlying the neurotoxicity of harmane and ET pathogenesis.
Keywords: treatment comparison

Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines

Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines for the article: Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases  (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells

Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives PFKFB1, 3, and 4 were further analyzed. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly in T-ALL cells. The 3 other family members, in contrast, were not or weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (15A and 15B) did not have any detectable effect on survival or cell cycle progression. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC.
Generation of the GC sensitive and resistant clones is described in Parson et al. FASEB J 2005 (Pubmed id 15637111). In brief GC sensitive clones were generated by limiting dilution subcloning from the GC sensitive T-ALL cell line CCRF-CEM-C7H2. To generate GC resistant clones the CCRF-CEM-C7H2 cell line was clutured in the presence of 10E-7 M dexametasone.

Huntington's disease: Gene expression changes caused by Hdh CAG mutation or 3-nitropropionic acid in striatal cells

Affymetrix MG430 2.0 expression levels of wild-type (STHdhQ7/Q7), 3NP-treated wild-type (STHdhQ7/Q7+3-NP), and mutant (STHdhQ111/Q111) striatal cells
Keywords: Pharmacological and genetic HD cell culture models

Search for specific biomarkers of IFN-beta bioactivity in patients with MS

We aimed to identify specific biomarkers of IFN-beta bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients who developed neutralizing antibodies (NAB) to IFN-beta. Nine genes followed patterns in gene expression over time similar to the MX1 and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In vitro experiments revealed specific induction of selected biomarkers by IFN-beta but not IFN-gamma, and several markers, in particular USP18 and HERC5, were significantly induced at lower IFN-beta concentrations and more selective than the MX1 as biomarkers of IFN-beta bioactivity. In addition, USP18 expression was deficient in MS patients compared with healthy controls (p=0.0004). We propose specific biomarkers that may be considered in addition to the MxA to evaluate IFN-beta bioactivity, and to further explore their implication in MS pathogenesis.

Dilated cardiomyopathy (DCM) is a common cause of heart failure and a leading cause of cardiac transplantation in western countries. The robust predictive expression profile of cardiomyopathic and NF hearts as well as the functional classification can help to identify promising candidates for DCM and may improve the early diagnosis of cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common cause of heart failure and a leading cause of cardiac transplantation in western countries. The robust predictive expression profile of cardiomyopathic and NF hearts as well as the functional classification can help to identify promising candidates for DCM and may improve the early diagnosis of
cardiomyopathy.

Determining the molecular events induced in the spleen during schistosome infection is an essential step in better understanding the immunopathogenesis of schistosomiasis and the mechanisms by which schistosomes modulate the host immune response.    The present study defines the transcriptional and cellular events occurring in the murine spleen during the progression of Schistosoma japonicum infection. Additionally, we compared and contrasted these results with those we have previously reported for the liver. Microarray analysis combined with flow cytometry and histochemistry demonstrated that transcriptional changes occurring in the spleen were closely related to changes in cellular composition. Additionally, the presence of alternatively activated macrophages, as indicated by up-regulation of Chi3l3 and Chi3l4 and expansion of F4/80+ macrophages, together with enhanced expression of the immunoregulatory genes ANXA1 and CAMP suggests the spleen may be an important site for the control of S. japonicum-induced immune responses.     The most striking difference between the transcriptional profiles of the infected liver and spleen was the contrasting expression of chemokines and cell adhesion molecules. Lymphocyte chemokines including the homeostatic chemokines, CXCL13, CCL19 and CCL21, were significantly down-regulated in the spleen but up-regulated in the liver. Eosinophil (CCL11, CCL24), neutrophil (CXCL1) and monocyte (CXCL14, CCL12) chemokines and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver but unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Co-ordinated expression of these genes probably contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver, thereby facilitating the development of hepatic granulomas and fibrosis.    Together these data provide, for the first time, a comprehensive overview of the molecular events occurring in the spleen during schistosomiasis and will substantially further our understanding of the local and systemic mechanisms driving the immunopathogenesis of this disease.

Using Gfi1b conditional mice, deletion of gfi1b in the hematopietic system was induced by injecting MxCre tg Gfi1bfl/fl mice with pIpC. 30 days after injection, Cd150 pos, Cd 48 neg, Lin neg Sca and c-kit pos stem cells were sortrted from Gfi1bfl/fl and Mxcre tg Gfi1bfl/fl mice and analysed. We used the mouse Affymetrix Gene ST Array.

Genome-wide measurement of gene expression changes with Bptf knockout in mouse embryonic fibroblasts

Gene expression frequently requires the action of chromatin remodeling complexes and it is assumed that these complexes have common gene targets across cell-types. Contrary to this belief, we show that Bptf, an essential and unique subunit of the Nucleosome Remodeling Factor (NURF), largely regulates cell-type-restricted gene expression across diverse cell-types. Unexpectedly, cell-type-restricted gene expression is accomplished through both physical and functional interactions between NURF and the ubiquitous multivalent factor Ctcf. We go on to show in these cell types, that Bptf influences the regulatory activity of Ctcf binding sites in a cell type-restricted manner. Detailed studies of Ctcf binding sites in embryonic stem cells (ESC) show that Bptf prevents Klf4 binding upstream of H2-K1, and facilitates RNA polymerase elongation through Ccnd1, to regulate genes important for embryonic development. In total, these studies provide mechanisms by which prominent chromatin remodeling complexes can maintain cell-type-restricted gene expression through ubiquitous factors like Ctcf.

Expression data of untreated or cisplatin-treated wildtype or Rb-/- MEFs

The absence of the Rb tumor suppressor gene changes levels/activities of transcription factors (e.g., E2F and p53) which alter gene expression patterns, related to cell cycle control and cellular response to DNA damage.  Cisplatin is a genotoxic chemotherapeutic agent and wildtype or Rb null cells have different sensitivities to cisplatin-induced cytotoxicity.
We used microarrays to compare global profiles of gene expression and distinct responses of wildtype and Rb null MEFs in response to cisplatin.

Multilineage dysplasia does not influence prognosis in patients with CEBPA mutated AML supporting the WHO proposal to classify these patients as a unique entity

By WHO 2008, CEBPA-mutated AML became a provisional subentity, but it remains to be clarified how CEBPAmut AML with multilineage dysplasia (MLD; ≥50% dysplastic cells in 2-3 lineages) but no other MDS-related feature should be classified. We investigated 108 CEBPAmut AML (15.7-87.6 years) for the impact of MLD and genetic features. MLD-positive patients differed from MLD-negative only by lower mean WBC counts (p=0.004), but not by other blood values, biologic characteristics, cytogenetic risk profiles, or additional molecular markers (NPM1mut, FLT3-ITD/TKD, RUNX1, MLL-PTD, IDH1/2). Biallelic CEBPAmut differed from wild-type-cases by differential expression of 213 genes, but did not differ significantly between MLD-positive/-negative patients. Survival outcomes were improved for females and those <60 years, intermediate versus adverse karyotypes (p=0.021), and for  biallelic versus monoallelic/homozygous CEBPAmut (p=0.060) in case of FLT3-ITD-negativity. In contrast, 2-year OS (MLD+: 56.5%; MLD-: 65.5%) and 2-year EFS (MLD+: 13.8 months; MLD-: 16.3 months) did not differ significantly between MLD-positive/-negative patients. By univariable Cox regression analysis, gender, age, WBC count and MRC-cytogenetic risk category only were prognostically relevant for OS, while MLD was  irrelevant. Therefore, CEBPAmut AML patients should be characterized only according to mut-status, cytogenetic risk groups, or additional mutations, whereas dysplasia is not relevant for this subtype.

This experiment was conducted to identify target genes of the peroxisome proliferator-activated receptor alpha (PPARa) in skeletal muscle of transgenic mice that overexpressed PPARa. The following abstract from the published manuscript describes the major findings of this work.

A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes.Finck BN, Bernal-Mizrachi C, Han DH, Coleman T, Sambandam N, LaRiviere LL, Holloszy JO, Semenkovich CF, Kelly DP.

The role of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in the development of insulin-resistant diabetes was evaluated using gain- and loss-of-function approaches. Transgenic mice overexpressing PPARalpha in muscle (MCK-PPARalpha mice) developed glucose intolerance despite being protected from diet-induced obesity. Conversely, PPARalpha null mice were protected from diet-induced insulin resistance in the context of obesity. In skeletal muscle, MCK-PPARalpha mice exhibited increased fatty acid oxidation rates, diminished AMP-activated protein kinase activity, and reduced insulin-stimulated glucose uptake without alterations in the phosphorylation status of key insulin-signaling proteins. These effects on muscle glucose uptake involved transcriptional repression of the GLUT4 gene. Pharmacologic inhibition of fatty acid oxidation or mitochondrial respiratory coupling prevented the effects of PPARalpha on GLUT4 expression and glucose homeostasis. These results identify PPARalpha-driven alterations in muscle fatty acid oxidation and energetics as a potential link between obesity and the development of glucose intolerance and insulin resistance.

Gene expression abnormalities in histologically normal breast epithelium of breast cancer patients

Normal-appearing epithelium of cancer patients can harbor occult genetic abnormalities. Data comprehensively comparing gene expression between histologically normal breast epithelium of breast cancer patients and cancer-free controls are limited. The present study compares global gene expression between these groups. We performed microarrays using RNA from microdissected histologically normal terminal ductal-lobular units (TDLU) from 2 groups: (i) cancer normal (CN) (TDLUs adjacent to untreated ER1 breast cancers (n = 14)) and (ii) reduction mammoplasty (RM) (TDLUs of age-matched women without breast disease (n = 15)). Cyber-T identi?ed differentially expressed genes. Quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), and comparison to independent microarray data including 6 carcinomas in situ (CIS), validated the results. Gene ontology (GO), UniProt and published literature evaluated gene function. About 127 probesets, corresponding to 105 genes, were differentially expressed between CN and RM (p < 0.0009, corresponding to FDR <0.10). 104/127 (82%) probesets were also differentially expressed between CIS and RM, nearly always (102/104 (98%)) in the same direction as in CN vs. RM. Two-thirds of the 105 genes were implicated previously in carcinogenesis. Overrepresented functional groups included transcription, G-protein coupled and chemokine receptor activity, the MAPK cascade and immediate early genes. Most genes in these categories were under-expressed in CN vs. RM. We conclude that global gene expression abnormalities exist in normal epithelium of breast cancer patients and are also present in early cancers. Thus, cancer-related pathways may be perturbed in normal epithelium. These abnormalities could be markers of disease risk, occult disease, or the tissue’s response to an existing tumor.

Thyroid hormone (TH) influences metabolic pathways by binding to specific receptors (TRs), which are conditional transcription factors. T3 works through TRs to induce fibroblast growth factor (FGF) 21, a peptide hormone that is usually induced in fasting and influences lipid and carbohydrate metabolism via local hepatic and systemic endocrine effects. While administered TH and FGF21 display overlapping actions, including reductions in serum lipids, current models suggest that these hormones act independently in vivo. Here, we examined mechanisms of TH regulation of FGF21 expression and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (T3) and the TRβ selective thyromimetic GC-1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRβ. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRβ1, which binds a TRE within intron 2 of FGF21. Gene expression profiles in wild type and FGF21 knockout mice are highly similar indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously involved in T3-dependent hepatic lipid and carbohydrate metabolic processes. Accordingly, T3-dependent effects upon serum lipids are maintained in the FGF21-/- background. Our findings suggest that T3 regulates genes involved in classical hepatic metabolic responses independently of FGF21.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with preiods of active disease followed by remission.
We performed a whole-genome transcriptional analysis of colonic biopsies from patients with histologically active and inactive UC, as well as non-inflammatory controls.

Expression data from postmortem human brain samples with and without FTLD-U

FTLD-U is the most common pathological correlate of the neurodegenerative dementia frontotemporal dementia
We used microarrays to perform global expression profiling of FTLD-U brain samples

Cbfb/Runx1-repression independent blockage of differentiation and accumulation of Csf2rb expressing cells by Cbfb-MYH11

It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. However, knockin mice expressing Cbfb-MYH11 (Cbfb+/MYH11) showed defects in primitive hematopoiesis not seen in Cbfb null (Cbfb-/-) embryos indicating that Cbfb-MYH11 has repression independent activities as well.
To identify gene expression changes associated with this novel activity, we compared the gene expression profile in the blood cells of Cbfb+/MYH11 and Cbfb-/- embryonic day 12.5 (E12.5) embryos with that of their wildtype littermates.

Genome-wide gene expression profile analysis in pulmonary sarcoidosis

We hypothesized that tissue genome-wide gene expression analysis, coupled with gene network analyses of differentially expressed genes, would provide novel insights into the pathogenesis of pulmonary sarcoidosis.

Wee1 Inhibition sensitizes prostate cancer cells to Hsp90 inhibitor to activate the intrinsic apoptotic pathway

Heat shock protein 90 (Hsp90) is an essential evolutionarily conserved molecular chaperone in eukaryotes. Cancer cells rely on Hsp90 to chaperone activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is most commonly cytostatic, and efforts to enhance the anti-tumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In this study, we show that dual inhibition of Wee1 tyrosine kinase and Hsp90 causes prostate cancer cells to undergo apoptosis. Gene-expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional down-regulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins as well as activated Akt was completely abrogated. Similar results were obtained in prostate cancer xenografts. These data establish a novel therapeutic strategy to enhance the efficacy of Hsp90 inhibitors in prostate cancer, and they provide a mechanistic rationale for stimulating the pro-apoptotic activity of Hsp90 inhibitors.
In order to explore the mechanism underlying the enhanced cell death caused by Wee1 inhibitorII and 17-AAG combination, we performed microarray analysis using PC3 cells treated with Wee1 inhibitorII alone, 17-AAG alone, or the two drugs in combination.

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery (BeadChip)

Protein Arginine MethylTransferase 5 (PRMT5) is known to mediate epigenetic control on chromatin and to functionally regulate components of the splicing machinery. In this study we show that selective deletion of PRMT5 in different organs leads to cell cycle arrest and apoptosis. At the molecular level, PRMT5 depletion results in reduced methylation of Sm proteins, aberrant constitutive splicing and in the Alternative Splicing (AS) of specific mRNAs. We identify Mdm4 as one of these mRNAs, which due to its weak 5’-Donor site, acts as a sensor of splicing defects and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in PRMT5 conditional knockout mice. Our data demonstrate a key role of PRMT5, together with p53, as guardians of the transcriptome. This will have fundamental implications in our understanding of PRMT5 activity, both in physiological conditions, as well as pathological conditions, including cancer and neurological diseases.

Microarray gene expression profiling of aortic genes of APOE-deficient mice receiving atherosclerosis treatment with the antioxidant vitamin E

Hypercholesterolemic APOE-deficient mice are a widely used experimental model of atherosclerosis and increased generation of reactive oxygen species (ROS) is a prominent feature of atherosclerosis development. To study the impact of ROS on atherogenesis, we treated APOE-deficient mice for 7 months with the antioxidant vitamin E (2000 IU/kg diet) and performed whole genome microarray gene expression profiling of aortic genes. Microarray gene expression profiling was performed of whole aortas isolated from vitamin E-treated APOE-deficient relative to untreated APOE-deficient mice with overt atherosclerosis, and nontransgenic B6 control mice. Microarray gene expression profiling revealed that vitamin E treatment prevented atherosclerosis-related gene expression changes of the aortic intima and media.

Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligandindependent signaling by the precursors of mature antigen receptors regulates development of B- and T-lymphocytes. Here we describe a basal signal controlling gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results from constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.

MiR-221 overexpression leads to activation of apoptosis, growth arrest and reduced invasivness in PCa cells. Interaction of miR-221 with potential target genes was analyzed by a genome wide expression profiling.. Regulation of selected genes and proteins identified in the gene array analysis was confirmed by Real Time RT-PCR assay (IRF1, IRF2 SOCS3, STAT1), and Western Blotting. In total, 282 genes were upregulated and 64 downregulated based on a more than 2-fold difference to untransfected PC-3 cells. Regulated genes are involved in apoptosis, hemostasis, oxidative stress response,  tumorigenesis and inflammation. We confirmed dysregulation of IRF-2 SOCS3, STAT1,IRF9. These results indicate that miR-221 overexpression might lead to activation of the JAK/STAT pathway and downregulation of miR-221 might contribute to tumorigenesis in PCa cells.

The experiment studied the effect of knockdown WTAP in HUVEC. two control samples and two WTAP knockdown samples.

Expression data from MMP-8 wild type and KO mice with or without arthritis

Rheumatoid arthritis is an autoimmune disease in which joint inflammation lead to progressive cartilage and bone destruction. Matrix metalloproteinases (MMP) implicated in homeostasis of extracellular matrix (ECM) play a central role in cartilage degradation. The aim of this study was to investigate the role of MMP-8 (collagenase-2) suppression in the K/BxN serum-transfer arthritis model.

Lubiprostone effects on small intestinal gene expression in wild type and Cftr-null mice

Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype.
Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray.
Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels.
Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion.

In the nervous system, neural stem cells (NSC) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3-/- mice have fewer NSC in vivo than wild type counterparts. NSC isolated from adult FoxO3-/- mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSC suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSC might have important implications for counteracting brain aging in long-lived species.

Lung;Non Small Cell Lung Cancer;Adenocarcinoma;Squamous Cell Carcinoma;Normal Lung.;other

This series contain 36 samples obtained from human lung tissue and includes the following:
7 Adenocarcinoma samples.
16 Squamous cell carcinoma samples.
1 AdenoSquamous sample.
2 Renal Metastasis.
1 Colon metastasis.
7 normal lung tissue adjacent to the tumors.
2 commercial normal lung RNA.

Identification of novel tissue-specific transcription arising from E-cadherin/CDH1 intron2: a novel protein isoform increases gastric cancer cell invasion and angiogenesis.

E-cadherin, a protein encoded by the CDH1 gene is the dominant epithelial cell adhesion molecule playing a crucial role in epithelial tissue polarity and structural integrity. The progression of 90% or more carcinomas is believed to be mediated by disruption of normal E-cadherin expression, subcellular localization or function. Despite the strong correlation between E-cadherin loss and malignancy the mechanism through how this occurs is not known in most sporadic and hereditary epithelial carcinomas. Previous works have shown the importance of CDH1 intron 2 sequences for proper gene and protein expression supporting the possibility of these being cis-modulators of E-cadherin expression/function. but when co-expressed it led to reduced cell-cell adhesiveness, increased invasion and angiogenesis. By expression array analysis, IFITM1 and IFI27 levels were found to be increased upon CDH1a overexpression. Importantly, CDH1a was found to be de novo expressed in gastric cancer cell lines when compared to normal stomach.
Results: In this sense we have searched for additional CDH1 transcripts arising from intron 2. From the four found, one (CDH1a) was demonstrated to be tissue specific and to be translated in vivo. When overexpressed in an E-cadherin negative context CDH1a replaced the canonic protein functions
Conclusions: We have demonstrated, for the first time, transcripts arising from CDH1 intron 2. One of these, CDH1a, was found to modulate E-cadherin function representing a novel mechanism underlying invasion and angiogenesis in epithelial cancers and opening the way for novel therapeutic approaches.

Under conditions of hormonal adjuvant treatment the estrogen receptor apoprotein supports breast cancer cell cycling through the retinoic acid receptor α1 apoprotein

Under conditions of hormonal adjuvant treatment the estrogen receptor apoprotein supports breast cancer cell cycling through the retinoic acid receptor α1 apoprotein.
Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using siRNA  inhibited basal proliferation by promoting cell cycle arrest. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition.

In this study that was specifically designed to identify early stages of glaucoma in DBA/2J mice, we used genome-wide expression profiling and a series of computational methods. Our methods successfully subdivided eyes with no detectable glaucoma by conventional assays into molecularly defined stages of disease. These stages represent a temporally ordered sequence of glaucoma states. Using an array of tools, we then determined networks and biological processes that are altered at these early stages. Our strategy proved very sensitive, suggesting that similar approaches will be valuable for uncovering early processes in other complex, later-onset diseases. Early changes included upregulation of both the complement cascade and endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in the complement component 1a gene (C1qa) were robustly protected from glaucoma with the protection being among the greatest reported. Similarly, inhibition of the endothelin system was strongly protective. Since EDN2 is potently vasoconstrictive and was produced by microglial/macrophages, our data provide a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early events such as the upregulation of the complement and endothelin pathways may provide effective new treatments for human glaucoma.

Exercise and transcriptional analysis in male eNOS Knockout Mice

There is cardiac dysfunction in male eNOS (-/-) with age and 50% mortality at 21M. It was of interest to investigate the gene expression profile of aged eNOS (-/-) male in comparison to (+/+) in order to explore the genetic markers and molecular mechanisms leading to heart failure. RNA was extracted from the left ventricle from male (-/-) (n=3) and (+/+) (n=4) at the age of 21M.
Keywords = eNOS knockout

Background & Aims: MiRNAs are small (~22 nucleotide), non-coding RNA molecules that regulate gene expression through imperfect complementarity with target messenger RNAs. The function of miRNA in mammalian organogenesis is largely unknown. Conditional loss-of-function of Dicer, the enzyme that processes precursor miRNA transcripts into their mature, active form, has been shown to cause severe defects in a number of organ systems. Here we address the role of Dicer in liver development and function. Methods: Mice lacking Dicer function in hepatocytes were generated using an Afp-Cre strain to drive deletion of a floxed Dicer allele. Deletion of the flox-dicer allele was confirmed by quantitative PCR. Decreased miRNA levels detected by quantitative RT-PCR and in situ hybridization confirmed loss of Dicer function. Gene expression microarray analysis was performed on liver RNA from P28 mutant and control mice. Liver sections from mutant and control mice ranging from embryonic stages through 3-4 months of age were examined and liver function tests were performed on adult mice. Results: Mice lacking hepatocyte Dicer function were born alive at the expected frequency, and had grossly normal appearance and behavior. Despite the loss of mature miRNA, hepatic function was normal, as reflected by normal blood gludose, albumin, cholesterol, and bilirubin. However, mutant mice between 2-4 months of age exhibit progressive hepatocyte damage, elevated ALT/AST, with evidence of balanced proliferation and apoptosis in the lobule. Microarray analysis indicates large-scale changes in gene expression, with increased expression of many miRNA targets, as well as imprinted genes. Conclusions: Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic physiology. However, miRNA deficiency results in hepatocyte apoptosis and balanced hepatocyte regeneration. Finally, microarray analysis of gene expression in mutant liver suggests a previously unrecognized role for Dicer in the repression of imprinted genes.

These studies are aimed at understanding gene expression chnages in a Her2 positive breast cancer cell line that has developed acquired resistance to lapatinib. Samples include SKBR3 parental and resistant (SKBR3-R) under basal conditions and in response to 0.1 and 1uM lapatinib treatment after 24 hours.

Expression data from mouse model using targeted deletion of hepatic RICTOR (Albumin-Cre Rictor LoxP/LoxP)

Recent work using mouse models has revealed that mTORC2, which unlike mTORC1 is not acutely sensitive to rapamycin, plays a key role in the regulation of organismal physiology. The substrates and pathways regulated by mTORC2 are at present relatively unknown
Using a mouse model with a targeted deletion of hepatic RICTOR, we investigated the loss of mTORC2 on the murine liver transcriptome

Control of 5mC and 5hmC by TET dioxygenases in human embryonic carcinoma cells [Microarray]

The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remains largely unknown. We depleted TET1, TET2, and TET3 by siRNA in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC and 5hmC patterns.  TET1 depletion yielded widespread reduction of 5hmC, while depletion of TET2 and TET3 reduced 5hmC at a subset of TET1 targets suggesting functional co-dependence.  TET2 or TET3-depletion also caused increased 5hmC, suggesting they play a major role in 5hmC removal.  All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion resulted in prolific hypermethylation.  Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction.  TET function was highly specific to chromatin environment:  5hmC maintenance by all TETs occurred at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3.  Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting the TETs normally promote 5hmC at these loci, and all three TETs are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes.  These results provide novel insight into the division of labor among TET proteins and reveal an important connection of TET activity with chromatin landscape and gene expression.

A PGC-1alpha-dependent decrease in mitochondrial oxidative metabolism in muscle of humans with inherited insulin resistance

We used microarrays to assess gene expression profiling of 6 patients with a mutation (Arg1174Gln) in the tyrosine kinase domain of the insulin receptor gene (INSR) and 10 matched healthy controls

Gene expression on peripheral blood mononuclear cells (PBMC) from SPARKS CHARMS juvenile idiopathic arthritis (JIA) cohort pre and post methotrexate therapy. This is the first study to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyze genetic variation in differentially expressed genes. We have identified a gene, which may contribute to genetic variability in MTX response in JIA.

SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart

A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks.  Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the “fetal gene program” in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction.  In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.

Gene expression profiling in cells with Ago1 knockdown (Affymetrix)

Argonautes, a family of highly evolutionarily conserved proteins, are the central platform for small RNA-mediated gene regulatory mechanisms which occur mainly in the cytoplasm. To understand a potential role of Argonaute 1 (Ago1) protein in the nucleus of mammalian cells in regulating gene transcription and epigenetics, we performed integrated analyses of Ago1 ChIP-sequencing (GSE40536) and gene expression profiling in cells depleted of Ago1. For gene expression profiling, we knocked down the expression of Ago1 by siRNA in PC-3 cells and compared gene expression profiles in the cells depleted of Ago1 and cells receiving control treatments. We found that Ago1 depletion resulted in more downregulated genes which were enriched in gene responsible for promoting cell cycle progression, DNA replication and repair, and response to endogenous stimuli. Integrated analyses of Ago1-chromosomal interactions and gene expression changes in response to Ago1 depletion reveal a significant correlation between Ago1-bound genes and genes altered by Ago1 depletion. These genes are significantly enriched in cancer-related pathways. Our data suggests a nuclear function of Ago1 in regulating gene transcription.

Musashi 2 regulates normal hematopoiesis and accelerates leukemogenesis (LK and MS12-inducible)

We demonstrate that Msi2 is the predominant form expressed in hematopoietic stem cells (HSC), and its knockdown leads to reduced engraftment and depletion of HSCs in vivo. Overexpression of Msi2 in a mouse model increases HSC cell cycle progression and cooperates with BCR-ABL1 to induce an aggressive leukemia. MSI2 is over-expressed in human myeloid leukemia, and expression levels directly correlate with decreased patient survival, thereby defining MSI2 expression as a novel prognostic marker in acute myeloid leukemia (AML). Depletion of MSI2 in human myeloid leukemia cells leads to decreased proliferation and apoptosis. These data implicate the MSI2 RNA binding protein in myeloid leukemogenesis and identify a novel potential target for therapy in AML.

Expression data from U373 cells expressing EGFP, MAML1-dn and DTX1-myc

Glioblastoma multiforme (GBM) is the most malignant and most common tumor of the central nervous system characterized by rapid growth and extensive tissue infiltration. GBM results in more years of life lost than any other cancer type. Notch signaling has been implicated in GBM pathogenesis through several modes of action. Inhibition of Notch leads to a reduction of cancer-initiating cells in gliomas and reduces proliferation and migration. Deltex1 (DTX1) is part of an alternative Notch signaling pathway distinct from the canonical MAML1/RBPJκ-mediated cascade. In this study, we show that DTX1 activates both the RTK/PI3K/PKB as well as the MAPK/ERK pathway. Moreover, we found the anti-apoptotic factor Mcl-1 to be induced by DTX1. In accordance with this, the clonogenic potential and proliferation rates of glioma cell lines correlated with DTX1 levels. DTX1 knock down mitigated the tumorigenic potential in vivo, and overexpression of DTX1 increased cell migration and invasion of tumor cells accompanied by an elevation of the pro-migratory factors PKBβ and Snail1. Microarray gene expression analysis identified a DTX1-specific transcriptional program - including microRNA-21 - which is distinct from the canonical Notch signaling. We propose the alternative Notch pathway via DTX1 as oncogenic factor in malignant glioma and found low DTX1 expression levels to correlate with prolonged survival of GBM and early breast cancer patients in open source databases.

Expression data from 18h-fasted WT versus KLF15-null mice: heart

We used microarray analysis to identify differences in gene expression levels in heart following an 18h (overnight) fast in WT control and KLF15-null mice

The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos.

Comparison of corneas from wildtype mice to those from mice under the influence of high doses of TGF-beta russe-affy-mouse-372894

Characteristic structural details of the cornea are transparency, the absence of blood vessels, and the presence of numerous sensory nerve endings. The corneal epithelium is one of the most densely innervated tissues of the body. The characteristics of the cornea are established during fetal development, and are lost in adult life when the cornea regenerates after injury. The common reaction of the cornea to injury is the formation of opaque scars, the ingrowth of blood vessels, and distinct changes in the innervation pattern. Scar formation of the cornea is critically modulated by the expression of transforming growth factor-beta (TGF-beta).
To identify genes that are important for corneal transparency, dense innervation and absence of blood vessels by comparing corneas from wildtype mice with those that are under the influence of high doses of TGF-beta.
Transparency, dense innervation, and absence of blood vessels in the cornea all depend on the expression of a critical set of genes that are not expressed when TGF-beta is present.
Mice were generated that overexpress TGF-beta under control of a strong lens-specific promoter. These mice developed opaque corneas that are vascularized and lack sensory nerves. In addition, these corneas were densely populated with cells expressing neural cell adhesion protein.
RNA was isolated from corneas of transgenic animals and wildtype littermates in order to analyze differentially expressed genes and to identify those that are only expressed in transparent, avascular, and densely innervated wildtype corneas.

We showed that diets containing 0.1% or 0.5% quercetin lowered the STZ-induced increase in blood glucose levels and improved plasma insulin levels.  A cluster analysis of the hepatic gene expressions showed that 0.5% quercetin diet suppressed STZ-induced alteration of gene expression.  Gene set enrichment analysis (GSEA) and quantitative RT-PCR analysis showed that the quercetin diets had their greatest suppressive effect on the STZ-induced elevation of expression of cyclin dependent kinase inhibitor p21(WAF1/Cip1) (Cdkn1a).
In this experiment, we determined the effect of quercetin on healthy control BALB/c mice that were fed the AIN93G diet containing 0, 0.1, 0.5 or 1% quercetin for 2 weeks. GSEA and one-way ANOVA did not detect any significant changes in hepatic gene expression in normal mice as a result of a quercetin diet.  Using a linear modeling approach and the empirical Bayes statistics, we found that Ubc were significantly reduced by both the 0.5% and 1% quercetin.

Hormone-Independence of Prostate Cancer Cells is Supported by the Androgen Receptor without Binding to Classical Response Elements

Treatment of late passage (LP50) LNCaP cells with R1881 (androgen) and AR shRNA identified a gene program controlled by androgen receptor in the absence of androgen.

Interleukin-27 priming of T cells controls Interleukin-17-production in trans via induction of Programmed cell death ligand 1

Interleukin (IL)-27 is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naïve T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)1-dependent manner. When co-cultured with naïve CD4+ T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, co-administration of naïve TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4+ T cells can restrict differentiation of Th17 cells in trans.

The immunoregulatory effect of macrophage-specific PPAR gamma deficiency on experimental inflammatory bowel disease

Inflammatory bowel disease (IBD) is a condition characterized by severe intestinal inflammation and immune cell activation.  The severity of the disease can be mitigated by compounds which activate peroxisome proliferator-activated receptor gamma (PPAR gamma), a receptor present widely in tissues involved in IBD pathogenesis.  Our objective was to assess the affect of macrophage-specific deficiency of PPAR gamma on peripheral and colonic immune populations and colonic gene expression in experimental IBD.  Macrophage-specific PPAR gamma-deficient mice (PPAR gamma flfl Lysozyme M Cre+) and control (PPAR gamma flfl Lysozyme M Cre-) littermates were treated with 2.5% dextran sodium sulfate (DSS) for 7 days.  Disease activity was recorded daily and immune cell populations in the blood, spleen, mesenteric lymph nodes (MLN), and lamina propria were examined by flow cytometry.  Colonic gene expression was assessed by real time PCR and microarray analyses.  Our findings show that macrophage PPAR r-deficiency significantly exacerbates DSS inflammation.   CD4+CD25+FoxP3+ regulatory T cells (T-regs) were significantly reduced in Cre+ mice, and MLN macrophages and CD40 expression were enhanced.  There were significant differences in the number colonic macrophages between Cre+ and Cre- mice, but those from Cre+ mice expressed more CD40, Ly6C, and TLR-4.  PPAR r-deficiency also increased the percent of CD8+ T cells in the lamina propria and enhanced colonic interferon gamma expression.  Our findings indicate that macrophage PPAR gamma deficiency augments the severity of DSS colitis by reducing peripheral T-regs and increasing colonic macrophage activation and T cell inflammation.

Platelets, genes, enzymes, metabolism, signal transduction, sickle cell disease

The platelet transcriptome reveals changes in arginine metabolic pathways in patients with sickle cell disease

In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide (NO) and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, suggesting that pathological platelet activation may contribute to sickle cell disease vasculopathy. Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate here the feasibility of comparative platelet transcriptome studies on clinical samples from single donors, by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by more than 10-fold increased expression compared to the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the current study included 82% or 70% of platelet abundant genes identified in two previous gene expression studies on non-amplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to 12 African American controls, at a 3-fold cut-off and 5% false discovery rate, we identified ~100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways using real time PCR and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. These studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

Effects of Rituximab on global gene expression profiles in the RA synovium

Objective: Rituximab displays therapeutic benefits in the treatment of rheumatoid arthritis (RA) patients resistant to TNF blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. In this study we investigated the global molecular effects of rituximab in synovial biopsies obtained from anti-TNF resistant RA patients before and after administration of the drug.

Methods: Paired synovial biopsies were obtained from the affected knee of anti-TNF resistant RA patients before (T0) and 12 weeks after initiation of rituximab therapy (T12). Total RNA was extracted, labeled according to standard Affymetrix procedures and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time PCR experiments were performed to confirm the differential expression of selected transcripts.

Results: According to paired Student’s t-tests, 549 out of 54,675 investigated probe sets were differentially expressed between T0 and T12. Pathway analysis revealed that genes down-regulated between T0 and T12 were significantly enriched in immunoglobulin genes, and genes involved in chemotaxis, leucocyte activation and immune responses (Gene Ontology annotations). By contrast, genes up-regulated between T0 and T12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (GSEA). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy.

Conclusion: Rituximab displays unique effects on global gene expression profiles in synovial tissue of RA patients. These observations open new perspectives in the understanding of the biological effects of the drug and in the selection of patients likely to benefit from this therapy.

The interferon induced transmembrane protein 1 (Ifitm1): detailed analysis on its assumed functions

Several functions have been suggested for the interferon induced transmembrane protein 1 (Iftitm1) gene in mammals. Originally it was identified as a member of a gene family that is highly inducible by type I and type II inteferons. Based on its expression during primordial germ CELl (PGC) specification it was suggested to be required for normal PGC migration. Ifitm1 targeted knockdown experiments in mouse embryos provided evidence that the gene might be necessary for normal somitogenesis. Finally, the complete deletion of the Ifitm gene cluster on mouse chromosome 7 revealed that the five deleted Ifitm1 genes are not essential for PCG migration and fertility. Here, we generated a novel targeted knockin allele of the Ifitm1 gene by replacing its coding region with a lacZ reporter gene to systematically reassess the suggested functions of this gene.
We find that during embryogenesis somites and in the adult the vertebral column appear normal in homozygous Ifitm1 knockout mice, strongly suggesting that Ifitm1 is not essential for normal segmentation of the paraxial mesoderm. In addition, homozygous Ifitm1 mutant mice are fertile, reproduce normally and have normal sperm quality. Furthermore, we show that the Ifitm1 loss-of-function allele does not cause immunological mutant phenotypes in unchallenged as well as in Listeria challenged homozygous mutant animals. To further focus on unique functional requirements of Ifitm1, we took advantage of the lacZ reporter gene in the Ifitm1 locus to analyze for the first time in detail and with high sensitivity its complex expression pattern in mouse embryos from E8.5 to E14.5 and in adult organs. The detailed description of the Ifitm1 expression domains will support the identification of organs that might be affected by the loss of Ifitm1 function.

Transcript profiles of DCs of PLOSL patients show abnormalities in pathways of actin bundling and immune response

This study was aimed to identify pathways associated with loss-of-function of the DAP12/TREM2 receptor complex and thus gain insight into pathogenesis of PLOSL (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy). Transcript profiles of PLOSL patients' DCs showed differential expression of genes involved in actin bundling and immune response, but also for the stability of myelin and bone remodeling.
Keywords: PLOSL patient samples vs. control samples

HD R6/1 transgenic mouse line brain hemispheres dissected.  RNA targets were created for transgenics and wildtypes at time points 18, 22 and 27 weeks.  Profiles and data analysis performed using the Bioconductor software and linear model contrasts using LIMMA on RMA probeset summarys.
Keywords: timecourse

Differential Cardiac Gene Regulation by Arg- and Gly389 Polymorphic Forms of the beta1-adrenergic Receptor

The beta1-adrenergic receptor (beta1AR; ADRB1) polymorphism Arg 389Gly is located in an intracellular loop and is associated with distinct human and mouse cardiovascular phenotypes. To test the hypothesis that beta1-Arg389 and beta1-Gly389 alleles could differentially couple to pathways beyond that of classic Gs-adenylyl cyclase (AC)/cAMP signaling, we performed comparative gene expression profile analyses on hearts from wildtype and transgenic mice that expressed either human beta1-Arg389 and beta1-Gly389 receptors, or AC5 adenyl cyclase, sampling at an early age and stage, prior to the onset of pathologic features. We observed substantial overlap of dysregulated genes across all three transgenic heart models, consistent with a shared coupling to cAMP-dependent regulation of cardiac processes and adaptive responses.  All three models up-regulated genes associated with RNA metabolism and translation, and down-regulated genes associated with mitochondria and energy metabolism, consistent with cAMP-driven increase in cardiac contractility, protein synthesis, and compensatory down-regulation of mitochondrial energy production. Both beta1AR transgenics activated additional genes associated with kinase-dependent pathways, and uniquely, beta1-Arg389 hearts caused up-regulation of genes associated with inflammation, programmed cell death, and extracellular matrix. These results substantially expand the scope of 7-transmembrane domain receptor signaling propagation beyond known cognate G-protein couplings. Moreover, they implicate alterations of a repertoire of processes evoked by a single amino acid variation in the cardiac beta1AR that might be exploited for genotype-specific heart failure diagnostics and therapeutics.
Keywords: Polymorphism, adrenergic, heart failure, beta-blocker, microarrays.

Critical Requirement of the DNA Methyltransferase, Dnmt1, for the Development and Function of Foxp3+ Regulatory T Cells

We investigated the role of DNMT1 in immune homeostasis by generating mice lacking DNMT1 in Foxp3+ T-regulatory (Treg) cells. These mice showed decreased peripheral Foxp3+ Tregs, complete loss of Foxp3+ Treg suppressive functions in vitro and in vivo, and died from autoimmunity by 3-4 weeks unless they received perinatal transfer of wild-type Tregs that prolonged their survival. Methylation of CpG-sites in the TSDR region of Foxp3 was unaffected by DNMT1 deletion, but microarray revealed more >500 proinflammatory and other genes were upregulated in DNMT1-/- Tregs. CD4-Cre-mediated DNMT1 deletion showed inability of conventional T cells to convert to Foxp3+ Treg under appropriate polarizing conditions. Hence, DNMT1 is absolutely necessary for maintenance of the gene program required for normal Treg development and function.

The mitogen-activated protein kinase (MAPK) p38alpha controls inflammatory responses and cell proliferation. Using mice carrying conditional p38alpha alleles, we investigated its function in postnatal development and tumorigenesis. When p38alpha is specifically deleted in the mouse embryo, fetuses develop to term but die shortly after birth, likely due to lung dysfunction. Fetal hematopoietic cells and embryonic fibroblasts deficient in p38alpha display increased proliferation, resulting from sustained activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway. Importantly, in chemical-induced liver cancer development, mice with liver-specific deletion of p38alpha show enhanced hepatocyte proliferation and tumor development that also correlates with JNK/c-Jun upregulation. Furthermore, increased proliferation of p38alpha-deficient hepatocytes and tumor cells is suppressed by inactivation of JNK or c-Jun.  These results reveal a novel mechanism whereby p38alpha negatively regulates cell proliferation through antagonizing the JNK/c-Jun pathway in multiple cell types and in liver cancer development.
We used microarrays to identifiy differental regulated genes by p38alpha in fetal liver cells

Comparison of transcripts from the anterior tibialis, gastrocnemius, and liver of glycogen synthase WT, GSL30 or KO mice

Anterior tibialis removed from 3-month old muscle glycogen synthase WT or knockout mouse. RNA was extracted using GibcoBRL TRIzol Reagent and a Quiagen RNeasy kit. Targets were produced using standard Affymetrix procedures from about 5ug of total RNA. GSM40057-GSM40063 AND GSM40956.

Liver removed from 3-month old muscle glycogen synthase WT or knockout mouse. RNA was extracted using GibcoBRL TRIzol Reagent and a Quiagen RNeasy kit. Targets were produced using standard Affymetrix procedures from about 10ug of total RNA. GSM40064-GSM40071.

Medial gastrocnemius removed from 3-month old muscle glycogen synthase WT or knockout mouse. RNA was extracted using GibcoBRL TRIzol Reagent and a Quiagen RNeasy kit. Targets were produced using standard Affymetrix procedures from about 5ug of total RNA. GSM40072-GSM40079.

Medial gastrocnemius removed from 8-month old muscle glycogen synthase WT or overexpressing mouse. RNA was extracted using GibcoBRL TRIzol Reagent and a Quiagen RNeasy kit. Targets were produced using standard Affymetrix procedures from about 5ug of total RNA.  GSM40080-GSM40089

Fibroblasts from normal histology lung tissue or UIP histology lung tissue

Pulmonary fibrosis is a chronic, progressive, and lethal interstitial lung disease. It is characterized by extracellular matrix deposition, fibroblast proliferation, and accumulation. Fibroblasts from normal or UIP histology were cultured and analyzed.
Keywords: Fibroblasts from normal histology lung tissue or UIP histology lung tissue

This series represents leukemic samples obtained from pediatric AML patients at diagnosis and control normal bone marrow samples.

Adipogenesis;adipocyte;early B-cell factor 1 (EBF1);commitment;differentiation;NIH-3T3;pparg;time-course

Differentiation of NIH-3T3 cells to adipocytes by PPARg or EBF1 over-expression.

NIH-3T3 cells transduced with either EBF1-, PPARg2- or empty vector were stimulated with hormones to initiate adipocyte differentiation. RNA extraction was done using TriZol at d0, d2, d4 and d10 after stimulation. Samples were handled according to standard affymetrix protocols.
Keywords = Adipogenesis

estrogen;gene expression;microarray;phenotypic anchoring;uterus;time-course

Phenotypic Anchoring of Gene Expression Changes during Estrogen-Induced Uterine Growth

Immature (19/20 days of age) Alpk:APfCD-1 mice were treated with arachis oil (AO) vehicle or 0.4mg/kg 17beta-estradiol (E2), via a single subcutaneous injection, and sacrificed at 1hr, 2hr, 4hr, 8hr, 24hr, 48hr, 72hr post dose.
Keywords = estrogen

Genome-wide identification of binding sites of Evi1 in human myeloid cell lines

The EVI1 gene codes for a transcription factor with important roles in development and leukemogenesis. Overexpression of EVI1 in acute myeloid leukemia (AML) is one of the worst prognostic factors in patients with and without 3q26 rearrangements. Evi1 acts in several pathways through the interaction with proteins with important functions in hematopoiesis; however, the role of Evi1 as a transcription factor is not well known, and only some Evi1 target genes have been identified in mice. Our aim was to investigate the pathways and direct target genes of EVI1. Differential expression profiles after EVI1 knockdown allowed us to identify 125 genes involved in cell growth, differentiation and signal transduction that could be related to EVI1. Moreover, we looked for potential EVI1 binding sites within the region 1000 bp upstream of the transcription start sites of all human genes. We selected a total of 70 genes from the bioinformatics search, genes related to EVI1 by literature, and genes differentially expressed in the expression array. ChIP in the TF1 and HEL cell lines with two EVI1 antibodies demonstrated that EVI1 binds to the proximal promoter regions of 18 of these genes, most of them involved in important differentiation and proliferation pathways, confirming the important role of EVI1. Interestingly, EVI1 binds to the proximal region of its promoter, suggesting that it could be regulating its own transcription. Further functional studies are in progress. These data provide a starting point for further studies aimed at uncovering the mechanism for EVI1-induced transformation leukemias.

Oligonucleotide microarrays were used to establish a profile for gene expression in wild-type airway epithelial cells after paramyxoviral infection.
Analysis was performed on mRNA isolated from SeV-infected primary-culture mouse tracheal epithelial cells that were maintained under physiologic conditions (air-liquid interface).

Series of samples studying effect of knock out Emx2 in urogenital epithelium of mouse embryos at E10.5.
Keywords: repeated sample

Satb1 and Satb2 are regulators of higher order chromatin in T cells and osteoblasts repectively. We were interested if Satb1 and Satb2 play a role in the regulation of gene expression in ES cells.

This SuperSeries is composed of the SubSeries listed below.

PPARg is a nuclear receptor that plays an important role in lipid metabolism, homeostasis and immunity. Microarray analysis of gene expression was performed in macrophages from WT and PPARg KO mice. Differentially expressed genes were selected for further analysis.

Effects of neonatal stress and morphine on murine hippocampal gene expression

Critically ill preterm infants experience multiple stressors while hospitalized. Morphine is commonly prescribed to ameliorate their pain and stress. We hypothesized that neonatal stress will have a dose-dependent effect on hippocampal gene expression, and these effects will be altered by morphine treatment. Male C57BL/6 mice were exposed to 5 treatment conditions between postnatal day 5 and 9: 1) Control, 2) mild stress + saline, 3) mild stress + morphine, 4) severe stress + saline and 5) severe stress + morphine. Hippocampal RNA was extracted and analyzed using Affymetrix Mouse Gene 1.0 ST Arrays. Single gene analysis and gene set analysis were used to compare groups with validation by qPCR. Stress resulted in enrichment of genes sets related to fear response, oxygen carrying capacity and NMDA receptor synthesis. Morphine downregulated gene sets related to immune function. Stress plus morphine resulted in enrichment of mitochondrial electron transport gene sets, and down-regulation of gene sets related to brain development and growth. We conclude that neonatal stress alone influences hippocampal gene expression, morphine alters a subset of stress-related changes in gene expression and influences other gene sets. Stress plus morphine show interaction effects not present with either stimulus alone. These changes may alter neurodevelopment.

In inflammatory diseases of the airway, a high level (estimated to be as high as 8 mM) of HOCl can be generated through a reaction catalyzed by the leukocyte granule enzyme myeloperoxidase (MPO). HOCl, a potent oxidative agent, causes extensive tissue injury through its reaction with various cellular substances, including thiols, nucleotides, and amines. In addition to its physiological source, HOCl can also be generated by chlorine gas inhalation from an accident or a potential terrorist attack. Despite the important role of HOCl-induced airway epithelial injury, the underlying molecular mechanism is largely unknown. In the present study, we found that HOCl induced dose-dependent toxicity in airway epithelial cells. By transcription profiling using GeneChip, we identified a battery of HOCl-inducible antioxidant genes, all of which have been reported previously to be regulated by nuclear factor erythroid-related factor 2 (Nrf2), a transcription factor that is critical to the lung antioxidant response. Consistent with this finding, Nrf2 was found to be activated time and dose dependently by HOCl. Although the epidermal growth factor receptor-MAPK pathway was also highly activated by HOCl, it was not involved in Nrf2 activation and Nrf2-dependent gene expression. Instead, HOCl-induced cellular oxidative stress appeared to lead directly to Nrf2 activation. To further understand the functional significance of Nrf2 activation, small interference RNA was used to knock down Nrf2 level by targeting Nrf2 or enhance nuclear accumulation of Nrf2 by targeting its endogenous inhibitor Keap1. By both methods, we conclude that Nrf2 directly protects airway epithelial cells from HOCl-induced toxicity.

Molecular profiles of dystrophin-deficient and normal murine muscle

Expression profiles of six skeletal muscle types in mdx, mdx5cv and wildtype mice.

Co-chaperone CCRP/DNAJC7-regulated gene regulation in the mouse liver

Co-chaperone protein CAR Cytoplasmic Retention Protein (CCRP) interacts with various nuclear receptors and determines their localization. However, there is limited information about in vivo role of CCRP especially in nuclear receptor-mediated gene regulation. We have generated CCRP global knockout (KO) mouse and have employed whole genome microarray analysis to reveal the role of CCRP in gene regulation in the mouse liver treated with phenobarbital (CAR activator) or non-treated. Male WT and KO mice were fasted for 24 h followed by the treatment with phenobarbital or vehicle PBS for 6h. Phenobarbital significantly induced or repressed  1302 and 2744 genes in WT and KO, respectively. Interestingly, cholesterol synthesizing genes were significantly up-regulated in KO even without treatment.

Constitutive activation of EGFR- and NF-kB-dependent pathways is a hallmark of cancer, yet signaling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced upon human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signaling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signaling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial-mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-kB activity that transmits pro-survival and invasive signals through EGFR signaling.
We used microarrays to detail the global programme of gene expression upon BCL-3 overexpression

Microarray analysis of the dynamics of gene expression plasticity in the supraoptic nucleus following dehydration

The hypothalamo-neurohypophyseal system (HNS) is crucial for osmoregulation. The HNS consists of the magnocellular neurons (MCNs) of the hypothalamus that have their axons terminating in the posterior pituitary. The HNS exerts its role in water balance via the release of the anti-diuretic hormone vasopressin (VP). Following dehydration VP is released into the general circulation. By binding to V2-type receptors located in the kidney, VP decrease the amount of water lost in urine. An osmotic stimulus also elicits an increase in VP gene expression and a functional remodelling of the entire HNS. We want to understand this plasticity in terms of the co-ordinated reprogramming of gene expression. We thus used Affymetrix U34a rat GeneChips to examine the dynamics of gene expression plasticity over the duration of a dehydration stimulus. Arrays were probed in triplicate with targets derived from SON of control rats, and rats dehydrated for 1 and for 3 days. Transcripts were thus identified that respond early to dehydration (up- or down-regulated after only 1day of dehydration), or that respond late (up- or down-regulated at 3 days of dehydration). 
Keywords: SON, Dehydration, clone

MYC is an early response regulator of human adipogenesis in adipose stem cells

MYC is induced early in human adipose stem cells in response to a standard MDIR adipogenic cocktail. The objective of this experiment was to identify key gene networks impacted by MYC loss-of-function in a mixed donor pool of human derived adipose stem cells.
Microarray analysis was used to identify up- and -down regulated genes in control vs MYC knockdown adipose stem cells in response to adipogenic stimulus

To determine whether the polyamide-Chl conjugate 1R-Chl would cause similar changes in global gene expression in K562 cells, affymetrix gene chip analysis was performed using 1R-Chl.  Through class comparison analysis, 1R-Chl affected the levels of transcription and genes of interest were determined.

Recent studies suggest that telomerase promotes cell growth by mechanisms that extend beyond the rescue of critically short telomeres. The in vitro model of mTert overexpressing MEFs recapitulates fundamental aspects of the growth-promoting effects of mTert in vivo. First, in Terc-proficient cells, mTert overexpression favors escape from replicative senescence and enhances anchorage-independent growth in response to oncogenic stress, which fits well with previous data showing that mTert overexpression promotes tumor formation. Second, in Terc-deficient cells, retroviral transduction with mTert results in a delayed onset of immortalization and impairs colony formation in response to oncogenic stress, which is in agreement with the inhibitory effect of mTert overexpression on tumorigenesis in a Terc null mouse background. To unravel the molecular targets of telomerase that impact on cell growth, we compared the transcriptome of MEFs, before and after mTert introduction. We found that ectopic expression of mTert was associated with detectable gene expression changes (greater than 1.5-fold; validated by qRT-PCR) of 26 transcripts. Analysis of the observed transcriptional changes indicates that ectopic expression of mTert suppresses in a coordinated manner functionally related genes with overlapping roles in growth arrest, resistance to transformation, and apoptosis. We show that the majority of the telomerase target genes are growth-inhibitory, transforming growth factor-beta (TGF-beta) -inducible genes and provide functional evidence for the potential of telomerase to abrogate TGF-beta -mediated growth inhibition. Thus, in line with the current view that the diversity of TGF-beta responses is not so much a consequence of the use of different signaling pathways but caused by different ways of reading the output from the same basic pathway, we propose that the telomerase status of a cell creates a gene expression pattern that determines how cells read growth inhibitory signals, among them signals propagated through the TGF-beta pathway.

Global gene expression analysis of gastric cancer by oligonucleotide microarrays

To gain molecular understanding of carcinogenesis, progression, and diversity of gastric cancer, 22 primary human advanced gastric cancer tissues and 8 noncancerous gastric tissues were analyzed by high-density oligonucleotide microarray in this study. Based on expression analysis of approximately 6800 genes, a two-way clustering algorithm successfully distinguished cancer tissues from noncancerous tissues. Subsequently, genes that were differentially expressed in cancer and noncancerous tissues were identified; 162 and 129 genes were highly expressed (P < 0.05) >2.5-fold in cancer tissues and noncancerous tissues, respectively. In cancer tissues, genes related to cell cycle, growth factor, cell motility, cell adhesion, and matrix remodeling were highly expressed. In noncancerous tissues, genes related to gastrointestinal-specific function and immune response were highly expressed. Furthermore, we identified several genes associated with lymph node metastasis including Oct-2 or histological types including Liver-Intestine Cadherin. These results provide not only a new molecular basis for understanding biological properties of gastric cancer, but also useful resources for future development of therapeutic targets and diagnostic markers for gastric cancer.

Gene expression profiling from memory P14 T cells with control or mutated ThPOK

We noticed that ThPOK repression is readily abrogated upon in vitro TCR stimulation of peripheral CD8 T cells. This observation prompted us to investigate a role of ThPOK in the CD8 T cell response to an acute viral infection. We observed that clonal expansion is significantly less in both primary and secondary CD8 T cell responses in the absence of functional ThPOK. To approach this mechanism, we carried out a microarray analysis for comparison of gene expression between ThPOKhd/hd and ThPOKwt/wt P14 memory T cells.

Gene expression profile studies have identified an interferon signature in whole blood or mononuclear cell samples from patients with systemic lupus erythematosus. This study was designed to determine whether specific lymphocyte and myeloid subsets freshly isolated from the blood of systemic lupus erythematosus patients demonstrated unique gene expression profiles compared to subsets isolated from healthy controls.
Keywords: patient compared to control

Expression data from activated and non-activated NTAL knockout, wild type and knockdown bone marrow derived mast cells

Non-T cell activation linker (NTAL, also called Lab or LAT2) is a transmembrane adaptor protein involved in the high affinity IgE receptor (FcεRI) signaling in mast cells. Studies with bone marrow-derived mast cells (BMMCs) from NTAL-/- mice indicated that NTAL is a negative regulator of FcεRI signaling. In contrast, depletion of NTAL by RNA interference in human or rat mast cells suggested its positive regulatory role. To determine whether compensatory developmental alterations are responsible for the observed discrepancies, we compared FcεRI-mediated signaling events in BMMCs isolated from NTAL-/- mice or BMMCs of wild type in which NTAL expression was decreased by RNA interference. We found that when compared to corresponding controls, NTAL-deficient cells, both knockouts (KOs) and knockdowns (KDs), exhibited enhanced degranulation, calcium mobilization, tyrosine phosphorylation of linker for activation of T cells (LAT) and ERK, depolymerization of filamentous actin and chemotaxis. These data indicate that NTAL in mouse BMMCs is a negative regulator of FcεRI signaling, independently of compensatory developmental alterations. To gain more insight into NTAL function, we examined gene expression profiles of resting and antigen-activated BMMCs with NTAL KO or KD and corresponding controls and identified several genes that were more than 1.8-fold differentially expressed in resting and FcεRI-activated NTAL-deficient cells, when compared to wild-type controls. We provide evidence that at least some of these genes could be involved in regulation of cholesterol-dependent events in chemotaxis towards antigen.

Expression data from mouse embryonic stem cells lacking the Alkbh1 gene

AlkB homolog 1 (ALKBH1) is one of nine members of the AlkB homologs in mammals. Most Alkbh1-deficient mice die during embryonic development, and survivors have severe defects in tissues originating from the ectodermal lineage. We hypothesized the phenotype to rely upon aberrant epigenetic regulation and provided evidence for ALKBH1 to be a histone H2A demethylase. We used a whole genome expression microarray to detail differentially expressed genes in embryonic stem cells lacking the Alkbh1 gene and identified distinct classes of up- and down-regulated genes during this process.

Effects of nicastrin (NCSTN) siRNA knockdown in HEK293 and HEK001 cells.

The goal of this study was to determine if knockdown of nicastrin induced a proinflammatory phenotype in HEK001 and HEK293 cells. Nicastrin (NCSTN) is a member of the gamma-secretase complex, and has been identified as the most frequently mutated gene in familial hidradenitis suppurativa. While much research has been done into the effects of PSEN1 and PSEN2 loss, less is known about isolated NCSTN haploinsufficiency. Two cell lines were knocked down with either NCSTN siRNA or an siRNA to luciferase in triplicate. RNA was extracted from drug selected knockdowns and profiled on the Illumina Human HT-12 v4 beadarray. Gene ontology analysis of differentially expressed genes revealed a proinflammatory and decreased proliferation signature in keratinocytes. HEK293 cells demonstrated expression signatures for decreased cholesterol synthesis and interferon-alpha signaling, as well as increased p53 signaling and caspase mediated cytoskeletal cleavage.

expression analysis, progesterone, TNFa, follicle assembly, ovary

Interactions Between Progesterone and Tumor Necrosis Factor-alpha in the Regulation of Primordial Follicle Assembly

Follicle assembly is the process by which groups or ‘nests’ of oocytes break down to form primordial follicles.  The size of the primordial follicle pool is the major determinant of the reproductive lifespan of a female.  Previously, progesterone (P4) has been shown to inhibit follicle assembly, while tumor necrosis factor alpha (TNF-alpha) has been shown to promote the apoptosis that is necessary for follicle assembly. The current study examines how TNF-alpha and progesterone interact to regulate primordial follicle assembly.  Ovaries were collected from newborn rats and placed in organ culture to examine the actions of P4 and TNF-alpha.  P4 was found to decrease primordial follicle assembly and increase the percentage of un-assembled oocytes both in vitro and in vivo.  TNF-alpha treatment did not change the proportion of assembled follicles in cultured ovaries, but did block the ability of P4 to inhibit follicle assembly.  Microarray analysis of  the ovarian transcriptome  revealed that progesterone treatment of the ovaries altered the expression of 513 genes with 132 only expressed after P4 treatment and 16 only expressed in control ovaries.  The majority of genes were up-regulated greater than 2-fold over control, with a small subset of 16 genes down-regulated.  Categories of genes affected by P4 are described including a group of extra-cellular signaling factors.  The progesterone receptors expressed at the time of follicle assembly included the surface membrane progesterone receptors PGRMC1, PGRMC2 and RDA288.  The nuclear genomic P4 receptor was not expressed at appreciable levels.  Progesterone increased the expression of several genes (TANK, NFkappaB, Bcl2l1 and Bcl2l2) involved in a signaling pathway that promotes cell survival and inhibits apoptosis.  Observations indicate that P4 acts through the surface membrane progesterone receptors to regulate primordial follicle assembly, and that TNF-alpha can over-ride the inhibitory actions of P4 on follicle assembly.  A major mechanism involved in the actions of P4 is an increase in cell survival genes and inhibition of the apoptosis pathway.  Observations provide insight into the hormonal regulation of primordial follicle assembly and lead to novel approaches to potentially manipulate follicle assembly and reproductive capacity.
Keywords: expression analysis, progesterone, TNFa, follicle assembly, ovary

Identification of phase-specific arthritis-related genes in mice

Rheumatoid arthritis (RA), one of the most common polygenic diseases, is characterized by a chronic, progressive inflammation mainly in joints and has an unknown etiology. Numerous studies have revealed the significance of cytokines TNF and IL-1 in the onset and progression of RA. Due to the complexity of interactions among different cytokines and immune cells, little is known about the precise molecular mechanisms underlying RA. In this study, oligonucleotide microarray analysis and a mouse model of RA, IL-1 receptor antagonist deficient mice were used to address this issue. Two hundred and ninety transcripts were found to be dysregulated greater than or equal to 2-fold in the diseased mice. Phase-specific gene expression changes were identified, including early increase and late decrease of heat shock protein coding genes and Cyr61. Moreover, common gene expression changes were also observed, especially the upregulation of paired-Ig-like receptor A (Pira) in both early and late phases of arthritis. We conclude that common and distinct gene expression change patterns that were identified globally may represent novel opportunities for better control of RA through early diagnosis and development of alternative therapeutic strategies.

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape mechanisms

Breast cancer metastasis to bone is a critical determinant of long-term survival after treatment of primary tumors. We used a mouse model of spontaneous bone metastasis to determine new molecular mechanisms. Differential transcriptome comparisons of primary and metastatic tumor cells revealed that a substantial set of genes suppressed in bone metastases were highly enriched for promoter elements for the type I interferon (IFN) regulatory factor, Irf7, itself suppressed in mouse and human metastases. The critical function of the Irf7 pathway was demonstrated by restoration of exogenous Irf7 or systemic interferon administration, which significantly reduced bone metastases and prolonged metastasis free survival. Using mice deficient in the type I receptor (Ifnar1-/-) or mature B, T and NK cell responses (NOD Scid IL-2rγ-/- mice) we demonstrated that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. Metastasis suppression correlated with decreased accumulation of myeloid-derived suppressor cells and increased CD4++, CD8 T cells and NK cells in the peripheral blood and was reversed by depletion of CD8+ cells and NK cells. Clinical importance of our findings was demonstrated as increased primary tumor Irf7 expression predicted prolonged bone and lung metastasis-free survival. Thus we report for the first time, a novel innate immune pathway, intrinsic to breast cancer cells, whose suppression in turn restricts systemic immunosurveillance to enable metastasis. This pathway may constitute a novel therapeutic target for restricting breast cancer metastases.

Effects of allergen challenge on airway epithelial cell gene expression

Allergen exposure induces the airway epithelium to produce chemoattractants, proallergic interleukins, matrix-modifying proteins, and proteins that influence the growth and activation state of airway structural cells. These proteins, in turn, contribute to the influx of inflammatory cells and changes in structure that characterize the asthmatic airway. To use the response of the airway epithelium to allergen to identify genes not previously associated with allergic responses, we compared gene expression in cytokeratin-positive cells before and after segmental allergen challenge. After challenge with concentrations of allergen in the clinically relevant range, 755 (6%) of the detectable sequences had geometric mean fold-changes in expression, with 95% confidence intervals that excluded unity. Using a prospectively defined conservative filtering algorithm, we identified 141 sequences as upregulated and eight as downregulated, with confirmation by conventional polymerase chain reaction in all 10 sequences studied. Using this approach, we identified asthma-associated sequences including interleukin (IL-)-3, IL-4, and IL-5 receptor subunits, the p65 component of nuclear factor-kappaB, and lipocortin. The genomic response of the human airway to concentrations of allergen in the clinically relevant range involves a greater number of genes than previously recognized, including many not previously associated with asthma that are differentially expressed after airway allergen exposure.
Keywords: Before and after allergen challenge

Transcription factor, development, photoreceptor, retina, neuron, differentiation, gene regulation, microarray, gene profiling, cell type comparison

Targeting of GFP to new-born rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors

Purpose: To investigate the gene regulatory networks during photoreceptor differentiation.
Special aims: To generate gene expression profiles of purified photoreceptors at distinct developmental stages and from different genetic backgrounds.
Background: Rod photoreceptor genesis spans a broad temporal window during retinal development. It starts as early as E12.5 and peaks at P0-P2. At E16.5, there are some early born rods but the peak of rod genesis does not occur. At P2, the majority of rod photoreceptors are born. At P6, rod specific structural/functional genes begin to express. At P10, Outer segments morphogenesis is taking place. At 4 weeks, retinal development is complete and retina is functional. Nrl is a rod specific transcription factor and one of the earliest markers of rod photoreceptors. Nrl promoter drives the expression of GFP exclusively to rod photoreceptors shortly after they exit cell cycle. In the Nrl-knockout background, the expression of GFP is detected in S-opsin positive cells, which suggested a cell fate transformation from rods to cones in the absence of Nrl.
Design: GFP positive photoreceptors from the WT-Gfp or Nrl-knockout-Gfp retina were enriched (purified) by FACS at five distinct developmental stages (E16, P2, P6, P10, and 4 weeks). Total RNA was extracted by Trizol reagent.  Around 50 ng of total RNA was used for linear amplification and biotin labeling followed Nugen kit protocol.  Fragmented cDNA was hybridized on Affymetrix mouse genomic expression array 430 2.0 and then scanned with the standard protocol. Four replicates were performed for each time point.
Conclusion: By comparing the gene expression profiles from different developmental stages, we can obtain novel insights into molecular events underlying photoreceptor differentiation.

Expression data from Rat liver 48 hours after treated with different toxic compounds.

Rat has been treated with different compounds with the purpose of investigating toxicological mechanisms. But toxic and non-toxic compounds has been administered. 3 toxic (ANIT, DMN, NMF) 3 non-tox (Caerulein, dinitrophenol(DNP), Rosiglitazone) in 5-plicates (30 arrays in all) and 9 untreated (control), 39 samples in all.
The array data was used to identify genes with biomarker potential for detecting toxic properties of compounds.

The expression of interferon-related genes was more enhanced in irradiated ATM-deficient mouse embryonic fibroblasts (MEFs) than in irradiated ATM wild-type MEFs.

GENE PROFILING AND PATHWAY ANALYSIS OF NASP EXPRESSION IN HELA CELLS

Background
NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or inhibited by siRNA.
Methods
Total RNA was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to oligonucleotide microarrays.
Results
From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 57 up-regulated and 11 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 68 up-regulated and 92 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response and cellular growth and cancer pathways (major “hubs”: TNF, FOS, EGR1, NFkB, IRF7, STAT1, IL6). Inhibition of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major “hubs”: E2F8, TP53, FGF, FSH, FST, hCG, NFkB, TRAF6).
Conclusions
This study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. Most of these genes have not been previously reported to be connected to expression levels of NASP. Gene ontology and protein network analysis identified general biological processes as well as individual genes/gene products and possible interaction networks. Networks with cancer-related functions had the highest significance. We found signaling pathways which were affected as a result of changed NASP expression. This analysis revealed that, despite some overlap, each reactive response was associated with a unique gene signature. These results place NASP in important cell regulatory networks and suggest a number of directions for further research.

Gene expression profiles of PIK3CA knockdown by shRNA in lung cancer cells

To gain insight into EMT-independent biological processes through which PI3K promotes invasion, RNA samples from 344SQ_p110α shRNA cells and 344SQ_scr cells were subjected to global transcriptional profiling.

Gata4 is required for postnatal cardiac function and protection from pressure overload-induced heart failure

An important event in the pathogenesis of heart failure is the development of pathological cardiac hypertrophy. In cultured cardiac cardiomyocytes, the transcription factor Gata4 is required for agonist-induced cardiomyocyte hypertrophy. We hypothesized that in the intact organism Gata4 is an important regulator of postnatal heart function and of the hypertrophic response of the heart to pathological stress. To test this hypothesis, we studied mice heterozygous for deletion of the second exon of Gata4 (G4D). At baseline, G4D mice had mild systolic and diastolic dysfunction associated with reduced heart weight and decreased cardiomyocyte number. After transverse aortic constriction (TAC), G4D mice developed overt heart failure and eccentric cardiac hypertrophy, associated with significantly increased fibrosis and cardiomyocyte apoptosis. Inhibition of apoptosis by overexpression of the insulin-like growth factor 1 receptor prevented TAC-induced heart failure in G4D mice. Unlike WT-TAC controls, G4D-TAC cardiomyocytes hypertrophied by increasing in length more than width. Gene expression profiling revealed upregulation of genes associated with apoptosis and
fibrosis, including members of the TGF? pathway. Our data demonstrate that Gata4 is essential for cardiac function in the postnatal heart. After pressure overload, Gata4 regulates the pattern of cardiomyocyte hypertrophy and protects the heart from load-induced failure.

human peripheral blood derived monocytes, LPS stimulation time-series

To evaluate gene expression in human peripheral blood derived monocytes over the course of an LPS stimulation time-series.

NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown.

To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype.

Chromatin Remodeling Enzyme Smarca5/Snf2h Regulates Cell Cycle Exit, Differentiation of the Lens Epithelium, and Denucleation of Lens Fiber Cells

Genome-wide approach to identify the cell-autonomous role of Snf2h in lens fiber cell terminal differentiation. Differential gene expression was analyzed in Snf2h lens-conditional knockout and wildtype newborn mouse eyeballs, with subsequent comparison of this data with the Brg1 lens-conditional knockout mouse eyes expression data (GSE25168).

Effect of a high fat diet on the hypothalami of mice susceptible to diet-induced obesity KOZAK2_ABI_HYP

The experimental goals of this study were to determine the differences in hypothalamus gene expression in genetically identical mice that have variability in their susceptibility towards diet-induced obesity following 6 weeks feeding a high fat diet, 2 weeks low fat diet and 6 weeks high fat diet.
Keywords: Comparative gene expression analysis

Gene expression profiling on innervated and denervated muscles of Smad4 KO and control mice.

To identify novel atrophy-related genes, which are controlled by BMP signaling, we performed gene expression profiling on innervated and 14 days denervated muscles of Smad4 knockout and control mice, focusing on genes that were differentially upregulated in denervated Smad4-/- muscles compared to controls. Among the different genes our attention was attracted by a gene that encodes for a novel f-box protein (Fbxo30) belonging to the SCF complex family of the ubiquitin ligases.
Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate Myostatin lead to important muscle growth in animals and humans. However, the signals and pathways responsible for this hypertrophy remain largely unknown. Here we find that BMP signaling, acting through Smad1/5/8, is the fundamental hypertrophic signal. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of Myostatin knockout and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 negatively regulates a novel gene (Fbxo30) that encodes an ubiquitin ligase, that is required for muscle loss. Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.

The experimental goals of this study were to determine differences in adipose tissue gene expression in genetically identical mice that have variability in their susceptibility towards diet-induced obesity following 4 weeks feeding a high saturated fat diet.
Keywords: comparative gene expression analysis

Comparative analysis of gene regulation by the transcription factor PPARα_mouse

Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on  species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.

Comparative analysis of gene regulation by the transcription factor PPARα_human

Gene knockdown and overexpression of 402C>G FOXL2 in COV434 and KGN cells

Despite their distinct biology, granulosa cell tumours (GCTs) are treated the same as other ovarian tumours.  Intriguingly, a recurring somatic mutation in the transcription factor Forkhead Box L2 (FOXL2) 402C>G has been found in nearly all GCTs examined.  This investigation aims to identify the pathogenicity of mutant FOXL2 by studying its altered transcriptional targets. The expression of mutant FOXL2 was reduced in the GCT cell line KGN, and wildtype and mutant FOXL2 were overexpressed in the GCT cell line COV434. Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2.      Comparisons were made between the transcriptomes of control cells and cells altered by FOXL2 knockdown and overexpression, to detect potential transcriptional targets of mutant FOXL2.

Expression data from well-differentiated human bronchial epithelial cells infected with H1N1 Influenza isolates

We used microarrays to compare the  gene expression profiles of different H1N1 isolates (seasonal and pandemic) in lung epithelial cells in vitro.

Human Whole Blood: Psoriatic Arthritis [PsA] vs. Cutaneous Psoriasis Without Arthritis [PsC] vs. Controls

Transcriptional profiling of human whole blood comparing PsA, PsC, and unaffected controls

The putative transcription factor, activity-dependent neuroprotective protein (ADNP) is a zinc finger and homeodomain - like profile containing protein. ADNP knockout mice die at day 9 of gestation. To reveal ADNP-associated pathways, a 22,690- Affymetrix probe array was used on ADNP knockout and control embryos.
References:
Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.
J Neurochem. 1999 Mar;72(3):1283-93.
PMID: 10037502 [PubMed - indexed for MEDLINE]
Cloning and characterization of the human activity-dependent neuroprotective protein.
J Biol Chem. 2001 Jan 5;276(1):708-14.
PMID: 11013255 [PubMed - indexed for MEDLINE]
Activity-dependent neuroprotective protein: a novel gene essential for brain formation.
Brain Res Dev Brain Res. 2003 Aug 12;144(1):83-90.
PMID: 12888219 [PubMed - indexed for MEDLINE]

ERG (Ets Related Gene) is an ETS transcription factor that was originally described for its role in a number of human cancers.  Our preliminary data demonstrate that ERG exhibits a highly EC restricted pattern of expression in cultured primary cells and several adult tissues including the heart, lung, and brain.  In response to inflammatory stimuli, such as TNF-alpha, we observed a marked reduction of ERG expression in EC.
To further define the role of ERG in the regulation of normal EC function we used RNA interference to knockdown ERG.  Knockdown of ERG in human umbilical vein EC (HUVEC) using siRNA was associated with the reduction of a number known ERG targets.

Male Wistar rats weighing 90-120 g were acclimatized for one week and fed standard laboratory chow, at which time the animals were divided into two groups.  Animals were then pair-fed for 8 weeks a regular laboratory chow and water “ad libitum” or Lieber-DeCarli diet (36% calories from ethanol). Control animals received the iso-caloric amount of dextrose to replace ethanol. After 8 weeks of differential feeding rats were euthanized, the pancreas immediately dissected and stored at -80?C until RNA isolation.  RNA expression was analyzed using Affymetrix RAE230A gene chips
Keywords: stress response, disease state analysis

Gene expression data from hCdc73 knock-down HEK293 cells under confluent and sparse condition.

The parafibromin/hCdc73 is a component of the PAFc, which controls RNA polymerase II-mediated general transcription. In parathyroid carcinoma and familial autosomal dominant hyperparathyroidism-jaw tumor (HPT-JT), hCdc73 mutations are heavily implicated, yet the underlying mechanism of its carcinogenic action is poorly understood. Here, we demonstrate that hCdc73 specifically controls mRNA stability of p53, and p53-mediated apoptosis. hCdc73 is associated with mature p53 mRNA in the cytoplasm and facilitates its degradation. Cytoplasmic hCdc73 physically interacts with eEF1Bγ and hSki8, and this interaction is required to bind and destabilize p53 mRNA. Furthermore, enhanced association of p53 mRNA with a cancer driven hCdc73(K34Q) mutant was also observed. As a result, reduced p53 expression as well as enhanced cell proliferation was acquired in the hCdc73 (K34Q) overexpressed cells. Altogether, our findings indicate that hCdc73 directly targets p53 mRNA to repress p53 expression, and aberrant regulation of this interaction may lead to tumor progression.

Lymphocytes are adversely affected during sepsis.  Some CD4+ splenocytes undergo apoptosis while others become Th2 polarized.  The molecular determinants of these phenotypic changes are not known.  Here we compare the transcriptional response of septic CD4 splenocytes to CD4 splenocytes from sham-manipulated animals 6h after sepsis and identify an early transcriptional component to the septic CD4+ splenocyte phenotype.
We used microarrays to detail the global program of gene expression underlying the sepsis-induced changes in CD4+ splenocyte phenotype.

Genome-Wide Profiling of Airway Epithelial Cells in Asthmatics, Smokers and Healthy Controls

We collected airway epithelial brushings for microarray analysis from 42 asthmatics and two control groups – 28 healthy subjects and 16 smokers.  A subgroup of 32 asthmatics completed a randomized placebo-controlled trial of fluticasone propionate in which collection of brushings was repeated after 1 week of treatment.
Keywords: disease state analysis, clinical trial

The transcription factor FOXM1 coordinates the expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression

The transcription factor FOXM1 coordinates the expression of cell cycle-related genes and plays a pivotal role in tumorigenesis and cancer progression. We have previously shown that FOXM1 acts downstream of 14-3-3ζ signaling, which correlates with a more aggressive tumor phenotype. However, the role that FOXM1 might play in engendering the resistance to endocrine treatments in estrogen receptor-positive (ER+) patients when tumor FOXM1 is high, has not been clearly defined.
To understand the role of FOXM1 in endocrine resistance and cancer aggressiveness, we analyzed FOXM1 protein expression by IHC in 501 ER-positive breast cancers; this revealed high FOXM1 expression in 20% of the tumors and a significantly reduced survival in these patients (p=0.003, log rank Mantel-Cox). We also mapped genome-wide FOXM1 binding events by chromatin immunoprecipitation, followed by high-throughput sequencing (ChIP-seq), in hormone-sensitive and resistant breast cancer cells after tamoxifen treatment. FOXM1 binding sites were enriched at the transcription start site of genes involved in cell cycle progression, maintenance of stem cell properties, and invasion and metastasis, all of which are correlated with the worst prognosis in ERα-positive patients treated with tamoxifen. Binding profiles were also integrated with gene expression data from cells before and after FOXM1 knockdown to highlight specific FOXM1 transcriptional networks. By modulating the levels of FOXM1 and newly discovered FOXM1-regulated genes [in breast cancer cells], we demonstrate that increased expression of FOXM1 was associated with an expansion of the cancer stem-like cell population and with increased cell invasiveness and resistance to endocrine treatments. Use of a selective FOXM1 inhibitor proved very effective in restoring endocrine therapy sensitivity and decreasing breast cancer aggressiveness.
Collectively, our findings uncover novel roles for FOXM1 and FOXM1-regulated genes in promoting cancer stem-like cell properties and therapy resistance, and highlight the relevance of FOXM1 as a therapeutic target to be considered for reducing invasiveness and enhancing breast cancer response to endocrine treatments.

Mice expressing a doxycycline-inducible dominant negative BMPR2 transgene expressed only in smooth muscle are activated for one or eight weeks, and compared to transactivator-only mice also fed doxycycline. All mice are 12 weeks of old at sacrifice.
Keywords: time course with SM22-rtTA x tetO7-BMPR2 transgene

Gene expression changes in brain vascularture in Notch3 knockout mice

Vascular smooth muscle cells (VSMC) are important for contraction, blood flow distribution and regulation of blood vessel diameter, but to what extent they contribute to the integrity of blood vessels and blood-brain barrier function is less well understood. In this report, we explored the impact of the progressive loss of VSMC in the Notch3-/- mouse on blood vessel integrity in the central nervous system
To explore the molecular consequences of the VSMC phenotype in Notch3-/- mice, we performed genome-wide transcriptional profiling of the brain vasculature in mutants and littermate controls using Affymetrix Mouse Genome 430 2.0 Array.

Involvement of transcription factor early growth response 4 (EGR4) in lung cancer bone metastasis

EGR4 (early growth response 4), a transcription factor, was previously shown to be up-regulated in lung cancer bone metastasis, compared with its expression in metastasis in other organs, using a multiorgan metastasis model of human small-cell lung cancer cells (SBC-5) in NK cell-depleted SCID mice (Kakiuchi S et al. Mol Cancer Res 2003). Here we show that EGR4 was essential for survival cancer cells, and migration ability. Next, in searching of downstream genes regulated by EGR4, we analyzed the changes in transcriptomes of SBC-5 cells at different time-points (24, 48 and 72 hr) when knockdown of EGR4 by siRNA. Lots of genes were identified either up-regulated or down-regulated in siEGR4 samples compared with control siRNA. Among these genes, there were genes with roles in metastasis or bone biology. The fact that EGR4 knockdown led to up-regulation or suppression of these genes also indicated dual roles of EGR4 in transcriptional regulation, which is consistent with previous reports: EGR4 may repress its own promoter (Zipfel PF et al. Biochim Biophys Acta. 1997) or activate certain inflammatory genes (Decker EL et al. Nucleic Acids Res. 2003; Wieland GD et al. J Cell Sci. 2005).

Transcriptional profiling of human breast cancer cell line LM2, a subline of MDA-MB-231 highly metastatic to lung when injected to nude mice, to identify the genes that are regulated after the metastasis gene metadherin is knocked down.
Keywords: Genetic modification

TFPI alpha and beta regulate mRNAs involved in cancer biology and in the immune system

Emerging evidences indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIa or TFPIb cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. Overexpression of TFPIa or TFPIb resulted in a 6.4- and 17-fold increase in TFPI protein levels, respectively. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIa or TFPIb, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor (EGFR), small GTPases, and the NFkB and JAK/STAT cascades. Integrated mRNA-miRNA analyses showed that up to 46 and 252 genes differentially expressed in cells overexpressing TFPIa or TFPIb, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes facilitating cancer cell growth and immunologic response, probably by signal transduction involving the EGFR pathway.

Similar to asthma, nematode infections are commonly associated with production of Th2 cytokines and hyporesponsive T cells.  This T cell phenotype has been reported to be induced by IL-4 dependent macrophages termed alternatively activated macrophages (AAM).  Additionally, AAM have been implicated in several pulmonary diseases, including allergic asthma.  This study examines the potential importance of AAM as immune regulatory cells in both infectious and noninfectious disease contexts. http://www.hopkins-genomics.org/asthma/asthma006/index.html

It has been found that fat oxidation is reduced in the skeletal muscle of obese humans.  This study aims to identify the mRNA of proteins involved in fat oxidation that may be reduced in obese and morbidly obese individuals.  Information gathered will help in understanding how obesity contributes to cardiovascular disease via insulin resistance.

Essential role of Jun family transcription factors in PU.1-induced leukemic stem cells

Knockdown of the transcription factor PU.1 (Spi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of PU.1 knockdown hematopoietic stem cells (HSC) in the preleukemic phase by linear amplification and genome-wide array analysis to identify transcriptional changes preceding malignant transformation. Hierarchical cluster analysis and principal component analysis clearly distinguished PU.1 knockdown from wildtype HSC. Jun family transcription factors c-Jun and JunB were among the top downregulated targets. Retroviral restoration of c-Jun expression in bone marrow cells of preleukemic mice partially rescued the PU.1-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to reduced clonogenic growth, loss of leukemic self-renewal capacity, and prevented leukemia in transplanted NOD-SCID mice. Examination of 305 AML patients confirmed the correlation between PU.1 and JunB downregulation and suggests its relevance in human disease. These results delineate a transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1-induced AML by blocking differentiation (c-Jun) and increasing self-renewal (JunB). Therefore, examination of disturbed gene expression in HSC can identify genes whose dysregulation is essential for leukemic stem cell function and are targets for therapeutic interventions.

Waldenström’s macroglobulinemia, expression profiling, microarrays, Affymetrix.

Gene expression profiling of B lymphocytes and plasma cells from WaldenstrÖm’s macroglobulinemia.

The tumoral clone of Waldenström’s macroglobulinemia (WM) shows a wide morphological heterogeneity which ranges from B-lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell-counterparts from CLL and MM, as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes down-regulated in WM-BL were IL4R, which plays a relevant role in CLL B cell survival, and BACH2 that participates in the development of class-switched PC. Interestingly, one of the up-regulated genes in WM-BL was IL6. A set of 4 genes was able to discriminate clonal B-lymphocytes from WM and CLL: LEF1 (WNT/ßcatenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5 which was overexpressed in WM-PC, and IRF4 and BLIMP1 which were underexpressed. In addition, three of the target genes activated by PAX5 -CD79, BLNK and SYK- were up-regulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell-counterpart.
Keywords: Waldenström’s macroglobulinemia, expression profiling, microarrays, Affymetrix.

Gene expression analysis of wild type and KAP1 KO splenic B cells

Chromatin remodeling is fundamental for B cell differentiation. Here, we explored the role in this process of KAP1, the cofactor of KRAB-ZFP transcriptional repressors. B lymphoid-specific Kap1 knockout mice displayed reduced numbers of mature B cells, lower steady-state levels of antibodies and accelerated rates of decay of neutralizing antibodies following viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an upregulation of PTEN, the enzymatic counter-actor of PIK3 signaling, and of genes encoding DNA damage response factors, cell-cycle regulators and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to a number of these genes, and controlled chromatin status at their promoters. Genome-wide, KAP1-binding sites avoided active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. This work thus reveals the role of KRAB/KAP1-mediated epigenetic regulation in B cell development and homeostasis.

Expression data from DOX(doxorubicin) -treated wild type or CHIP knockout C57BL/6J mice at day 5

The clinical application of doxorubicin as a broad-spectrum anti-tumor antibiotic is limited greatly by its cardiotoxicity. Various mechanisms have been studied, but little is known about whether genes or pathways relevant with energy metabolism contributes to doxorubicin-induced cardiomyopathy or not.
We used microarrays to detail the global expression profiling of acute cardiomyopathy induced by doxorubicin in wild type or CHIP knockout C57BL/6J mice and discovered distinct classes of changed genes during this process.

Lymphocyte gene expression data from moderate stage HD patients and controls

Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease modifying therapies. For Huntington's disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared to controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV et al. (2005) Proc Natl Acad Sci U S A 102: 11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.
Keywords: RNA biomarker screen

PIAS3 induction of apoptosis in non-small cell lung cancer cells is p53-independent and has STAT3-independent mediator.

Protein inhibitor of activated STAT3 (PIAS3) is an endogenous inhibitor of STAT3 that negatively regulates STAT3 transcriptional activity and cell growth and demonstrates limited expression in the majority of human squamous cell carcinomas of the lung. In the present study we sought to determine if PIAS3 inhibits cell growth in non-small cell lung cancer (NSCLC) cell lines by induction of apoptosis and further determine the dependence of PIAS3 activity on p53 status by using both wild-type and p53-null cells. Our results demonstrate that over-expression of PIAS3 promotes caspase 3 activation and PARP cleavage. Furthermore, the expression of pro-survival family members Bcl-xL and Bcl-2 is decreased. These effects were observed after both transient and regulated expression of exogenous PIAS3 and were independent of p53 status. Furthermore, while p53 can promote apoptosis by inhibition of STAT3 activity, PIAS3 inhibition of STAT3 activity was also p53 independent. Microarray experiments were performed to further investigate the STAT3-dependence of PIAS3-induced apoptosis by comparing the apoptotic gene expression signature induced by PIAS3 over-expression with that induced by STAT3 siRNA. The results showed that a subset of apoptotic genes, including CIDEC and DAPK2, were uniquely expressed only after PIAS3 expression. Thus, PIAS3 may represent a promising lung cancer therapeutic target because of its p53-independent efficacy as well as its potential to synergize with direct STAT3 inhibitors.

MHC-I overexpression in muscle biopsies is a hallmark of inflammatory myopathies.However the mechanisms of MHC-I overexpression in each disease is not well understood. Microarray analysis from MHC-I-microdissected myofibers showed a differential expression signature in each inflammatory myopathy. Innate immunity and IFN-I pathways are upregulated vs healthy controls, specifically in dermatomyositis (DM).

Differential Expression of Ove26(Diabetic) vs FVB(Nondiabetic) mice

Objective – Previous studies showed that genetic deletion or pharmacological blockade of the Receptor for Advanced Glycation Endproducts (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy (DN).  To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to DN in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function.

Research Design and Methods – We bred OVE26 mice with homozygous RAGE knock out (RKO) mice and examined structural changes associated with DN and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Affymetrix Mouse 430.2 microarrays were used to measure the differential expression of OVE26vsFVB(WT) mice.   Transcriptional changes in the TGF-β1 and Plasminogen activator inhibitor 1 gene products were measured by pcr to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice.

Results - Deletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE.  Increased transcription of the genes for Plasminogen activator inhibitor 1, TGF-β1, TGF-β induced, α1- (IV) collagen observed in OVE26 renal cortex significantly reduced in OVE26 RKO kidney cortex.  ROCK1 activity was significantly lower in OVE26 RKO compared to OVE26 kidney cortex.

Conclusions - These data provide compelling evidence for critical roles for RAGE in the pathogenesis of DN and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice

To determine the effect of consumption of a quercetin-rich diet on obesity and dysregulated hepatic gene expression, C56BL/6J mice were fed for 20 weeks on control or a Western diet high in fat, cholesterol and sucrose, both with or without 0.05% quercetin. Chronic dietary intake of quercetin reduced body weight gain and visceral and liver fat accumulation, and improved hyperglyceamia, hyperinsulinaemia, dyslipidaemia in mice fed a Western-style diet.
Feeding a Western-style diet altered expression of genes related to inflammatory responses, lipid metabolism and oxidative phosphorylation in C57BL/6J mice after 20 weeks. The results from exhaustive gene expression analysis showed that quercetin minimally influenced hepatic gene expression in mice fed the Western diet. The gene screening results (GSEA) were consistent with the notion that it did improve mitochondrial function to some extent. Quantitative RT-PCR analysis indicated that quercetin did influence important regulators of fat accumulation and metabolic disorders. Our results suggest that quercetin reduces fat accumulation presumably through decreasing oxidative stress and increasing PPARα expression, and the following improvement of gene expression related to steatosis in the liver.

genetic modification (effect of gene knock-in, stable transfection)

XBP1 confers estrogen independence and antiestrogen resistance in breast cancer cell lines

Human X-box binding protein-1 (XBP1) is an alternatively spliced transcription factor that participates in the unfolded protein response (UPR), a stress signaling pathway that allows cells to survive the accumulation of unfolded proteins in the endoplasmic reticulum lumen.  We have previously demonstrated that XBP1 expression is increased in antiestrogen-resistant breast cancer cell lines, and is co-expressed with estrogen receptor alpha (ER) in breast tumors.  The purpose of this study is to investigate the role of XBP1 and the UPR in estrogen and antiestrogen responsiveness in breast cancer. Overexpression of spliced XBP1 (XBP1(S)) in ER-positive breast cancer cells leads to estrogen-independent growth and reduced sensitivity to growth inhibition induced by the antiestrogens Tamoxifen and Faslodex in a manner independent of functional p53. Data from gene expression microarray analyses imply that XBP1(S) acts through regulating the expression of ER, the anti-apoptotic gene BCL2, and several other genes associated with control of the cell cycle and apoptosis.
Keywords: genetic modification (effect of gene knock-in, stable transfection)

To identify which miR-148b targets were involved in tumorigenesis, a microarray analysis was performed for miR-148b over-expressing cells versus controls and 129 (49 up and 80 down) modulated genes were revealed.

Inhibition of Interleukin-6 (IL-6) Signaling and Tumor Growth by a Human Neutralizing Anti-IL-6 Antibody with a Prolonged Half-Life

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a major role in responding to injury or infection as well as immune response, inflammation, and hematopoiesis.  High levels of circulating IL-6 are observed in many tumor types and are associated with poor outcomes.  We show that knockdown of IL-6 or IL-6 receptor (IL-6R) inhibits IL-6 signaling and cell viability.  In contrast, over-expression of IL-6 enhances tumor growth in vitro and in vivo, thereby supporting the role of IL-6 in tumorigenesis.  We developed a human monoclonal antibody against human IL-6 (MEDI5117) that bears Fc mutations (YTE) to extend its half-life.  We tested this antibody in several cancer cell lines that secrete high levels of IL-6, soluble IL-6R, and express gp130.  High constitutive pSTAT3 (phosphorylated signal transducer and activator of transcription 3) activation is seen in several of these cell lines, suggesting autocrine growth stimulation by IL-6. Treating these cell lines with MEDI5117 effectively blocked phosphorylation of STAT3 and inhibited IL-6-induced cell proliferation.  In vivo, MEDI5117 suppressed the growth of multiple cancer xenograft models and specifically modulated IL-6 signaling and downstream gene expression.  Combining MEDI5117 with chemotherapy or gefitinib demonstrated significantly enhanced anti-tumor activities in vivo.  Taken together, our data suggest that IL-6 signaling contributes to tumor growth, thereby supporting the development of MEDI5117 as a therapy to treat solid tumors.

Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here, we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared with nevi or metastatic lesions. Neuregulin 1-treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma.

Paclitaxel resistant MDA-MB-231 Cells and resensitization with bexarotene treatment

Acquired drug resistance represents a major challenge in chemo-therapy treatment for various types of cancers.  We have found that the retinoid X receptor–selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating chemo-resistant cancer cells. The goal of this microarray study was to understand the mechanism of bexarotene’s role in overcoming acquired drug resistance using human breast cancer cells MDA-MB-231 as a model system and paclitaxel as model compound.  After MDA-MB-231 cells were repeatedly treated with paclitaxel for 8 cycles with each cycle including a 3-day treatment with 30 nM paclitaxel and followed by a 7-day exposure to control medium, MDA cells resistant to paclitaxel were developed and their growth was no longer inhibited by paclitaxel treatment.  Those MDA cells with acquired drug resistance, when treated with paclitaxel and bexarotene in combination, could regain their sensitivity and their growth were again inhibited.  Therefore, RNA samples from parental MDA-MB-231 cells, paclitaxel-resistant MDA cells treated with vehicle, paclitaxel alone or in combination with bexarotene, were used for perform global gene expression profiling with Affymetrix HG-U133A gene chips.

This experiment was designed to study if there are differences in gene expression in the adipose tissue of women affected by polycystic ovary syndrome (PCOS) compared to non-hyperandrogenic women. PCOS is the most common endocrinopathy in women of reproductive age, and is characterized by hyperandrogenism and chronic anovulation. This disease is frequently associated with obesity, insulin resistance, and defects in insulin secretion, predisposing these women to type 2 diabetes, atherosclerosis, and cardiovascular disease.

We have applied high-density oligonucleotide arrays to omental adipose tissue samples obtained from eight morbidly obese PCOS patients and seven morbidly obese non-PCOS women at the time of bariatric surgery.

Deletion of DUSP5 affects the expression of a subset of TPA-inducible genes

This experiment was designed to determine if deletion of the dual-specificity MAP kinase phosphatase encoded by DUSP5 had any effect on the pattern of gene expression in cells treated with the phorbol ester tumour promoter TPA. Sex matched primary WT and DUSP5 knockout MEFs were isolated from littermate embryos and cultured. Cells were then exposed to TPA for 0, 1 and 3h before isolation of RNA prior to microarray hybrdisation and data analysis. Our results show that the transcript levels of a subset of TPA-inducible genes are altered in cells that lack DUSP5.

Developmental influence of the cellular prion protein on the gene expression profile in mouse hippocampus

A key event in the pathogenic process of prion diseases is the conversion of the cellular prion protein (PrPC) to an abnormal and protease-resistant isoform (PrPSc). Mice lacking PrP are resistant to prion infection, and down-regulation of PrPC during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrPC during prion diseases. However, the silencing of a protein that is widely expressed throughout the CNS could be detrimental to brain homeostasis. The physiological role of PrPC remains still unclear, but several putative functions have been proposed. Among these, several lines of evidence support PrPC function in neuronal development and maintenance.
To assess the influence of PrPC on gene expression profile during development in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus, at two different developmental stages: newborn (4-day-old) and adult (3-month-old) mice, both from Prnp+/+ and Prnp0/0 animals. The comparison of the different datasets allowed us to identify “commonly” co-regulated genes and “uniquely” de-regulated genes during postnatal development in these animal models.  The lack of PrPC during neuronal development affected several biological pathways, among which the most representative were cell signaling, cell-cell communication and transduction process. In addition, the absence of PrPC influenced genes involved in calcium homeostasis, nervous system development, synaptic transmission and cell adhesion. There was only a moderate alteration of the gene expression profile during neuronal development in the animal models we studied. PrPC deficiency does not lead to a dramatic alteration of gene expression profile, and produces moderate altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrPC levels as a therapeutic approach to prion diseases.

Induction of pathogenic Th17 cells by salt inducible kinase SGK-1 (SGK-1 KO)

Th17 cells are highly proinflammatory cells that are critical for clearing extracellular pathogens like fungal infections and for induction of multiple autoimmune diseases1. IL-23 plays a critical role in stabilizing and endowing Th17 cells with pathogenic effector functions2. Previous studies have shown that IL-23 signaling reinforces the Th17 phenotype by increasing expression of IL-23 receptor (IL-23R)3. However, the precise molecular mechanism by which IL-23 sustains the Th17 response and induces pathogenic effector functions has not been elucidated. Here, we used unbiased transcriptional profiling of developing Th17 cells to construct a model of their signaling network and identify major nodes that regulate Th17 development. We identified serum glucocorticoid kinase-1 (SGK1), as an essential node downstream of IL-23 signaling, critical for regulating IL-23R expression and for stabilizing the Th17 cell phenotype by deactivation of Foxo1, a direct repressor of IL-23R expression. A serine-threonine kinase homologous to AKT4, SGK1 has been associated with cell cycle and apoptosis, and has been shown to govern Na+ transport and homeostasis5, 6 7, 8. We here show that a modest increase in salt (NaCl) concentration induces SGK1 expression, promotes IL-23R expression and enhances Th17 cell differentiation in vitro and in vivo, ultimately accelerating the development of autoimmunity. The loss of SGK1 resulted in abrogation of Na+-mediated Th17 differentiation in an IL-23-dependent manner. These data indicate that SGK1 is a critical regulator for the induction of pathogenic Th17 cells and provides a molecular insight by which an environmental factor such as a high salt diet could trigger Th17 development and promote tissue inflammation.

substance use, major depression, SLC6A4, AUTS2, CAPN2, ELN, lymphoblast;disease state analysis

Transcriptional Profiling of Subjects from the Iowa Adoption Studies

Transcriptional profiling has been used to identify gene expression patterns indicative of general medical illnesses such as atherosclerosis.    However, whether these methods can identify common psychiatric disorders has not been established.  To answer this question with respect to nicotine use, we used genome wide expression profiling lymphoblast cell lines from six actively smoking Iowa Adoption Studies subjects and nine ?clean? control subjects, followed by real-time PCR (RT-PCR) of gene expression patterns in lymphoblast derived RNA from 94 subjects in the Iowa Adoption Studies.  As compared to those from controls without a history of smoking (n=9), the expression levels of 579 of 29,098 genes were significantly up-regulated and expression levels of 584 of 29,098 genes were significantly down-regulated in lymphoblast lines from currently smoking subjects (n=6).  RT-PCR confirmation of four select RNA levels confirmed the validity of the overall profile and revealed highly significant relationships between the expression of some of these transcripts and 1) major depression, 2) antisocial personality, 3) nicotine dependence and 4) cannabis dependence.  We conclude that the use of expression profiling may contribute significant insights into the biology of complex behavioral disorders.
Keywords:  substance use, major depression, SLC6A4, AUTS2, CAPN2, ELN, lymphoblast

Alpha-synuclein deficiency affects brain Foxp1 expression and ultrasonic vocalization

Alpha-synuclein is an abundant protein implicated in synaptic function and plasticity, but the molecular mechanism of its action is not understood. Missense mutations and gene duplication/triplication events result in Parkinson's disease, a neurodegenerative disorder of old age with impaired movement and emotion control. Here, we systematically investigated the striatal as well as the cerebellar transcriptome profile of alpha-synuclein-deficient mice via a genome-wide microarray survey in order to gain hypothesis-free molecular insights into the physiological function of alpha-synuclein. A genotype-dependent, specific and strong downregulation of forkhead box P1 (Foxp1) transcript levels was observed in all brain regions from postnatal age until old age and could be validated by qPCR. In view of the co-localization and heterodimer formation of FOXP1 with FOXP2, a transcription factor with a well established role for vocalization, and the reported regulation of both alpha-synuclein and FOXP2 expression during avian song learning, we performed a detailed assessment of mouse movements and vocalizations in the postnatal period. While there was no difference in isolation-induced behavioral activity in these animals, the alpha-synuclein-deficient mice exhibited an increased production of isolation-induced ultrasonic vocalizations (USVs). This phenotype might also reflect the reduced expression of the anxiety-related GABA-A receptor subunit gamma 2 (Gabrg2) we observed. Taken together, we identified an early behavioral consequence of alpha-synuclein deficiency and accompanying molecular changes, which supports the notion that the neural connectivity of sound or emotion control systems is affected.

Ablation of Cdh1 and Trp53 in the uterus reveals novel mechanisms controlling tumor microenvironment in endometrial cancer.

Because TP53 mutation and CDH1 inactivation are the most common abnormalities found in human type II endometrial carcinomas, the contribution of dysfunctional TRP53 and CDH1 in the tumor microenvironment to induce type II endometrial cancer was characterized using mouse as a model. The results of our analysis revealed that conditional deletion of Cdh1 and Trp53 in the uterus regulated most of the genes categorized by their involvement in inflammatory responses, immune cell trafficking, cellular movement, cell-to-cell signaling and interaction and cellular growth and proliferation.

Aryl hydrocarbon receptor senses bacterial pigmented virulence factors and orchestrates anti-bacterial defenses (part 1)

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR not only evolved to sense pollutants from the environment, but also insults of microbial origin. We demonstrate that bacterial pigmented virulence factors, namely the phenazines pyocyanin and 1-hydroxyphenazine from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, are ligands of AhR. AhR activation leads to degradation of these virulence factors and regulated cytokine and chemokine production. The relevance of AhR to host defense is underlined by heightened susceptibility of AhR-deficient mice, both to P. aeruginosa and M. tuberculosis. Our data demonstrate that AhR senses distinct bacterial virulence factors and controls anti-bacterial responses. We provide evidence for a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigmented virulence factors as a new class of pathogen-associated molecular patterns.

Gene expression variation induced by loss of the CD146 mRNA in MDA-MB-231

Metastasis is a complex process involving loss of adhesion, migration, invasion and proliferation of cancer cells. Cell adhesion molecules play a pivotal role in this phenomenon by regulating cell-cell and cell-matrix interactions. CD146 (MCAM) is associated with advanced tumor stage in melanoma, prostate and ovarian cancers.
For this study, the MDA-MB-231 cell line was used as a prototypic mesenchymal and invasive cell line, spontaneously expressing high levels of CD146. Using whole-genome DNA microarrays, we investigated genes for which expression was modified by CD146 down-regulation, obtained by siRNA or shRNA technology

QK-knockdown U87 transduced with miR-20a or a scrambled non-targeting microRNA (miR-NT)

Identify potential miR-20a regulated mRNAs and linked pathways in the setting of QK knockdown by comparing the transcriptional profiles of shQK-transduced human U87 cells together with miR-20a or a scrambled miRNA control (miR-NT)

Gene expression data for shRNA PTPN1 knockdown vs. Non-silencing in the classical Hodgkin lymphoma-derived cell line KM-H2

Conditional activation of B-catenin in adult mouse epidermis for 0, 1, or 7 days

Expression of deltaNB-cateninER allows the time and duration of B-catenin activation to be controlled precisely through the application of 4OHT.  We have shown that B-catenin activation in adult mouse epidermis stimulates de novo hair follicles and the formation of a new bulge and melanocyte niche.  We utilize Affymetrix Mouse Genome 430A 2.0 oligonucleotide arrays to investigate the differential regulation of RNA in skin from 7 week old female deltaNB-cateninER transgenic mice following 0, 1, or 7 days B-catenin activation in the epidermis.  RNA from age and sex matched wild-type littermates was used as a control.
Keywords = Mouse

Expression data from C57BL/6J and C57BL6/J Sarm-deficient mice uninfected or infected with vesicular stomatitis virus (VSV)

Sarm-deficient mice are protected from VSV encephalitis and death. Microarray was done to examine genes contributing to this phenotype

Monocytes were isolated from 30 ml of whole blood from each of 19 women, 10 with high BMD and 9 with low BMD, using monocyte negative isolation kit from Dynal Biotech Inc. Total RNA was extracted from monocytes using Qiagen RNeasy Mini Kit. Targets were produced for each subject using standard Affymetrix procedures from about 4ug of total RNA. Hybridization was made for each subject. Comparison was performed between 10 high BMD and 9 low BMD subjects.

MCF10A cells expressing HER2 and HER3 and grown in three-dimensional cultures

The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival. We provide gene expression profile of the mammary epithelial cells MCF10A expressing HER2, HER3 or HER2/HER3 and grown in three-dimensional cultures for 15 days in the presence of heregulin, a known HER3-ligand that stabilizes and activates the HER2/HER3 heterodimer.

Series contains four arrays - two wildtype, two null.  The experiment studied the effect of knocking out SOCS3 in mouse ES cells.

The specific deletion of Rb gene in epidermis leads to altered proliferation and differentiation, but not to the development of spontaneous tumors. Our previous data have demonstrated the existence of a functional compensation of Rb loss by Rbl1 (p107) in as the phenotypic differences with respect to controls are intensified. However, the possible evolution of this aggravated phenotype, in particular in relationship with tumorigenesis, has not been evaluated due to the premature death of the double deficient mice. We have now investigated whether p107 can also act as a tumor suppressor in pRb-deficient epidermis using different experimental approaches. We found spontaneous tumor development in doubly-deficient skin grafts. Moreover, Rb-deficient keratinocytes are susceptible to Ha-ras-induced transformation, and this susceptibility is enhanced by p107 loss. Further functional analyses, including microarray gene expression profiling, indicated that the loss of p107, in the absence of pRb, produces the reduction of p53-dependent pro-apoptotic signals. Overall, our data demonstrate that p107 behaves as a tumor suppressor in epidermis in the absence of pRb and suggest novel tumor-suppressive roles for p107 in the context of functional p53 and activated Ras
Keywords: epidermis, tumorigenesis, pRb, p107, Ras, p53, apoptosis

Identifying the downstream effectors of E2F7 in squamous cell carcinoma (SCC) cells

Advanced head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 40%. We have previously shown that E2F7 may contribute to drug sensitivity. In the present study, we conducted an –omics screen to identify factors that were responsible for E2F7-dependent resistance to anthracyclines by HNSCC. We provide, in vitro, in vivo and patient data that identifies the existence of a novel E2F7/Sphingosine kinase 1/Sphingosine-1-phosphate/AKT axis that regulates sensitivity to anthracyclines in HNSCC. Specifically, we show that E2F7-dependent sensitivity to doxorubicin occurs via induction of the sphingosine kinase 1/AKT axis.  We also show that pharmacological inhibition of Sphingosine kinase 1 or AKT sensitizes SCC cells to the cytotoxic actions of doxorubicin in vitro and in vivo. Combined, these findings highlight a novel mechanism through which SCC cells acquire resistance to anthracyclines and identify specific pharmacological combinations that could be used to treat SCC.

Expression Profiling Identifies Novel Gene Targets and Functions for Pdx1 in the Duodenum of Mature Mice

Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, less is known about the function of Pdx1 in the small intestine.
We aim to investigate the role of Pdx1 in mature proximal small intestine by profiling the expression of genes differentially regulated in response to Pdx1 inactivation restricted to the intestinal epithelium in mice.

Gene expression analysis of cMyc-deficient CD8 T cells retrovirally overexpressing cMyc or AP4

To determine functional overlap between cMyc and AP4 in CD8+ T cell priming, we retrovirally expressed cMyc or AP4 in cMyc-deficient CD8+ T cells and examined gene expression after activation.

Methylphenidate exposure induces dopamine neuron loss and activation of microglia in the basal ganglia of mice

Background: Methylphenidate (MPH) a psychostimulant prescribed to manage ADHD symptoms, has been increasingly prescribed to children not meeting the strict criteria for ADHD. It has also been misused as a “cognitive enhancer” and as an alternative to other psychostimulants. Here, we investigate whether chronic or acute administration of methylphenidate in mice at either 1mg/kg or 10mg/kg dosing, affects cell number and gene expression in the basal ganglion.
Methodology/Principal findings: Through the use of stereological counting methods, we observed a significant reduction (~20%) in dopamine neuron numbers in the substantia nigra pars compacta (SNpc) following chronic administration of 10mg/kg MPH. This dosage of MPH also induced a significant increase in the number of morphologically-activated SNpc microglia. Additionally, exposure to either 1mg/kg or 10 mg/kg MPH increased the sensitivity of SNpc dopamine neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced oxidative stress. Unbiased gene screening employing Affymetrix GeneChip® HT MG-430 PM revealed changes in 115 and 54 genes in the substantia nigra (SN) of mice exposed to 1mg/kg and 10mg/kg MPH doses, respectively. Decreases in the mRNA levels of gdnf, dat1, vmat2 and th in the substantia nigra (SN) was observed with both acute and chronic dosing of 10mg/kg MPH. We also found an increase in mRNA levels of the pro-inflammatory genes il-6 and tnf-α in the striatum; although these were seen only at an acute dose of 10mg/kg and not following chronic dosing.
Conclusion: Collectively our results suggest that chronic MPH usage in mice, especially at prolonged higher doses, has long-term neurodegenerative consequences.

Expression data from skin fibroblasts derived from Setleis Syndrome patients and normal controls

Setleis Syndrome is a rare type of facial ectodermal dysplasia characterized by an aged leonine appearance with puckered skin about the eyes, absent eyelashes on both lids or multiple rows on the upper lids and none on the lower lids, eyebrows that slant sharply upward laterally, and a rubbery feel of the nose and chin. Some of the patients showed bilateral temporal marks superficially like forceps marks and like the lesions seen in focal facial dermal dysplasia. We have evidence that Setleis Syndrome is caused by nonsense mutations in the gene coding for the small bHLH transcription factor known as TWIST2  in Puerto Rican and Omani patients.

We performed expression microarray analysis of RNA samples derived from skin fibroblasts grown from skin biopsies of Setleis Syndrome patients and normal controls in order to identify genes potentially involved in facial development and the pathogenesis of Setleis Syndrome.

HIVE, HIV, AIDS, Microarray, neuroimmunity, neuroinflammatory, neurodegeneration

Patterns of gene dysregulation in the frontal cortex of patients with HIV encephalitis

The neurodegenerative process in HIV encephalitis (HIVE) is associated with extensive damage to the dendritic and synaptic structure that often leads to cognitive impairment. Several mechanisms might be at play, including release of neurotoxins, oxidative stress and decreased activity of neurotrophic factors. Furthermore, HIV-mediated dysregulation of genes involved in neuronal maintenance might play an important role. For this purpose, cRNA was prepared from the brains of 17 AIDS patients for analysis with the Affymetrix Human U95Av2 GeneChip and analyzed with the GeneSpring Expression Analysis Software. Out of 12,625 genes analyzed, 74 were downregulated and 59 were upregulated compared to controls. Initial alternative analysis of RNA was performed by ribonuclease protection assay (RPA). In cases with HIVE, downregulated genes included neuronal molecules involved in synaptic plasticity and transmission (ion channels, synaptogyrin, synapsin II), cell cycle (p35, p39, CDC-L2, CDC42, PAK1) and signaling molecules (PI3K, Ras-Raf-MEK1), transcription factors and cytoskeletal components (MAP-1B, MAP-2, tubulin, adducin-2). Upregulated genes included those involved in neuroimmune (IgG, MHC, β2microglobulin) and anti-viral responses (interferon-inducible molecules), transcription (STAT1, OLIG2, Pax-6) and signaling modulation (MEK3, EphB1) of the cytoskeleton (myosin, aduccin-3, radixin, dystrobrevin). Taken together, this study suggests that HIV proteins released from infected macrophages might not only induce a neuroinflammatory response, but also may promote neurodegeneration by interfering with neuronal transcription of genes involved in regulating signaling and cytoskeletal molecules important in maintaining synapto-dendritic functioning and integrity.
Keywords: HIVE, HIV, AIDS, Microarray, neuroimmunity, neuroinflammatory, neurodegeneration

The reduced form of Coenzyme Q10 decreases the expression of LPS-sensitive genes in human THP-1 cells

Monocytes are key players in inflammatory processes which are triggered by lipopolysaccharide (LPS), the major outer membrane component of gram-negative bacteria. The present study in human monocytic THP-1 cells was designed in order to identify LPS-inducible genes which are down-regulated by the reduced form of CoQ10 (ubiquinol, Q10H2). For this purpose, THP-1 cells were incubated with 10 µM Q10H2 for 24 h. Subsequently, cells were stimulated for 4 h with 1µg/ml LPS and the resulting gene expression levels were determined using microarrays. 14 LPS-inducible genes were identified to be significantly (p < 0.05) down-regulated by Q10H2 pre-treatment between a factor of 1.32 and 1.65. The strongest effect of Q10H2 incubation was found for the nuclear receptor coactivator 2 gene (NCOA2). Gene Ontology (GO) terms revealed for the Q10H2-sensitive genes an involvement in e.g. signal transduction processes (CENTD1, NCOA2, PSD3, PPP2R5C), transcriptional regulation (NCOA2, POU2F1, ETV3) and cell proliferation pathways (CCDC100, EPS15). In conclusion, we provide evidence in THP-1 cells that the reduced form of CoQ10 (Q10H2) modulates LPS-induced gene expression.

Profiling gene expression in Vax2 knockout compared with wild type mice

Total RNA samples from Vax2 knockout mouse eyes (at least two  biological replicates) were profiled by gene expression. As control we used total RNA from  wild type  eyes. The analysis was carried out at five different developmental stages: E10.5, E12.5, E16.5, P8, and P60.

Expression data from serum-starved control and CDK8 depleted cells following serum stimulation

The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). CDK8, the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein. Here we show that CDK8 is predominantly a positive regulator of gene expression within the serum response network, as it is required for expression of several members of the AP-1 and EGR family of oncogenic transcription factors (e.g. FOS, JUN, EGR1-3). Mechanistic studies demonstrate that CDK8 is not required for recruitment of RNAPII and promoter escape at these loci. Instead, CDK8 depletion leads to the appearance of slower elongation complexes carrying hypophosphorylated RNAPII. We show that CDK8-Mediator regulates precise steps in the assembly of a functional elongation complex, including the recruitment of P-TEFb and BRD4, but is dispensable for recruitment of SPT5 and FACT. Furthermore, CDK8-Mediator specifically interacts with P-TEFb. Thus, we uncovered a novel role for CDK8 in transcriptional regulation that may contribute to its oncogenic effects.
We used microarrays to analyze the expression of immediate early genes in response to serum in cells with and without the Mediator associated kinase, CDK8.

Organ - specific expression profiles of rat treated with targretin, 9-cis retinoic acid, and 4-hydroxyphenylretinamide

A rexinoid, targretin (TRG) and two retinoids, 9-cis retinoic acid (9cRA) and 4-hydroxyphenylretinamide (4HPR) were examined for their effects on gene expression in rat mammary gland, liver, and lung tissues. The chemopreventive effects of these agents have largely been attributed to their ability to interact with retinoic acid receptors (RAR) and/or retinoid X-receptors (RXR). TRG interacts with the RXR receptors. 9cRA interacts with both the RAR and RXR receptors, while 4HPR has a moderate affinity primarily for RAR gamma. Based on previously performed mammary chemoprevention studies, TRG (150mg/Kg diet), 9cRA (100 mg/Kg diet), and 4HPR (792 mg/Kg diet), were administrated to rats continually in their diet for 7 days. Tissue - and agent - specific expression differences were determined by comparing tissues from treated rats with those form rats administered a control diet. There were significantly more changes seen associated with TRG than 9cRA or 4HPR. Only a limited number of expression changes were found with 4HPR treatment. For each organ, TRG and 9cRA treated tissues clustered closely together, whereas 4HPR treated tissues clustered with the tissues from the control diet group. In contrast to 9cRA treatment, TRG treatment altered genes that involved fatty acid metabolism and modulation of various cytochromes P450 in the liver clearly demonstrating the very disparate nature of these two retinoids. These expression signatures could provide useful pharmacodynamic biomarkers for retinoids’ treatment and chemoprevention.

The LEDGF transcript from the PSIP1 gene was knocked down using RNAi technology. The resulting 293T derived cell line (293TsiLL) was compared to a control cell line (293TsiScram) using microarray analysis. Genes identified as being modulated by LEDGF were preferential targets of HIV integration.
Keywords: gene knockdown effects

Gene expression profiling in tibial muscular dystrophy and control skeletal muscle

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. To identify these components, we used gene expression profiling of muscle biopsies from TMD patients and healthy controls.

The human SYT-SSX fusion protein was expressed in a developmentally dependent fashion in murine myoblasts.  Tumors harvested from mice in adolescence were compared to normal mouse skeletal muscle samples.
Keywords: tumor versus normal muscle tissue

juvenile rheumatoid arthritis (JRA);peripheral blood mononuclear cells (PBMC);synovial fluid mononuclear cells (SFMC);other

Biotinylated cRNA was synthesized from total RNA (Enzo; Farmingdale, NY) and processed according to the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix; Santa Clara, CA).  57 samples: 5 pauciarticular PBMC, 15 polyarticular PBMC, 11 control PMBC, 6 JSpA PBMC, 5 pauciarticular SFMC, 10 polyarticular PBMC, 5 JSpA SFMC classified by course.
Keywords = juvenile rheumatoid arthritis (JRA)

Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures

Doxorubicin is considered one of the most potent established chemotherapeutics in the treatment of liposarcoma; however, the response rates usually below 30%, are still disappointing. This study was performed to identify gene expression changes in liposarcoma after doxorubicin treatment. Cells of 19 primary human liposarcoma were harvested intraoperatively and brought into cell culture. Cells were incubated with doxorubicin for 24 h, RNA was isolated and differential gene expression was analysed by the microarray technique.
We used microarrays to detail the global  gene expression changes following doxorubicin treatment of primary liposarcoma cell cultures

Gene expression analysis of macrophages from ankylosing spondylitis patients reveals interferon-gamma dysregulation

OBJECTIVE: To determine whether macrophages, a type of cell implicated in the pathogenesis of ankylosing spondylitis (AS), exhibit a characteristic gene expression pattern. METHODS: Macrophages were derived from the peripheral blood of 8 AS patients (median disease duration 13 years [range <1-43 years]) and 9 healthy control subjects over 7 days with the use of granulocyte-macrophage colony-stimulating factor. Cells were stimulated for 24 hours with interferon-gamma (IFNgamma; 100 units/ml), were left untreated for 24 hours, or were treated for 3 hours with lipopolysaccharide (LPS; 10 ng/ml). RNA was isolated and examined by microarray and real-time quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: Microarray analysis revealed 198 probe sets detecting the differential expression of 141 unique genes in untreated macrophages from AS patients compared with healthy controls. Clustering and principal components analysis clearly distinguished AS patients and controls. Of the differentially expressed genes, 78 (55%) were IFN-regulated, and their relative expression indicated a reverse IFN signature in AS patient macrophages, where IFNgamma-up-regulated genes were underexpressed and down-regulated genes were overexpressed. Treatment of macrophages with exogenous IFNgamma normalized the expression of these genes between patients and controls. In addition, the messenger RNA encoded by the IFNgamma gene was approximately 2-fold lower in AS patient macrophages at baseline (P = 0.004) and was poorly responsive to LPS (P = 0.018), as compared with healthy controls. CONCLUSIONS: Our findings reveal consistent differences in gene expression in macrophages from AS patients, with evidence of a striking reverse IFN signature. Together with poor expression and responsiveness of the IFNgamma gene, these results suggest that there may be a relative defect in IFNgamma gene regulation, with autocrine consequences and implications for disease pathogenesis.

Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Recent studies including our own identified that the mitotic kinase, maternal embryonic leucine-zipper kinase (MELK), is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we provide evidence that the kinase activity of MELK is essential for the action of MELK in GSCs and vital for GBM growth. We utilized in silico structure-based analysis for protein-compound interaction to predict that a recently identified small molecule, Compound 1 (C1), binds to the kinase-active site of MELK protein and eliminates MELK kinase activity in nanomolar concentrations. When treated with C1, GSCs undergo mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed using MELK shRNA-mediated knockdown. C1 treatment strongly induces tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuates growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.

The effect of Crem absence on gene expression in mouse adrenal glands.

CREM (cAMP responsive element modulator) together with CREB and ATF-1 belong to the CREB family of transcriptional factors, that respond to cyclic AMP signaling and bind to cAMP responsive element (CRE) sites in promoters of selected genes. CREM can produce isoforms that have either activating or repressing functions, depending on the transcription of specific exons. In adrenal glands it is involved in the regulation of expression of genes for steroid hormone synthesis.
In this dataset, we include the expression data obtained from wild-type and Crem knock-out mouse adrenal glands.

CREM (cAMP responsive element modulator) together with CREB and ATF-1 belong to the CREB family of transcriptional factors, that respond to cyclic AMP signaling and bind to cAMP responsive element (CRE) sites in promoters of selected genes. CREM can produce isoforms that have either activating or repressing functions, depending on the transcription of specific exons. In testis, it is involved in the regulation of spermatogenesis.
In this dataset, we include the expression data obtained from wild-type and Crem knock-out mouse testis.

Rat Retinal Müller cells from diabetic rats (diabetes duration 6 months) compared to Rat Retinal Müller cells from healthy rats.  Diabetes was induced by streptozotozine.  Diabetic rats were treated with small doses of insulin to prevent catabolism.

The hematopoietic stem cell in chronic phase CML is characterized by a transcriptional profile resembling normal myeloid progenitor cells and reflecting loss of quiescence

We found that composition of cell subsets within the CD34+ cell population is markedly altered in chronic phase (CP) chronic myeloid leukemia (CML). Specifically, proportions and absolute cell counts of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) are significantly greater in comparison to normal bone marrow whereas absolute numbers of hematopoietic stem cells (HSC) are equal. To understand the basis for this, we performed gene expression profiling (Affymetrix HU-133A 2.0) of the distinct CD34+ cell subsets from six patients with CP CML and five healthy donors. Euclidean distance analysis revealed a remarkable transcriptional similarity between the CML patients' HSC and normal progenitors, especially CMP. CP CML HSC were transcriptionally more similar to their progeny than normal HSC to theirs, suggesting a more mature phenotype. Hence, the greatest differences between CP CML patients and normal donors were apparent in HSC including downregulation of genes encoding adhesion molecules, transcription factors, regulators of stem-cell fate and inhibitors of cell proliferation in CP CML. Impaired adhesive and migratory capacities were functionally corroborated by fibronectin detachment analysis and transwell assays, respectively. Based on our findings we propose a loss of quiescence of the CML HSC on detachment from the niche leading to expansion of myeloid progenitors.

The effects of Hmgb1 depletion on gene expression in murine cells

High Mobility Group Box 1 (HMGB1) is a highly abundant and evolutionarily conserved non-histone chromatin component with the ability to bind and bend DNA. The protein is involved in fundamental nuclear processes including nucleosome sliding, transcription, replication, V(D)J recombination and DNA transposition. To assess the functional impact of HMGB1 on transcription we performed gene expression profile analyses of mouse embryonic fibroblasts, wild-type or knockout for the Hmgb1 gene

We used microarrays to detail the global programme of gene expression underlying palate development by persistent expression in R26Pax3 mice and identified distinct classes of up-regulated and down-regulated genes during this process.
Keywords: palate development

Differentially expressed genes of CD11b+Gr-1+ immature myeloid cells (IMCs) in the bone marrow and colonic tumor setting of histidine decarboxylase (HDC)-KO mice were examined by microarray (Affymetrix Mouse 430.2 array). Myeloid differentiation-related candidate genes were sought to be isolated and functionally studied.

HGF has been reported to have both positive and negative effects on carcinogenesis. Here we show that the loss of c-Met signaling in hepatocytes enhanced rather than suppressed the early stages of chemical hepatocarcinogenesis. c-Met conditional knockout mice (c-metfl/fl, AlbCre+/-; MetLivKO) treated with N-nitrosodiethylamine (DEN) developed significantly more and bigger tumors and with a shorter latency as compared with control (wt/wt, AlbCre+/-; Cre-Ctrl) mice. Accelerated tumor development was associated with increased rate of cell proliferation and prolonged activation of epidermal growth factor receptor (EGFR) signaling. MetLivKO livers treated with DEN also displayed elevated lipid peroxidation, decreased ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and upregulation of superoxide dismutase 1 (Sod1) and heat shock protein 70 (Hsp70), all consistent with increased oxidative stress. Likewise, gene expression profiling performed at 3 and 5 months after DEN treatment revealed upregulation of genes associated with cell proliferation and stress responses in c-Met mutant livers. The negative effects of c-Met-deficiency were reversed by chronic oral administration of anti-oxidant N-acetylcysteine (NAC). NAC blocked the EGFR activation and reduced the DEN-initiated hepatocarcinogenesis to the levels of Cre-Ctrl mice. These results argue that intact HGF/c-Met signaling is essential for maintaining normal redox homeostasis in the liver and has tumor suppressor effect(s) during the early stages of DEN-induced hepatocarcinogenesis.
Keywords: compound treatment design

Gene expression profile in MCF7 breast cancer cells after siRNA knock down of estrogen receptor alpha (ESR1)

Affymetrix microarray data was generated from MCF7 breast cancer cells treated in vitro with siRNAs against estrogen receptor alpha (ESR1).

POU4F1 is associated with t(8;21) AML and contributes directly to its unique transcriptional signature

POU4F1 is associated with t(8;21) acute myeloid leukemia (AML) and contributes directly to its unique transcriptional signature

Gata3 acts alongside Cdx2 to promote trophoblast gene expression downstream of Tead4 during mouse development

The first lineage decisions during mouse development lead to establishment of embryonic and extraembryonic tissues. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4 and promoting trophoblast fate at the blastocyst stage. Here we show that the transcription factor Gata3 is coexpressed with Cdx2 in the blastocyst and that overexpression of Gata3 in embryonic stem cells is sufficient to induce expression of trophoblast genes. Gata3 expression in the blastocyst does not depend on Cdx2, nor do Gata3 overexpressing cell lines require Cdx2 for expression of a subset of trophoblast genes. In the embryo, expression of Gata3, like Cdx2, depends on Tead4, and expression of both factors becomes restricted to nascent trophoblast by an Oct4-independent mechanism. These observations place Tead4 at the top of a trophoblast hierarchy, with Gata3 and Cdx2 acting downstream to induce expression of common and independent targets in this lineage.

Dioxins are ubiquitous environmental poisons that are developmentally toxic. Exposure of mouse E14 tooth germs to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to reduced tooth size and deformation of cuspal morphology implying the induction of a variety of biological responses on both cellular and molecular levels.
To verify such responses at the gene level, mouse embryonic cap staged tooth germs were cultured for 24 h with/without 1 µM TCDD.

A Myc transcriptional program that is independent of EMT drives a poor prognosis tumor-propagating phenotype in HER2+ breast cancer

The HER2 (ERBB2) and MYC genes are commonly amplified genes in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self renewal and tumour propagating capability of cells transformed with Her2 and c-Myc. Co-expression of both oncogenes in cultured cells led to a pronounced activation of a c-Myc transcriptional signature and acquisition of a self renewing phenotype independent of an EMT programme or regulation of cancer stem cell markers. We show that HER2 and c-MYC are frequently co-amplified in a clinical breast cancer cohort and that co-amplification is strongly associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in patients receiving adjuvant chemotherapy (but not targeted anti-HER2 therapy), MYC amplification is associated with a poor outcome in HER2+ breast cancer patients. These findings demonstrate the importance of molecular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have important diagnostic and therapeutic consequences for the clinical management of HER2+ breast cancer.

Gene expression profiling of temporal lobes of wfs1 deficient mice

Aim of present study was to describe the changes induced deletion of the Wfs1 gene in the temporal lobe of mice. Mutant mice were backcrossed to two different genomic backgrounds in order to exclude confounding foreign genomic background influence. Samples from temporal lobes were analyzed by using Affymetrix Genechips, expression profiles were functionally annotated by using GSEA and Ingenuity Pathway Analysis. We found that Wfs1 mutant mice are significantly smaller (20.9 ± 1.6 g) than their wild-type counterparts (31.0 ± 0.6g, p < 0.0001). Interestingly, genechip analysis identified growth hormone transcripts up-regulated and functional analysis found appropriate pathways activated. Moreover, we found significant increase in the level of IGF1 in the plasma of wfs1 mutant mice. Taken together, wfs1 mutation induces growth retardation whereas the growth hormone pathway is activated. Further studies are needed to describe biochemical and molecular details of the growth hormone axis in the wfs1 mutant mice.

Expression data from human amnion mesenchymal cells treated with interleukin-1-beta for 1hr and 8hr

Premature birth continues to be a challenging pregnancy complication, and a body of literature indicates that inflammation can contribute to premature delivery by converting a receptive uterine environment to a hostile one.  Cytokines have been demonstrated to provoke up-regulation of inflammatory genes (e.g. interleukin-1, 6, and 8, tumor necrosis factor-alpha, cyclooxygenase-2, and microsomal prostaglandin E synthase-1).
Using monolayer cultures of human amnion mesenchymal cells (AMCs) as a model, we aimed to detail the global programme of gene expression that occurs in response to cytokine challenge.

Microarray Analysis of Retinal Gene Expression in the DBA/2J Model of Glaucoma

Purpose: The DBA/2J mouse is a model for secondary angle-closure glaucoma due to iris atrophy and pigment dispersion, which ultimately leads to increased intraocular pressure (IOP). We sought to correlate changes in retinal gene expression with glaucoma-like pathology by performing microarray analysis of retinal RNA from DBA/2J mice at 3 months before disease onset, and at 8 months, after IOP elevation. Methods: IOP was monitored monthly in DBA/2J animals by Tono-Pen and animals with normal (3 months) or elevated IOP (8 months) were identified. RNA was prepared from 3 individual retinas at each age, and the RNA was amplified and used to generate biotin-labeled probe for high density mouse Affymetrix arrays (U430.2). A subset of genes was selected for confirmation by quantitative RT-PCR using independent retina samples from DBA/2J animals at 3, 5 and 8 months of age, and compared to retinas from C57BL/6J control animals at 3 and 8 months. Results: There were changes in expression of 68 genes, with 32 genes increasing and 36 genes decreasing at 8 months versus 3 months. Upregulated genes were associated with immune response, glial activation, signaling and gene expression, while down-regulated genes included multiple crystallin genes. Significant changes in 9 upregulated genes and 2 downregulated genes were confirmed by quantitative RT-PCR, with some showing changes in expression by 5 months. Conclusions: DBA/2J retina shows evidence for glial activation and an immune-related response following IOP elevation, similar to what has been reported following acute elevation of IOP in other models.
Keywords: retina, glaucoma, DBA/2J, elevated intraocular pressure

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced inter-patient heterogeneity. To characterize RA at the molecular level and to uncover key pathomechanisms, we performed whole-genome gene expression analyses. Synovial tissues from rheumatoid arthritis patients were compared to those from to normal donors.

Lentiviral shRNA knockdown of PTK7 in lung adenocarcinoma cell lines NCI-H1299 and NCI-H2009

PTK7 was identified from a meta-analysis of 1905 non-small-cell lung cancer (NSCLC) samples across 12 datasets to be one of seven genes commonly up-regulated in lung adenocarcinoma (ADC). Using ADC cell lines NCI-H1299 and NCI-H2009, disruption of PTK7 resulted in decreased cell viability and induction of apoptosis. A xenotransplantation model of the cell lines with PTK7 knock-down also resulted in decreased tumor burden. We assayed gene expression in these cell lines after PTK7 knock-down by shRNA to uncover deregulated pathways and genes.

FOXA1 is a transcription factor which aids AR function in prostate. There is controversary over the effect of high FOXA1 level has on prostate cancer so we forced the overexpression in the LNCaP prostate cancer cell line.

Skeletal muscle gene expression after myostatin knockout in mature mice

RNA from 5 mice with postdevelopmental knockout of myostatin and 5 mice with normal myostatin expression was analyzed with comprehensive oligonucleotide microarrays. Myostatin depletion affected the expression of several hundred genes at nominal P < 0.01, but fewer than a hundred effects were statistically significant according to a more stringent criterion (false discovery rate < 5%). Most of the effects were less than 1.5-fold in magnitude. In contrast to previously-reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at lower levels in the myostatin-deficient muscles, and this led to reduced tissue collagen levels as reflected by hydroxyproline content. Myostatin knockout tended to down-regulate the expression of sets of genes with promoter motifs for Smad3, Smad4, myogenin, NF-κB, serum response factor, and numerous other transcription factors. Main conclusions: in mature muscle, myostatin is a key transcriptional regulator of collagen genes, but not genes encoding contractile proteins or genes encoding proteins involved in energy metabolism.

Tyrosine kinase-activating mutations in Met have been observed in hereditary papillary renal carcinomas as well as in other cancers. These mutations have been examined in several in vitro systems, where they cause constitutive Met activation, focus formation, and cell motility, and are tumorigenic in xenografts. To determine the influence of these mutations on tumorigenesis in vivo, we generated mice with targeted mutations in the murine met locus. Five mouse lines with mutant Met were created: wt, D1226N, Y1228C, M1248T, and M1248T/L1193V. These mouse lines develop a wide range of sarcomas, lymphomas, and carcinomas. To determine if mutationally activated forms of Met have altered signaling specificity we utilized gene expression analysis. Mouse embryonic fibroblasts (MEFs) were isolated from the mutant Met lines and here we present data for wild type and M1248T (M833). Our results indicate that Met activating mutations have unique gene expression signatures.
Keywords: mutant analysis

Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors [gene expression]

Cell type-specific master transcription factors (MTFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that all three subunits of the ubiquitous heterotrimeric CCAAT-binding NF-Y complex are required for the maintenance of embryonic stem cell (ESC) identity, and establish NF-Y as a novel component of the core pluripotency network. Genome-wide occupancy and transcriptomic analyses in ESCs and neurons reveal that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with MTFs. Mechanistically, NF-Y's distinctive DNA-binding mode promotes MTF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a novel function for NF-Y in promoting chromatin accessibility, and suggest that other proteins with analogous structural and DNA-binding properties may function in similar ways.

Expression profiles in normal and cancerous cervix tissue samples

Dioxin exposure of human CD34+ hemopoietic cells induces gene expression modulation that recapitulates its in vivo clinical and biological effects

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic disease, diabetes, infertility and cancer. We analysed the in vitro TCDD effects on human CD34+ cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 253 differentially modulated probe sets, identifying 217 well-characterized genes. A large part of these were associated with cell adhesion and/or angiogenesis and with transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway, were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34+ cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems.

Expression data from human liver with or without type 2 diabetes

The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secreted proteins, termed hepatokines.
To identify hepatic secretory proteins involved in insulin resistance, we performed liver biopsies in humans with or without type 2 diabetes and conducted a comprehensive analysis of gene expression profiles.

PCGF6, a polycomb group protein, regulates mesodermal lineage differentiation in murine ES cells and functions in iPS reprogramming

Polycomb group (PcG) proteins comprise a large group of evolutionary conserved factors with essential roles for embryonic development and adult stem cell function. PcG proteins constitute two main multiprotein polycomb repressive complexes (PRC1 and PRC2) that operate in a hierarchical manner to silence gene expression. Functionally distinct PRC1 complexes are defined by Polycomb group RING finger protein (PCGF) paralogs. So far, six PCGF paralogs (PCGF1-6) have been identified but paralog-specific functions are not well understood. In our studies, we observed that Pcgf6 showed the highest expression level in undifferentiated murine embryonic stem cells (ESCs), blastocysts and testes. When ESCs differentiated, Pcgf6 expression strongly declined. To further investigate Pcgf6 biology, we established dox-inducible shRNA knockdown (KD) ESCs. Following Pcgf6 KD in ESCs the expression of pluripotency genes decreased, while mesodermal- and spermatogenesis-specific genes were de-repressed. Concomitantly with the elevated expression of mesodermal lineage markers, Pcgf6 KD ESCs showed increased hemangioblastic and hematopoietic activities. Finally, PCGF6 replaced SOX2 but not KLF4 or c-MYC in the generation of germline-competent iPS cells. Forced expression of Pcgf6 in OSKM-driven reprogramming increases iPS efficiency while Pcgf6 KD reduces the formation of ESC-like colonies. Together, these analyses show that Pcgf6 is non-redundantly involved in maintaining the pluripotent nature of ESCs and functions in iPS reprogramming.

Expression profiling of the murine neural crest precursor cell line, JoMa1

JoMa1 cells are pluripotent precursor cells, derived from the neural crest of mice transgenic for tamoxifen-inducible c-Myc. Following transfection with a cDNA encoding for MYCN, cells become immortlized even in the absence of tamoxifen.

The E-protein transcription factors E2A and HEB play important roles at several stages of hematopoiesis. However, the exact mechanism for theire action and the main targets in the LY6D negative common lymphoid progentior (CLP) compartment remains unknown. By adressing this question, we will gain important infromation regarding the early events leading to B-cell specification.

Genome-wide gene expression analysis of the whole blood leukocyte transcriptional response to endotoxin treatment

Several inflammatory diseases respond to TNFα inhibitors, implicating TNFα signaling in the pathogenesis of these conditions. Here, we set out to map the systemic genome-wide transcriptome influenced by TNFα release. We performed an intravenous endotoxin challenge in 16 healthy subjects, half of whom were pretreated with the soluble TNF receptor fusion protein, etanercept. Whole-blood leukocyte total RNA was isolated using the PAXgeneTM tube and PAXgeneTM RNA isolation system (PreAnalytiXTM, Qiagen) as described by the manufacturer. Total RNA yield and purity (260nm:280nm) were determined spectrophotometrically. The integrity of the re-suspended total RNA was determined using the RNA Nano Chip Kit on the Bioanalyzer 2100 and the 2100 Expert software (Agilent). To increase the sensitivity of our gene expression assay, the overload of globin mRNA of whole blood samples was reduced by applying the human GLOBINclear kit (Ambion/Appied Biosystems). Synthesis, amplification and purification of anti-sense RNA was performed using 300 ng of enriched mRNA per sample and the Illumina TotalPrep RNA Amplification Kit (Ambion/Applied Biosystems) following the Illumina Sentrix Array Matrix expression protocol. A total of 750 ng biotinylated cRNA was hybridized onto the HumanHT-12v3 expression BeadChips (Illumina). The BeadChips were scanned according to the protocol described in the Illumina Whole Genome Gene Expression for BeadStation Manual v3.2, Revision A using scanning software BeadScan 3.5.31. The GenomeStudio® Data Analysis Software (Illumina) was used for data collection. Missing values were imputed by using the k-nearest-neighbor algorithm (k=15). The raw scan and imputed data were read using the lumi available through Bioconductor. This was carried out using the R statistical package (version 2.10.1; R Foundation for Statistical Computing, Vienna, Austria). Quality control checks of the raw non-normalized data included visualization of the similarity matrix and hierarchical clustering analysis. Samples for one placebo-treated individual did not pass our quality control checks, and thus were removed from subsequent normalization and analysis. Total RNA isolated from a collection of human tissue-derived cell lines was used as an internal control; this sample also was omitted from subsequent data normalization. Variance stabilizing normalization (Biconductor library vsn) was applied to all other samples. All subsequent analyses were performed on vsn-transformed intensity values.

Gene expression profiling of PBMC from normal individuals, chronic hepatitis B carriers and hepatocellular carcinoma patients

Aberrant gene expression analysis between peripheral blood mononuclear cell (PBMC) samples from healthy individuals and patients with chronic hepatitis B carriers and HCC were identified using Affymetrix gene arrays.

Polyglutamine disorders, Spinocerebellar ataxia type 7 (SCA7), Huntington’s disease (HD), Neuronal dysfunction, Retinal degeneration

Molecular pathways involved in neuronal degeneration of polyglutamine mouse models

To elucidate the pathways involved in the SCA7 retinal degeneration, we used DNA GeneChips and compared the gene expression profiles of the mutant mice (R7E) and the control ones (R7N). We performed these experiments at the onset stage of the disease (3w) as well as on the moderate one (9w) in order to correlate the transcriptional deregulations with the phenotype progression.
We also compared the R6/2 mice vs wild type (wt) ones to identify between the deregulated transcripts the ones that were also differentially expressed in the R7E vs R7N mice. These common changes are likely caused by polyQ expansion independently of the protein context.

Prognostic Significance and Gene Expression Profiles of p53 Mutations in Microsatellite Stable Stage III Colorectal Adenocarcinoma

Clinical Significance: Understanding the differences in colorectal cancer (CRC) aggressiveness and clinical outcomes in relation to tumor stage and different molecular subsets is at most important for designing treatment regimens. However, molecular signatures for specific phenotypic subsets that predict the aggressiveness and clinical outcomes of CRC, specifically in advanced disease stage are lacking. Therefore, for the first time, the current study has identified a set of molecular markers that are associated with aggressive Stage III CRCs that exhibited microsatellite stable and mutant p53 phenotypic features. These findings might aid in designing aggressive treatment regimens and help to provide insights into the development of novel therapeutic targets.
Results: Increased incidence of p53 mutations in MSS CRCs (58%) was associated with higher CRC-specific mortality than MSS-p53 wild-type phenotypes (log-rank, P=0.025; and hazard ratio, 2.52; 95% confidence interval, 1.25-5.08). Of 49 down-regulated genes, LPAR6, PDLIM3 and PLAT and of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8, were confirmed by qNPA, qRT-PCR, and IHC platforms.  Conclusions: p53 mutations are associated with poor prognosis of Stage III microsatellite stable CRCs.

microarray was done on Heart tissue from ko and wt
MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the dosage of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals is unknown, and reliable prediction of mRNA targets remains problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation.  Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

Comparison of mRNA expression in circulating T-cells from patients with severe asthma

Comparison of mRNA expression showed widespread changes in the circulating CD8+ but not CD4+ T-cells from patients with severe asthma. No changes were observed in the CD4+ and CD8+ T-cells in non-severe asthmatics versus healthy controls. Bioinformatics analysis showed that the changes in CD8+ T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs, we also observed widespread changes in expression of non-coding RNA species including natural antisense, pseudogenes, intronic long ncRNAs and long intergenic long ncRNAs in CD8+ T-cells from severe asthmatics. Measurement of the miRNA expression profile showed selective down-regulation of miR-28-5p in CD8+ T-cells and reduction of miR-146a and miR-146b in both CD4+ and CD8+ T-cells.

Differentially expressed genes and transcript isoforms in mouse NSCs expressing miR-128

We identify a brain-specific microRNA—miR-128—that represses Nonsense Mediated mRNA Decay (NMD) and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. We employed exon arrays for this analysis, as this platform detects expression levels of individual exons and thus allows detection of not only differentially expressed transcripts (DETs), but also alternative isoform transcripts (AITs). The latter is particularly relevant to our study because alternative RNA processing events (e.g., RNA splicing, alternative promoter usage, and alternative polyadenylation-site usage) often place a translation termination codon in a premature context and thereby trigger NMD.

Atrial Identity Is Determined by A COUP-TFII Regulatory Network (RNA)

Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP- TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T-tubules.
We used microarrays to examine the molecular profile of cardiomyocyte-specific COUP-TFII knockout adult atria in comparison with that of normal atria.

Transcriptome profiling of fenretinide-treated  and untreated CD34+ cells from 4 CML patients

Imatinib, as the first-line agent of chronic myeloid leukemia (CML), is ineffective in eradicating CML stem/progenitor cells, thus unable to prevent late relapse. Here we present data indicating that fenretinide preferentially targets CD34+ CML cells and enhances the efficacy of imatinib in CML. As tested by colony forming cell assays, both number and size of total colonies derived from CD34+ CML cells were significantly reduced by fenretinide, and by combining fenretinide with imatinib. In particular, colonies derived from erythroid progenitors and those derived from more primitive pluripotent progenitor cells were highly sensitive to fenretinide/fenretinide plus immtinib. Further data showed that fenretinide was able to induce apoptosis in CD34+ CML cells which were refractory to imatinib. Through transcriptome analysis and followed by molecular validation, we further showed that apoptosis induced by fenretinide in CD34+ CML cells was mediated by complex mechanisms of stress responses, probably triggered by elevated levels of intracellular reactive oxygen species. Thus, fenretinide combines with imatinib may represent a new strategy for the treatment of CML, in which fenretinide targets primitive CD34+ CML cells whereas imatinib targets leukemic blasts. This strategy may eventually reduce the risk of relapse and probably resistant as well in CML patients.

Comparison of HNF4 null mouse embryonic livers with control mouse embryonic livers

To study the role of hepatic nuclear factor alpha (HNF4a in hepatogenesis, we used loxP-Cre technology to eliminate it from developing mouse livers.
A comparison of control and experimental livers revealed that hepatocytes lacking HNF4a_failed to fully differentiate.  Specifically, HNF4a null liver cells failed to express genes that are markers of mature hepatocytes including Gys2, Pck1, and G6pc.  These cells also failed to form normal cellular junctions, which are critical for establishment of the hepatic epithelium [Parviz et al. (2003) Nat.Genet. 34, 292-96].  Because HNF4a functions as a transcription factor, we hypothesized that it regulates liver development by controlling the expression of genes whose products are essential in executing cellular differentiation and morphogenesis.  To identify these genes, we compared expression profiles between HNF4a null and wild type E18.5 fetal livers using Affymetrix gene arrays.  We identified 564 genes whose expression was decreased by at least 2.5-fold and 34 genes whose expression was increased by at least 2.5-fold when HNF4a was eliminated from hepatocytes.  These represent genes involved in diverse molecular pathways, reflecting the pleiotropic phenotype of HNF4a null livers. Our goal was to use the information from the gene arrays to define the mechanisms through which HNF4a controls the generation of the hepatic epithelium. We identified 27 genes from our list of downregulated genes with either defined or predicted roles in cellular junctions and/or adhesion.  Most striking was that loss of HNF4a altered the expression of genes encoding proteins involved in virtually all types of cellular junctions including tight junctions (Cldn1, Cldn-2, Cldn-12, Ocln, F11r/Jam1, Cxadr, Crb3), adherens junctions (Cdh1), desmosomes (Dsc2, Pkp2, Krt2-8), and gap junctions (Gjb1, Gjb2).  Using immunoblotting and immunohistochemistry, we found that the expression of proteins representing each of these junction types was reduced or absent.  Analysis of these genes for the presence of putative HNF4a binding sites revealed that 25 of 27 contain such sites.  We have used chromatin immunoprecipitation to confirm that HNF4a occupies several of these sites in vivo, including Cdh1, Cldn1, and F11r/Jam1.  From these data, we conclude that HNF4a is a central mediator of the fully differentiated hepatocyte gene expression program and that it orchestrates the expression of cell junction and adhesion proteins required for establishing the hepatic epithelium.

To define changes in gene expression resulting from loss of hnf4 in the small intestine.

Aging Liver Profiles of Long Lived Pit-1 Mutant Mouse (Snell) and Wild Type Controls

To compare and contrast genetic signatures from livers of young and aged Snell dwarf mice with their wild type controls.
Keywords: Aging

Comparison of gene expression in alveolar macrophages of normal non-smokers and normal smokers.

Overexpression of the Apoptotic Cell Removal Receptor, MERTK, in Alveolar Macrophages of Cigarette Smokers

Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AM) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AM obtained by bronchoalveolar lavage of normal non-smokers (n=11) and phenotypic normal smokers (n=13, 36 ± 6 pack-yr) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER Tyrosine Kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AM of smokers vs nonsmokers by TaqMan RT-PCR, immunohistochemistry, Western and flow analysis. Smoker AM had up-regulation of MERTK mRNA levels (smoker vs non-smoker, 3.6-fold by microarray, p<0.003; 9.5-fold by TaqMan RT-PCR, p<0.02). Immunohistochemistry demonstrated a qualitative increase in MERTK protein expression on AM of smokers. Increased protein expression of MERTK on AM of smokers was confirmed by Western and flow analyses (p< 0.007 and p< 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AM, and its expression is up-regulated in AM of cigarette smokers. This may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease (COPD), a disorder associated with dysregulated apoptosis of lung parenchymal cells.
Keywords: Comparison of gene expression in alveolar macrophages of normal non-smokers and normal smokers.

Childhood Absence Epilepsy, Juvenile Absence Epilepsy, Idiopathic Generalised Epilepsy, gene expression, twin study, monozygotic twins

Gene expression analysis in absence epilepsy using a monozygotic twin design

Objective:
To identify genes involved in idiopathic absence epilepsies by analysing gene expression using a monozygotic (MZ) twin design.
Methods:
Genome-wide gene expression in lymphoblastoid cell lines was determined using microarrays derived from five discordant and four concordant MZ twin pairs with idiopathic absence epilepsies and five unaffected MZ twin pairs.  Gene expression was analysed using three strategies: discordant MZ twins were compared as matched pairs, MZ twins concordant for epilepsy were compared to control MZ twins, and a singleton design of affected versus unaffected MZ twin individuals was used irrespective of twin pairing.  An overlapping gene list was generated from these analyses.  Dysregulation of genes recognised from the microarray experiment were validated using quantitative real time PCR (qRT-PCR) in the twin sample and in an independent sample of 18 sporadic absence cases and 24 healthy controls.
Results:
Sixty-five probe sets were identified from the microarray analysis strategies.  Sixteen genes were chosen for validation and nine of these genes confirmed by qRT-PCR in the twin sample.  Differential expression of the immediate early gene EGR1 and RCN2, coding for the calcium-binding protein Reticulocalbin 2, was re-confirmed by qRT-PCR in the independent sample.
Interpretation:
Using a unique sample of discordant MZ twins, our study identified genes with altered expression, which suggest novel mechanisms in idiopathic absence epilepsy.  Dysregulation of EGR1 and RCN2 might represent common transcriptional alterations in idiopathic absence epilepsy.

Genome-wide analysis of gene expression patterns in mir-122 knockout mice livers

To invesigate the physiology roles of mir-122 in liver, we performed expression profiling of mir-122 knockout mice and the control B6/129 mice.

Gene expression signature predictive of rapid response to imatinib mesylate in Gastrointestinal Stromal Tumor (GIST)

Gene expression profiling using oligonucleotide microarrays performed on tumor samples obtained before and after imatinib mesylate (IM) therapy.  Rapid responding (to IM) samples were compared to non-responding/stable disease samples as measured by CT scan measurements to identify a gene signature that can predict rapid response to IM.

Effect of Tgfbr2 disruption on gene expression in the aorta of Fbn1 wild-type and Fbn1C1039G mice

We used microarrays to characterize the global changes in gene expression within the ascending aorta of mice due to conditional disruption of TGF-β signaling in smooth muscle and/or due to heterozygous fibrillin-1 mutation.

Expression analysis of wild-type and Tfam heterozygous knockout (Tfam+/-) murine embryonic fibroblasts (MEFs).

The goal of this analysis was to utilize microarray profiling to identify basal alterations in gene expression in response to TFAM depletion and mtDNA stress.
Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein, TFAM (transcription factor A,mitochondrial), regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)–IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signaling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

Chemotherapy induced dynamic gene expression changes in vivo are prognostic in ovarian cancer

Carboplatin and paclitaxel are the most widely prescribed chemotherapeutic agents for ovarian cancer. Not all patients respond to treatment, so there is a need for biomarkers that reliably predict resistance in ovarian tumors. Expression of such biomarkers may be dynamically controlled. Gene expression was assessed for a period of 14 days after treatment with carboplatin or combined carboplatin-paclitaxel in xenografts from two ovarian cancer models: chemosensitive serous adenocarcinoma derived OV1002 and slow growing, chemoresistant HOX424 of clear cell origin. Tumour volume reduction was observed in both cell lines post treatment, with a more prominent effect in OV1002, which subsided in late time points. In OV1002, hierarchical clustering classified differentially expressed genes into four time-related patterns, upregulated and downregulated groups for each early and late expressed genes. Upregulated genes were involved in DNA repair, cell cycle and apoptosis, while downregulated groups were involved in oxygen consuming metabolic processes and apoptosis control. Carboplatin-paclitaxel treatment triggered a more comprehensive response. HOX424 responded only to the combined treatment, while the observed reduction in tumour growth was limited. Several apoptosis and cell cycle genes were upregulated, while Wnt signaling was downregulated in the exclusively late expression pattern observed in this cell line. Late downregulated gene groups post carboplatin-taxane treatment were capable of predicting overall survival in two independent clinical datesets. Pathways overrepresented in these clusters were also predictive of outcome. This longitudinal gene expression study may help characterization of chemotherapy response, identification of resistance biomarkers and guiding timing of biopsies.

In order to study the effects of Kdm2b binding at CpG islands, Kdm2b was knocked down in mouse embryonic stem cells using shRNA and gene expression profiled using Affymetrix arrays
Depletion of KDM2B affects the gene expression of a subset of polycomb targets

B-Raf inhibitors induce epithelial differentiation in BRAF mutant colorectal cancer cells

Mutations in the BRAF proto-oncogene, which encodes the B-Raf kinase, are associated with more aggressive, less-differentiated and therapy-resistant colorectal cancers (CRC). However, the molecular mechanisms responsible for these correlations remain unknown. Here, we report the characterization of human isogenic CRC cell line models (Caco-2, HT29, Colo-205) in which we modulate the expression of the B-RafV600E oncoprotein either by conditional cDNA or shRNA expression. Using these models in conventional and three dimensional tissue culture systems, we demonstrate that genetic depletion of endogenous B-RafV600E decreases cellular motility and invasion, while it induces hallmarks of differentiated epithelia such as the formation of functional adherens and tight junctions. Importantly, these effects are recapitulated by exposing these lines to B-Raf (PLX4720, vemurafenib, dabrafenib) or MEK inhibitors (trametinib). Furthermore, loss of endogenous B-RafV600E in HT29 xenografts does not only stall tumor growth, but also induces epithelial structures with marked expression of Cdx-2, a prognostic marker and master regulator of intestinal morphogenesis. By performing the first transcriptome profiles of B-Raf inhibitor treated 3D cultures of a primary adenocarcinoma and a metastasis derived CRC cell line, we establish functional links between B-RafV600E and proteins of known and potentially new prognostic relevance. We propose that B-Raf/MEK/ERK pathway inhibitors could be used to induce CRC differentiation and thereby to limit metastatic disease.

breast cancer / tamoxifen monotherapy (whole tissue tumor biopsies)

dataset of 60 patients with ER-positive primary breast cancer and treated with tamoxifen monotherapy for 5 years. Data were generated from whole tissue sections of breast cancers.
Sample_keyword: breast cancer, tamoxifen, recurrence

breast cancer / tamoxifen monotherapy (microdissected tumor biopsies)

dataset of 60 patients with ER-positive primary breast cancer and treated with tamoxifen monotherapy for 5 years. Data were generated from LCMed cancer cells.

A study of rat femoral fracture healing in young (6 weeks old at fracture), adult (26 weeks old at fracture), and old (52 weeks old at fracture) rats.  Samples were collected at time of surgery (intact controls) and at 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks after fracture.  Samples were the mid third of the femoral length including the external callus, cortical bone and marrow elements.  Fracture was stabilized with an intramedullary rod prior to fracture with a Bonnarens and Einhorn device.

Reversible phosphorylation is a critical step in the control of cellular signaling events. The protein phosphatase 1 (PP1) regulates neuronal proteins by mediating their de-phosphorylation. PP1 selectivity and subcellular localization is conferred via association with a panel of interacting proteins. We have extensively characterized the association of PP1 with inhibitor-1, an interacting protein widely expressed in brain and heart. Inhibitor-1 deletion results in a persistently active PP1 phosphatase in vitro. Inhibitor-1 overexpression results in increased transformation of cultured cells and enhances the rate of learning and memory in mouse models through largely unknown mechanisms. Significantly, inhibitor-1 activation is cAMP-sensitive. The genes regulated by inhibitor-1/PP1 signals have not been characterized.  Identification of these genes is expected to provide key insights into the role of inhibitor-1/PP1 escape from cellular homeostasis and regulation of neuronal learning and memory.
We will determine gene expression patterns in hippocampus of wild-type and inhibitor-1 knock-out mice. The results of these studies of constitutive expression will be used as a baseline for future studies aimed to determine the role cAMP-mediated gene expression in I-1 knock-out mice.
We hypothesize that the genes regulated by the inhibitor-1/PP1 holoenzyme are critical to cellular growth commitment including resistance to growth arrest and neuronal learning and memory
We will examine the role of inhibitor-1 in regulating hippocampal gene expression using total RNA isolated from whole hippocampi of inhibitor-1 knockout (I-1-/-) animals and parental C57Bl6 controls under non-stimulated conditions. Animals will be sacrificed by a standard protocol. Hippocampal tissue will be rapidly dissected from several animals, flash frozen within 1 minute of extraction, pooled, and total RNA will be processed immediately using TriZol.

Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation, development and DNA damage responses. Although two homologous Drosophila YY family members (pleiohomeotic (pho)) and pleiohomeotic-like (phol)) are redundant, the functional significance of a recently described mammalian YY1-like gene (YY2) is unknown. Using microarray and gene set enrichment analysis (GSEA), we found that lentiviral constructs containing short hairpin loop YY1- and YY2-specific inhibitory RNAs (shYY1 and shYY2) caused significant changes in both redundant and distinguishable expression patterns. Ribosomal protein genes were the most significant gene set up-regulated by both shYY1 and shYY2, although combined shYY1/shYY2 knockdowns were not additive. In contrast, shYY2 reversed anti-proliferative effects of shYY1 on E2F target genes, and shYY2 particularly altered UV damage response, platelet-specific genes and mitochondrial function genes. The most YY2-specific gene was the platelet glycoprotein CD36 whose ligand is thrombospondin - a key UV response gene. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Taken together, our studies show that YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously thought to uniquely respond to YY1. Functions of thrombospondin and CD36 in inflammation, atherogenesis, innate immunity and malaria pathogenesis reveal new potential regulatory roles for YY1 and YY2.

Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction

By using high-density oligonucleotide arrays, we profiled gene expression in reward-related brain regions of rats that developed escalated cocaine intake after extended access to cocaine (6 h per day). Rats allowed restricted daily access to cocaine (only 1 h) that displayed a stable level of cocaine intake and cocaine naive rats were used for controls. Four analysis methods were compared: Affymetrix microarray suite 4 and microarray suite 5, which use perfect-match-minus-mismatch models, and dchip and rma, which use perfect-match-only models to generate expression values. Results were validated by RT-PCR in individual animals from an independent replication of the experiment. A small number of genes was associated with escalated cocaine intake (ESC genes). Unexpectedly, of the brain regions examined [prefrontal cortex, nucleus accumbens, septum, lateral hypothalamus (LH), amygdala, and ventral tegmental area], the LH was the most transcriptionally responsive in escalation of cocaine intake. Most of the ESC genes identified are also expressed during synaptogenesis and synaptic plasticity and include genes that code for several presynaptic and postsynaptic proteins involved in neurotransmission. These results suggest that LH intrinsic circuitry undergoes a structural reorganization during escalation of cocaine use. This remodeling of LH circuitry could contribute to the chronic deficit in reward function that has been hypothesized to drive the transition to drug addiction. Results also support the value of using multiple analysis strategies to identify the most robust changes in gene expression and to compensate for the biases that affect each strategy.
Keywords: cocaine addiction

Comparison of PBMC transcriptional profiles in healthy subjects, patients with Crohn's Disease, and patients with Ulcerative Colitis
Keywords: Disease state

Integration of BRCA1-mediated miRNA and mRNA signatures reveal miR-146a, miR-99b and miR-205 regulation of the TRAF2 and NFkB pathways (mRNA dataset)

BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to the DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on the HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA-mRNA relationships associated to the presence of the BRCA1 gene. Our results demonstrate that integration of mRNA and miRNA associated to BRCA1 expression was useful to discover new miRNA-gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.

Global gene expression data analysis in Zfx-deficient murine ESC and HSC

Zfx controls the self-renewal of embryonic and hematopoietic stem cells

Stem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult bone marrow HSC, but did not affect erythromyeloid progenitors or fetal HSC. In both ESC and HSC, Zfx activated a common set of direct target genes. In addition, the loss of Zfx resulted in the induction of immediate-early and/or stress-inducible genes in both SC types but not in their differentiated progeny. These studies identify the first shared transcriptional regulator of ESC and HSC, suggesting a common molecular basis of self-renewal in embryonic and adult SC.
Keywords: Global gene expression data analysis in Zfx-deficient murine ESC and HSC

Che-1 is a RNA Polymerase II binding protein involved in the regulation of gene transcription. Che-1 emerges as an important adaptor that connects transcriptional regulation, cell-cycle progression, checkpoint control, and apoptosis.
We have observed that Che-1 depletion sensitizes cells to chemotherapy. We used microarrays analysis to investigate classes of genes regulated by Che-1.

Expression data from murine colon tissue after exposure to 4% DSS for 6 days DSS followed by 4 days of water or 1% L-arginine

L-Arginine (L-Arg) is the substrate for both inducible nitric oxide synthase and arginase, which are upregulated in human IBD and in mouse colitis models. We have found that L-Arg supplementation enhances wound restitution in vitro, and improves the clinical parameters of weight loss, survival, and colon weight/length, in dextran sulfate sodium (DSS) induced murine colitis. Our aim was to further identify the potential mechanisms underlying the clinical benefit of L-Arg supplementation.

The glycopeptide antibiotic vancomycin (VCM) represents one of the last lines of defense against methicillin-resistant Staphylococcus aureus infections. However, vancomycin is nephrotoxic, but the mechanism of toxicity is still unclear.
The goal of this study was twofold: (1) gain insights into the molecular mechanisms of vancomycin nephrotoxicity at the genomic level and (2) evaluate potential biomarkers (gene expression profiles) of vancomycin-induced kidney injury

Male Sprague-Dawley albino rats (Charles River) initially weighting approximately 200 grams were randomly assigned to one of the following:  1) olanzapine (2 mg/kg/day, I.P.) (N=20) or 2) Saline (N=20) and administered olanzapine or saline for 21 days.
Frontal cortex (N=4/group) was dissected and homogenized for microarray, QPCR and western blotting following previous methods (Fatemi et al. 2003; Brooks et al. 2003).
RNA isolation, cDNA preparation, microarray hybridization and QPCR followed previous methods (Brooks et al. 2003).

Gene expression profile of ATF6a and ATF6b knockout mice with dextran sulfate sodium (DSS)-induced colitis

To understand the role of ATF6a and ATF6b in non-canonical endoplasmic reticulum stress response pathway, we have employed whole genome  microarray expression profiling to identify the genes regulated by ATF6s. WIild type or, ER stress mediators,ATF6a or ATF6b-knockout mice were treated with 3% DSS for 3days. Genes responsible for DSS in each mediator-dependent manner were extracted and categorized by Gene Ontology. Among them, expression of three cell structure-related genes (Muc2, Muc3, Muc4) was quantified in the RNA samples from another mice treated with DSS by real-time PCR.

We used microarrays to assess transcriptional changes in regulatory T cells upon deletion of PTEN.

Gene expression in satellite cell-derived primary myoblasts islolated from Gata4-loxP mice. Myoblasts were infected with nLacZ for control (Wt), Cre recombinase to knockout GATA4 (KO), or GATA4 expression vector to overexpress GATA4 (OE). Infected myoblasts were cultured in growth medium (day 0) then differentiated into myotubes in differentiation medium for 3 days (day 3).

Gene expression analysis of hormone treated MCF7 breast cancer cells in the presence or absence of cycloheximide

Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and more than 1500 putative secondary target genes of estradiol in MCF7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide.
Keywords: drug treatment

Aims/hypothesis Due to their ability to regulate various signalling pathways (cytokines, hormones, growth factors), the suppressor of cytokine signalling (SOCS) proteins are thought to be promising therapeutic targets for metabolic and inflammatory disorders. Hence, their role in vivo has to be precisely determined.
Methods We generated transgenic mice constitutively expressing SOCS-3 in skeletal muscle to define whether the sole expression of SOCS-3 is sufficient to induce metabolic disorders and whether SOCS-3 is implicated in physiological roles distinct from metabolism.
Results We demonstrate here that chronic expression of SOCS-3 in skeletal muscle leads to overweight in mice and worsening of high-fat diet-induced systemic insulin resistance. Counter intuitively, insulin sensitivity in muscle of transgenic mice appears to be unaltered. However, following constitutive SOCS-3 expression, several genes had deregulated expression, among them other members of the SOCS family. This could maintain the insulin signal into skeletal muscle. Interestingly, we found that SOCS-3 interacts with calcineurin, which has been implicated in muscle contractility. In SOCS-3 transgenic muscle, this leads to delocalisation of calcineurin to the fibre periphery. Relevant to this finding, SOCS-3 transgenic animals had dilatation of the sarcoplasmic reticulum associated with swollen mitochondria and  decreased voluntary activity.
Conclusions/interpretation Our results show that constitutive SOCS-3 expression in skeletal muscle is not in itself sufficient to induce the establishment of metabolic disorders such as diabetes. In contrast, we reveal a novel role of SOCS-3, which appears to be important for muscle integrity and locomotor activity.
(Patricia Lebrun et al, Diabetologia, 2009)

Gene expression in the mouse embryonic small intestine in the presence or absence of E-cadherin

Gene expression was compared between E18.5 E-cadherin conditional knockout (cKO) small intestine and E18.5 control mouse small intestine.

Expression data from advanced stage of murine pancreatic ductal adenocarcinoma (PDAC) and control pancreas

We used microarrays to detail the global gene expression signature of PDAC and to identify distinct up- and down-regulated transcripts in these tumors compared to control pancreas. We also established from this dataset the metabolic signature of PDAC in order to define new metabolic therapeutic target for pancreatic cancer.

Human ischemic cardiomyopathy, idiopathic cardiomyopathy, and nonfailing controls

Left ventricular myocardium was snap-frozen at time of cardiac transplantation from patients with advanced idiopathic or ischemic cardiomyopathy, or at time of harvest from unused donor heart that serve as a nonfailing control.  No subjects received mechanical support devices.
Keywords: disease state analysis (case:control)

27-Hydroxycholesterol links cholesterol and breast cancer pathophysiology.

The cholesterol metabolite and SERM, 27HC, is the signaling molecule that links cholesterol to breast cancer pathophysiology
Hypercholesterolemia is a risk factor for breast cancer, and patients taking statins demonstrate lower breast cancer incidence and decreased breast cancer recurrence, data that highlights the potential importance of the recent finding that 27-Hydroxycholesterol (27HC), a primary metabolite of cholesterol, acts as a selective estrogen receptor modulator (SERM). The goal of this study was to evaluate the impact of 27HC on breast cancer pathophysiology. Elevation of 27HC in murine models increased tumor growth in an estrogen receptor dependent manner. Importantly, a high cholesterol diet decreased the time to tumor onset and increased tumor growth, and this response required presence of CYP27A1. Within human breast cancer samples, CYP27A1 expression increasesd with grade, in addition to being highly expressed in tumor associated macrophages. Finally 27HC increases metastasis to the lung. The findings herein strongly support a role for 27HC in breast cancer pathophysiology, providing support for the exploration of potential chemopreventative benefits of lower cholesterol diets, and pharmacological inhibitors of HMG-CoA reductase or CYP27A1.

analyzed changes in cytokine/chemokine production and gene expression levels in, human peripheral blood mononuclear cells upon teratment with 15µM,2,4-benzenetriol
Keywords: treatment with chemical compound

In situ synthesized oligo arrays, U74Av2, from Affymetrix were used to measure differential gene expression in RNA samples generated from the liver (GSE864) and spleen (GSE865) of Nrf2 knockout and wildtype mice at 5 months of age.

RNA of Kidney was extracted using a Quiagen RNeasy kit. Targets were produced using standard Affymetrix procedures from about 8ug of total RNA.

H3K27 Methyltransferase PRC2 Represses Wnt Genes to Facilitate Adipogenesis

The Wnt/b-catenin signaling inhibits adipogenesis. Genome-wide profiling studies have revealed the enrichment of histone H3K27 methyltransferase PRC2 on Wnt genes. However, the functional significance of such a direct link between the two types of developmental regulators in mammalian cells, and the role of PRC2 in adipogenesis, remain unclear. Here we show PRC2 and its H3K27 methyltransferase activity are required for adipogenesis. PRC2 directly represses Wnt1, 6, 10a and 10b genes in preadipocytes and during adipogenesis. Deletion of the enzymatic Ezh2 subunit of PRC2 eliminates H3K27me3 on Wnt promoters and de-represses Wnt expression, which leads to activation of Wnt/b-catenin signaling and inhibition of adipogenesis. Ectopic expression of the wild type Ezh2, but not the enzymatically inactive F667I mutant, prevents the loss of H3K27me3 and the defects in adipogenesis in Ezh2-/- preadipocytes. The adipogenesis defects in Ezh2-/- cells can be rescued by expression of adipogenic transcription factors PPARa, C/EBPb, or inhibitors of Wnt/b-catenin signaling. Interestingly, Ezh2-/- cells show marked increase of H3K27 acetylation globally as well as on Wnt promoters. These results indicate that H3K27 methyltransferase PRC2 directly represses Wnt genes to facilitate adipogenesis, and suggest that acetylation and trimethylation on H3K27 play opposing roles in regulating Wnt expression.

PTSD - Posttraumatic stress disorder.
33 samples taken from PMBCs of survivors of psychological trauma, in two time points: in ER, few hours after the truma, and four months later. Some of the patients devepled chronic PTSD (17 samples) and others recovered and set to be the Control group (16 samples). This is the normalized active genes: 4512 probes from U95A chip.
The raw data is available in series GSE845.
Samples are labeled with 3 tags: P/C for PTSD or Control, ER/M4 - for time point of sample, D/ND for Decrement or Non-decrement symptoms over time. (e.g. sample 23C-M4-D-Norm was taken 4 months after trauma from patient 23 which belongs to the control group and showed decrease in symptoms) . Samples include the expression value, the GeneBank accession number and Affymetrix indication of valid calls.

Expression data from embryonic stem cells following siRNA transfection of UPS members [self_renewal]

While transcriptional regulation of stem cell self-renewal and differentiation has been extensively studied, only a small number of studies have addressed the roles for post-translational modifications in these processes.  A key mechanism of post-translational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Using UPS-targeted RNAi screens, we identify novel regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme, Psmd14, and the E3 ligase, Fbxw7, and characterize their importance in ES cell pluripotency and cellular reprogramming.

mouse;kidney;low phosphate diet;Hyp;Phex;X-linked hypophosphatemia;repeat sample

Mouse Kidney - Effect of Low Phosphate Diet in Normal and Hyp Mice

This is a study of the change in gene expression in mouse kidney after feeding control (1.0% P) or low phosphate diet (0.03% P) for 3 or 5 days to normal or Hyp (X-linked hypophosphatemic) mice at 5 weeks of age.  The mice were C57BL/6J.  Normal wild-type mice, hemizygous Hyp (Hyp/Y) male mice, or heterozygous (Hyp/+) female mice were used.  The gene is dominant, so that the two genders are equally affected with low renal retention of phosphate and severity of their bone disease.  RNA from 3 mice were pooled for each microarray.  The arrays were processed as described in the Affymetrix GeneChip Expression Analysis Technical Manual (copyright 2001, Affymetrix, Inc., Santa Clara, CA), rev. 1, Part number 701021.  The data for each array were scaled to an average signal value of 500 for all genes on the array.
Keywords = mouse

Transcriptome analyses in normal prostate epithelial cells following exposure to low-dose cadmium

BACKGROUND: Cadmium is implicated in prostate carcinogenesis, but its oncogenic action remains unclear.
OBJECTIVES: In this study we aimed to decipher changes in cell growth and the transcriptome in an immortalized human normal prostate epithelial cell line (NPrEC) following exposure to low-dose Cd.
METHODS: Synchronized NPrEC cells were exposed to different doses of Cd and assayed for cell viability and cell-cycle progression. We investigated changes in transcriptome by global profiling and used Ingenuity Pathways Analysis software to develop propositions about functional connections among differentially expressed genes. A neutralizing antibody was used to negate the effect of Cd-induced up-regulation of tumor necrosis factor (TNF) in NPrEC cells.
RESULTS: Exposure of NPrEC to 2.5 μM Cd enhanced cell viability and accelerated cell-cycle progression. Global expression profiling identified 48 genes that exhibited ≥ 1.5-fold changes in expression after 4, 8, 16, and 32 hr of Cd treatment. Pathway analyses inferred a functional connection among 35 of these genes in one major network, with TNF as the most prominent node. Fourteen of the 35 genes are related to TNF, and 11 exhibited an average of > 2-fold changes in gene expression. Real-time reverse transcriptase-polymerase chain reaction confirmed the up-regulation of 7 of the 11 genes (ADAM8, EDN1, IL8, IL24, IL13RA2, COX2/PTGS2, and SERPINB2) and uncovered a 28-fold transient increase in TNF expression in Cd-treated NPrEC cells. A TNF-neutralizing antibody effectively blocked Cd-induced elevations in the expression of these genes.
CONCLUSIONS: Noncytotoxic, low-dose Cd has growth-promoting effects on NPrEC cells and induces transient overexpression of TNF, leading to up-regulation of genes with oncogenic and immunomodulation functions.
KEY WORDS: carcinogenesis, cytokine, global expression profiling, heavy metals, immune response, inflammation, Ingenuity Pathway Analysis, knowledge-based analysis, prostate cancer.

Runx1 is a tumor suppressor gene in the mouse gastrointestinal tract

The Runx1 transcription factor plays an important role in tissue homeostasis through its effects on stem/progenitor cell populations and differentiation. The effect of Runx1 on epithelial differentiation of the secretory cell lineage of the colon was recently demonstrated. This study aimed to examine the role of Runx1 in tumor development in epithelial cells of the gastrointestinal tract. Conditional knockout mice were generated that lacked Runx1 expression in epithelial cells of the GI tract. These mice were crossed onto the ApcMin background, sacrificed, and their intestinal tumor phenotypes were compared with ApcMin Runx1 wildtype control mice. Apc-wildtype Runx1-mutant mice were also examined for tumor development. Colons from Runx1 knockout and wildtype mice were used for genome-wide mRNA expression analyses followed by gene-specific quantitative RT-PCR of whole colon and colon epithelium, to identify Runx1 target genes. Runx1 deficiency in intestinal epithelial cells significantly enhanced tumorigenesis in ApcMin mice. Notably, epithelial Runx1 deficiency in Apc-wildtype mice was sufficient to cause tumor development. Absence of Runx1 was associated with global changes in expression of genes involved in inflammation and intestinal metabolism, and with gene sets indicative of metastatic phenotype and poor prognosis. Gene-specific analysis of Runx1 deficient colon epithelium revealed increased expression of genes linked to an expansion of the stem/progenitor cell population. These results identify Runx1 as a novel tumor suppressor gene for gastrointestinal tumors and support a role for Runx1 in maintaining the balance between the intestinal stem/progenitor cell population and epithelial differentiation of the GI tract.

Genome-wide expression profile of human trabecular meshwork cultured cells, non-glaucomatous and POAG tissue

The goal of this study was to contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells, to that of control and primary open angle glaucoma (POAG) HTM tissues.

Total RNA from cultured HTM cells and HTM tissue dissected from control and POAG anterior segments fixed in RNA later™ was linearly amplified with the OvationTM Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using the GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by Quantitative real-time PCR in nonamplified RNA.

Cultured HTM cells retained the expression of some genes characteristic of the HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants, paraoxonase 3 and ceruloplasmin.

Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma.

Nuclear orphan receptor TAK1/TR4-deficient mice are protected against obesity-linked inflammation, hepatic steatosis, and insulin resistance

The nuclear receptor TAK1/TR4/NR2C2 is expressed in several tissues that are important in the control of energy homeostasis. TAK1-deficient (TAK1-/-) mice are resistant to the development of age- and high fat diet (HFD)-induced metabolic syndrome. Biochemical analysis showed significantly lower hepatic triglyceride levels and reduced lipid accumulation in adipose tissue in TAK1-/- mice compared to wild type (WT) mice. Gene expression profiling analysis revealed that the expression of several genes encoding proteins involved in lipid uptake and triglyceride synthesis and storage, including Cidea, Cidec, Mogat1, and CD36, was greatly decreased in the liver of TAK1-/- mice. Moreover, TAK1-/- mice exhibit reduced infiltration of inflammatory cells and expression of inflammatory genes in adipose tissue and were resistant to the development of glucose intolerance and insulin resistance. TAK1-/- mice consume more oxygen and produce more carbon dioxide than WT mice suggesting a higher rate of energy expenditure. Together, these results indicate that TAK1 plays a critical role in the regulation of energy and lipid homeostasis and potentiates the development of metabolic syndrome. Our study suggests that TAK1 might provide a novel therapeutic target in the management of metabolic syndrome.

Colon cancer cell lines with partial sensitivity to the BRAF inhibitor PLX4720 were grown in increasing concentration of the drug to develop acquired resistance.  Gene expression was performed for comparison of the resistant clones to the parental lines.

Human inositol polyphosphate multikinase regulates transcript-selective nuclear mRNA export to preserve genome integrity

Messenger (m)RNA export from the nucleus is essential for eukaryotic gene expression. Here, we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enriched for functions in genome duplication and repair, controlled by inositol polyphosphate multikinase (IPMK), an enzyme catalyzing inositol polyphosphate and phosphoinositide turnover. We studied transcripts encoding RAD51, a protein essential for DNA repair by homologous recombination (HR), to characterize the mechanism underlying IPMK-regulated mRNA export. IPMK depletion or catalytic inactivation selectively decreases the nuclear export of RAD51 mRNA, and RAD51 protein abundance, thereby impairing HR. Recognition of a sequence motif in the untranslated region of RAD51 transcripts by the mRNA export factor ALY requires IPMK. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an IPMK product, restores ALY recognition in IPMK-depleted cell extracts, suggesting a mechanism underlying transcript selection. Our findings implicate IPMK in a transcript-selective mRNA export pathway controlled by phosphoinositide turnover that preserves genome integrity in humans.

Tumorigenesis is characterised by changes in transcriptional regulation and the androgen receptor (AR) has been identified as a key driver in prostate cancer. In this study, we show that the hexosamine biosynthetic pathway (HBP) genes are overexpressed in clinical prostate cancer and androgen-regulated in cell-lines. HBP senses metabolic status of the cell and produces an essential substrate for O-GlcNAc transferase (OGT), which regulates target proteins via glycosylation. Using immunohistochemistry, we found that OGT is up-regulated in the protein level in prostate cancer (n=1987) and its expression correlates with high Gleason Score, pT and pN stages and biochemical recurrence (for all, p<0.0001). Both a small molecule inhibitor and siRNAs targeting OGT decreased prostate cancer cell growth. Microarray profiling revealed that the principal effects of the OGT inhibitor in prostate cancer cells are on cell cycle progression and DNA replication. We identified MYC as a candidate upstream regulator of these genes and found that OGT inhibitor caused a dose-dependent loss of c-MYC protein but not mRNA in cell lines. Finally, we observed a statistically significant co-expression between c-MYC and OGT in human prostate cancer samples (n=1306, p=0.0012).

To investigate genes that might influence resistance to infection through IGF1R, we screened human breast cancer-derived OCUB-M cells transfected with expression vector encoding IGF1R using microarray analysis.
Keywords: overexpression comparison

Targeting the Phosphoinositide 3-Kinase p110α Isoform Impairs Cell Proliferation, Survival and Tumor Growth in Small Cell Lung Cancer

Purpose: The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study we investigated the potential of targeting the catalytic class IA PI3K isoforms in small cell lung cancer (SCLC), which is the most aggressive of all lung cancer types.  Experimental Design: The expression of PI3K isoforms in patient specimens was analyzed. The effects on SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by selective inhibitors or down-regulation by small interfering RNA.  Results: Over-expression of the PI3K isoforms p110α and p110β was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110α with RNA interference (RNAi) or selective pharmacological inhibitors resulted in strongly affected cell proliferation of SCLC cells in vitro and in vivo, while targeting p110β was less effective. Inhibition of p110α also resulted in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt and components of the mammalian target of rapamycin (mTOR) pathway, such as the ribosomal S6 protein, and the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). A DNA microarray analysis revealed that p110α inhibition profoundly affected the balance of pro- and anti-apoptotic Bcl-2 family proteins. Finally, p110α inhibition led to impaired SCLC tumor formation and vascularization in vivo.  Conclusion: Together our data demonstrate the key involvement of the PI3K isoform p110α in multiple tumor-promoting processes in SCLC.

Despite over 3,000 articles published on dystrophin in the last 15 years, the reasons underlying the progression of the human disease, differential muscle involvement, and disparate phenotypes in different species are not understood. The present experiment employed a screen of 12,488 mRNAs in 16-wk-old mouse mdx muscle at a time when the skeletal muscle is avoiding severe dystrophic pathophysiology, despite the absence of a functional dystrophin protein. A number of transcripts whose levels differed between the mdx and human Duchenne muscular dystrophy were noted. A fourfold decrease in myostatin mRNA in the mdx muscle was noted. Differential upregulation of actin-related protein 2/3 (subunit 4), beta-thymosin, calponin, mast cell chymase, and guanidinoacetate methyltransferase mRNA in the more benign mdx was also observed. Transcripts for oxidative and glycolytic enzymes in mdx muscle were not downregulated. These discrepancies could provide candidates for salvage pathways that maintain skeletal muscle integrity in the absence of a functional dystrophin protein in mdx skeletal muscle.

lung, Chlamydia pneumoniae, mice, myd88, TLR, infection, knock out

MyD88-dependent changes in the pulmonary transcriptome after infection with Chlamydia pneumoniae

Chlamydia pneumoniae, an obligate intracellular bacterium, causes pneumonia in humans and mice. Toll-like receptors and the key adaptor molecule MyD88 play a critical role in inducing immunity against this microorganism and are crucial to survive the infection. To explore the influence of MyD88 on induction of immune responses in vivo on a genome wide level, WT or MyD88-/- mice were infected with C. pneumoniae upon anesthesia and the pulmonary transcriptome was analyzed three days later by microarrays. We find that the infection induced the transcription of 360 genes and repressed 18 genes in WT mice. Of these, 221 genes were not or weakly induced in lungs of MyD88-/- mice. This cluster contains primarily genes encoding for chemokines, cytokines and other immune effector molecules. Genes induced by interferons were abundant in a cluster of 102 genes which were only partially MyD88-dependent. Interestingly, a set of 37 genes were induced more strongly in MyD88-/- mice and most of them are involved in the regulation of cellular replication. In summary, ex vivo analysis of the pulmonary transcriptome upon infection with C. pneumoniae demonstrated a major impact of MyD88 on inflammatory responses but not on interferon-type responses, and identified MyD88-independent genes involved in cellular replication
Keywords: lung, Chlamydia pneumoniae, mice, myd88, TLR, infection, knock out

Estrogen-regulated gene expression in human osteosarcoma cells stably transfected with estrogen receptor alpha or beta

Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable to those in osteoblasts, and we characterized their response to estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the ca. 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48 h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.

This study aims to investigate age-specific, time-dependent changes in gene expression that may underlie the priming effect of early-life seizures by looking at the sequence of gene expression patterns in the hippocampus at various times following Kainate induced seizures at postnatal day (P) 15.

Analysis of Foxp3 ablated peripheral regulatory T cells. Regulatory T cells require the expression of the transcription factor Foxp3 for thymic development. It is not known whether continuous expression of Foxp3 is required for the maintained function of mature regulatory T cells in the periphery. Results indicate changes to the regulatory T cell developmental program in the absence of Foxp3.

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases.
Keywords: treated vs control

Uterine Epithelial Cells Specific Estrogen Receptor alpha-Dependent Gene Expression Profiles in Response to Estrogen

Estrogens stimulate hypertrophy and hyperplasia in the uterus and exert their activity through estrogen receptor α (ERα). A uterine epithelial ERα conditional knockout mouse model (Wnt7aCre+;Esr1f/f or cKO) demonstrated that ERα in the epithelial cells was dispensable for an early uterine proliferative response to 17β-estradiol (E2), but required for subsequent uterine biological responses. We compared the gene expression profile in the uterus after E2 treatment in the cKO samples with WT samples. We found that approximately 25% of the genes differentially expressed at 2 h were epithelial ERα independent, as they were preserved in the cKO, indicating they are mediated from the stroma and sufficient to promote initial proliferative responses. However, more than 90% of the differentially expressed transcripts at 24 h were absent in the cKO, indicating the majority of later transcriptional regulation required epithelial ERα and suggesting the loss of regulation of these later transcripts results in the blunted growth response 3 days after treatment. These transcription profiles correlate with our previous biological responses, in which the initial proliferative response is independent of epithelial ERα but dependent on stromal ERα, yet epithelial ERα is essential for subsequent tissue responsiveness. These analyses are now allowing for in vivo determination of the cell specific actions of ERα in the female reproductive tract.

Knockdown of breast cancer master regulators: siRNA targeting PTTG1 and SPDEF in MCF-7 cells.

Genome-wide association studies for breast cancer have identified over 80 different risk regions in the genome, with the FGFR2 locus consistently identified as the most strongly associated locus. However, we know little about the mechanisms by which the FGFR2 locus mediates risk or the pathways in which multiple risk loci may combine to cause disease.  Here we use a systems biology approach to elucidate the regulatory networks operating in breast cancer and examine the role of FGFR2 in mediating risk. Using model systems we identify FGFR2-regulated genes and, combining variant set enrichment and eQTL analysis, show that these are preferentially linked to breast cancer risk loci. Our results support the concept that cancer-risk associated genes cluster in pathways

NANOG-OCT4-SOX2 Regulatory Module in Human Embryonic Stem Cells (dataset 1)

YAP knockdown in HUVEC elicits proliferation and cell cycle preogression defects. YAP deficient cells caused arrest in G1 and defects in S-phase entry. The microarray analysis was conducted to identify potential YAP targets that are involved in HUVEC cell cycle regulation

miRNA regulate gene expression at the post-transcriptionnal level. To gain further insight into this process, we analysed by Affymetrix microarray, the transcriptome of Dicer WT or Dicer deleted mouse CD4 T cells.

Sepsis gene expression profiling: murine splenic compared with hepatic responses

The aim of this series of experiments was to gain experience in sepsis gene expression profiling in a well-accepted model of murine polymicrobial abdominal sepsis and begin characterizing (in the parlance of genomics) the sepsis "transcriptome." DESIGN: Prospective animal study. SETTING: University-based animal research facility.SUBJECTS C57BL/6 mice. INTERVENTIONS: After induction of general anesthesia, cecal ligation and puncture were performed to induce peritonitis and polymicrobial sepsis. The control group had sham laparotomy only. Three samples of spleen and liver were collected from septic and sham animals at 24 hrs after laparotomy. Changes in expression were measured for 588 annotated mouse genes by using a commercially available complementary DNA microarray kit. MEASUREMENTS AND MAIN RESULTS: Broad-scale gene expression profiles were characterized for septic liver and spleen and compared with sham controls. The analytical tools used included commercially available software packages and a novel analysis program. Very little overlap was observed in the septic gene expression profiles of these two organs. Most of the genes identified have previously been linked to regulation of the inflammatory response; importantly, however, some have not. In addition, hierarchical cluster analysis showed that cecal ligation and puncture at 24 hrs induced coordinate expression of genes that alter cell signaling and survival pathways in spleen, consistent with previously published reports of sepsis-induced splenocyte apoptosis. The current limitations of microarray analysis as reflected in these studies are also discussed. CONCLUSIONS: Microarray technology provides a powerful new tool for rapidly analyzing tissue-specific changes in gene expression induced by sepsis in animal models.

Peripheral Blood Mononuclear Cells of Rhesus Macaques Infected with SIVmac251

Using the Affymetrix HuGeneFL GeneChip, the global expression patterns of genes in the peripheral blood mononuclear cells (PBMCs) of rhesus macaques, infected with SIVmac251 and exhibiting rapid, typical, or slow rates of disease progression, were examined. Assessments of the change in gene expression (fold change), the temporal coordination of gene expression (self-organizing map analysis), and the similarities and significant differences in gene expression across the groups were performed on samples taken before infection and 3 and 7 weeks postinfection. An upregulation of the p27 interferon-inducible gene and of genes associated with cellular activation and immune response was observed in all three groups. Rapidly progressing animals exhibited a modest number of genes with a change in expression of 3-fold or greater, typically progressing animals exhibited the greatest number, and slowly progressing animals exhibited the fewest. Self-organizing map cluster analysis indicated that rapidly progressing animals exhibited the least coordinated gene expression over the three study time points, typically progressing animals exhibited a moderate degree, and animals with slow progression exhibited the most coordinated gene expression. Mann-Whitney U analysis indicated that differences in gene expression were most pronounced between the rapidly and slowly progressing groups and least pronounced between the rapidly and typically progressing animals. These observations elucidate distinct features of gene expression in animals with different rates of disease progression.

Mast cell expression data from murine aryl hydrocarbon receptor (AhR) null, AhR_d and normal control mice

We found that AhR, a unique chemical sensor, is critical in controlling mast cell differentiation, growth and function in vitro and in vivo. This study seeks to further the understanding of the mechanisms of how AhR control mast cell homeostasis.

Genome-wide expression analysis of VprBP knockdown in DU145 cells

VprBP has been implicated in transcriptionally silent chromatin formation and cell cycle regulation, but the molecular basis underlying such effects remains unclear. To investigate the role of VprBP on gene regulation, genme-wide gene expression analysis is carried out in DU145 cells expressing either control shRNA or VprBP shRNA.

PAX5 overexpression is not enough to reestablish the mature B-cell phenotype in classical Hodgkin lymphoma

In lymphomas derived from mature B cells the expression of the transcription factor PAX5 is maintained whereas classical Hodgkin lymphoma displays significantly reduced PAX5 expression despite its derivation from mature B cells. To elucidate the functional role of PAX5 in classical Hodgkin lymphoma, we re-established the PAX5 expression in the Hodgkin cell line L428 with and without epigenetic modulation. To this end, we stably transfected the Hodgkin cell line L428 with an inducible PAX5 expression construct. Although the overexpressed PAX5 was transcriptionally active as demonstrated by synthetic reporter constructs, no induction of the B-cell phenotype was achieved. PAX5 chromatin immunoprecipitation with subsequent next generation sequencing in B-cell lines and the PAX5 overexpressing L428 cell line showed different binding patterns. Since epigenetic restrictions might affect PAX5 binding, combined DNA demethylation and histone acetylation was performed. However, no re-expression of B-cell genes was observed also under these conditions. Thus, PAX5 is not sufficient for the re-activation of the B-cell program in Hodgkin cells despite epigenetic opening of the chromatin. This clearly indicates that the repression of the B-cell identity of the Hodgkin cells is caused and secured by complex molecular mechanisms.

The non-steroidal anti-inflammatory drugs Sulindac sulfide and Diclofenac induce apoptosis and differentiation in human acute myeloid leukemia cells through an AP-1 dependent pathway

Acute myeloid leukemia is a heterogeneous disease with regard to the underlying genetic and molecular pathophysiology. Non-steroidal anti-inflammatory drugs (NSAIDs) exert significant anti-proliferative effects in various malignant cells in vitro and in vivo. Hence, these agents can be utilized to study potential disease specific anti-proliferative pathways. In this study, a total number of 42 bone marrow derived CD34+ cells from de novo AML patients and the AML cell lines THP-1 and HL-60 were treated with the NSAIDs Sulindac sulfide and Diclofenac. We examined viability, apoptosis, differentiation and addressed the molecular mechanisms involved. We found a consistent induction of apoptosis and to some extent myeloid differentiation in NSAID treated AML cells. Comprehensive protein and gene expression profiling of Diclofenac treated AML cells revealed transcriptional activation of GADD45α and its downstream MAPK/JNK pathway as well as increased protein levels of the Caspase-3 precursor. This points towards a role of the c-Jun NH2-terminal kinase (JNK) in NSAID mediated apoptosis. This was dependent on JNK activity as addition of a specific JNK-inhibitor abrogated apoptosis. Furthermore, the AP-1 transcription factor family members’ c-Jun, JunB and Fra-2 were transcriptionally activated in NSAID treated AML cells. Re-expression of these transcription factors led to activation of GADD45α with induction of apoptosis. Mechanistically, we demonstrate that NSAIDs induce apoptosis in AML through a novel pathway involving increased expression of AP-1 heterodimers, which by itself is sufficient to induce GADD45α expression with consecutive activation of JNK and induction of apoptosis.

The Histone H2B Monoubiquitination Regulatory Pathway is Required for Differentiation of Multipotent Stem Cells

Extensive changes in post-translational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs), various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSC. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSC cells and plays a central role in controlling stem cell differentiation.

Gene expression profiling in arterial tissue from type 2 diabetic patients

Genexpression profiling of wild type, HIF-1α knockdown, or HIF-2α knockdown HepG2 tumor spheroids

10 days old tumor spheroids were processed for RNA isolation using the Quiagen RNeasy Micro Kit and cDNA synthesis was done using the Ambion WT Expression Kit. Wt, HIF-1α k/d and HIF-2α k/d samples were compared to each other.

Dominant Th1 and Minimal Th17 Skewing in Discoid Lupus Revealed by Transcriptomic Comparison with Psoriasis

Discoid lupus erythematosus (DLE) is the most common skin manifestation of lupus. Despite its high frequency in systemic lupus in addition to cases without extracutaneous manifestations, targeted treatments for DLE are lacking, likely because of a dearth of knowledge of the molecular landscape of DLE skin. Here, we profiled the transcriptome of DLE skin in order to identify signaling pathways and cellular signatures that may be targeted for treatment purposes. Further comparison of the DLE transcriptome with that of psoriasis, a useful reference given our extensive knowledge of molecular pathways in this disease, provided a framework to identify potential therapeutic targets. Although a growing body of data support a role for IL-17 and T helper type 17 (Th17) cells in systemic lupus, we show a relative enrichment of IFN-γ-associated genes without that for IL-17-associated genes in DLE. Extraction of T cells from the skin of DLE patients identified a predominance of IFN-γ-producing Th1 cells and an absence of IL-17-producing Th17 cells, complementing the results from whole-skin transcriptomic analyses. These data therefore support investigations into treatments for DLE that target Th1 cells or the IFN-γ signaling pathway.

The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis

Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from DSS-induced murine colitis. While tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal Disease Activity Index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation

Transcriptome Analysis Identifies Fn14, a TNF Superfamily Receptor Member, as a Therapeutic Target in Alcoholic Hepatitis

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease and occurs in patients with excessive alcohol intake It is characterized by marked hepatocellular damage, steatosis and pericellular fibrosis. Patients with severe AH have a poor short-term prognosis. Unfortunately, current therapies (i.e. corticosteroids and pentoxyphylline) are not effective in many patients and novel targeted therapies are urgently needed. The development of such therapies is hampered by a poor knowledge of the underlying molecular mechanisms. Based on studies from animal models, TNF alfa was proposed to play a pivotal role in the mechanisms of AH.   Consequently, drugs interfering TNF alfa were tested in these patients. The results were disappointing due to an increased incidence of severe infections. Unluckily, there are not experimental models that mimic the main findings of AH in humans. To overcome this limitation, translational studies with human samples are required. We previously analyzed samples from patients with biopsy-proven AH. In these previous studies, we identified CXC chemokines as a potential therapeutic target for these patients. We expanded these previous observations by performing a high-throughout transcriptome analysis.

KRAP (Ki-ras-induced actin-interacting protein) is a cytoskeleton-associated protein and a ubiquitous protein among tissues, originally identified as a cancer-related molecule.  KRAP-deficient (KRAP-/-) mice show enhanced metabolic rate, decreased adiposity, improved glucose tolerance, hypoinsulinemia and hypoleptinemia.  KRAP-/- mice are also protected against high-fat diet-induced obesity and insulin resistance despite of hyperphagia.

Expression profiles in aortas isolated from mouse strains C3H.2/HeJ and C57.2Bl/6. Mice were were either on a high fat diet or normal diet for 0, 4, 10, 24, or 40 weeks.

Microarray gene expression data from Hdh knock-out, wild-type and knock-in embryonic stem cells

Huntington's disease (HD) features a unique disease-initiating mechanism hypothesized to entail an impact of the CAG repeat encoded polyglutamine region on the full-length huntingtin protein, with dominant effects that are continuous with CAG size, in a simple gain of function. To evaluate these predictions, we generated a series of heterozygous Hdh CAG knock-in mouse embryonic stem (ES) cell lines, with 18, 48, 89, 109 CAGs, and found that a continuous analytic strategy efficiently identified, from genome-wide datasets, 73 genes and 172 pathways whose expression varied continuously with CAG length. The CAG-correlated genes were distinct from the set of 754 genes that distinguished huntingtin null ES cells from wild-type controls, and CAG-correlated pathways did not display a one-to-one correspondence with the 238 pathways altered in huntingtin null ES cells. Rather, the genes that varied with CAG size were either members of the same pathways as altered genes in huntingtin null cells or were members of unique pathways related to these pathways. These findings falsified a gain of function/loss of function proposal but were consistent with the simple gain of novel function mechanism hypothesis. The dominant CAG correlated gene expression changes conformed to the genetic features of the HD initiating mechanism and were system-wide and inter-related with pathways perturbed by lack of full-length huntingtin function, urging system-wide approaches for the discovery and validation of potential modulating factors, in the search for effective HD therapeutics.

Expression data from Ribonuclease Inhibitor (RI/Rnh1) deficient  E9.5 yolksac

Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However the entire biological role for Rnh1 is unknown. We generated Rnh1 knock out mice by homologous recombination. Here we studied differential gene expression from wild type (Rnh1 +/+), Heterozygous (Rnh1+/-) and Knock out (Rnh1-/-) yolk sacs isolated from embryonic day 9.5 (E9.5).
We used microarrays to study global gene expression regulated by Rnh1 in yolk sacs.

Mn-SOD;manganese superoxide dismutase;knockout;heart failure;heart;muscle;parallel sample

To investigate the pathological significance of excess oxidative stress in the heart, we generated heart/muscle-specific manganese superoxide dismutase (MnSOD) -deficient mice on a C57BL/6 background using the Cre-loxP system uder the control of the muscle reatin kinase (MCK) promoter. The mutant mice developed progressive congestive heart failure with dilated cardiomyopathy and physical disability and died with a median survival rate of 15.4 ± 4.0 weeks. The pathological changes in our model were compatible with human cardiac aging. To investigate the transcriptional alterations in the mutant heart, we carried out an Affymetrix microarray analysis for comparison between the mutant and normal heart at 16 weeks of age.
Keywords = Mn-SOD

Lymph node T cells isolated from Ezh2fl/fl or Ezh2 deficient mice

Dysregulated Expression of Lipid Storage and Membrane Dynamics Factors  in Tia1 Knock-Out Mouse Nervous Tissue

During cell stress, the transcription and translation of immediate early genes are prioritized, while most other mRNAs are stored away in stress granules or degraded in P-bodies. TIA-1 is an mRNA binding protein that needs to translocate from the nucleus to seed the formation of stress granules in the cytoplasm. Since other stress granule components such as TDP-43, FUS, ATXN2, SMN, MAPT, HNRNPA2B1 and HNRNPA1 are crucial for the motor neuron diseases ALS / SMA and for the frontotemporal dementia FTD, here we studied mouse nervous tissue to identify mRNAs with selective dependence on Tia1 deletion. Transcriptome profiling with oligonucleotide microarrays in comparison of spinal cord and cerebellum, together with independent validation in quantitative reverse transcriptase PCR and immunoblots demonstrated several strong and consistent dysregulations. In agreement with previously reported TIA1 knock-down effects, cell cycle and apoptosis regulators were affected markedly with expression changes up to 2-fold, exhibiting increased levels for Cdkn1a, Ccnf, and Tprkb versus decreased levels for Bid and Inca1 transcripts. Novel and surprisingly strong expression alterations were detected for fat storage and membrane trafficking factors, with prominent above 3-fold upregulations of Plin4, Wdfy1, Tbc1d24, and Pnpla2, versus a 0.5 -fold??  downregulation of Cntn4 transcript, encoding an axonal membrane adhesion factor with established haploinsufficiency. In comparison subtle effects on the RNA processing machinery included up to 1.2-fold upregulations of Dcp1b and Tial1. The effect on lipid dynamics factors is noteworthy, since also the gene deletion of Tardbp (encoding TDP-43) and Atxn2 led to fat metabolism phenotypes in mouse. In conclusion, genetic ablation of the stress granule nucleator TIA-1 has a novel major effect on mRNAs encoding lipid homeostasis factors in the brain.

Gene Expression Profiling of Prostate Benign and Malignant Tissue

We profiled genome-wide gene expression of human prostate benign and malignant tissue to identify potential biomarkers and immunotherapy targets.
We stratified malignant specimens according to their TMPRSS2:ERG gene fusion status.

Compare gene expression patterns in Chsy-1 wt and ko mouse 13.5 days embryo forelimbs

Transcriptional profiling of 13.5 day mouse embryo forelimbs. Gene expression comparison done between wild type and Chsy-1 gene knockout mice.

Expression data from patients with global developmental delay and autistic features and normal controls

We analyzed the whole-genome mRNA expression profiling of cultured lymphoblastoid cell lines (LCLs) from patients (n=2), carriers (n=2), and unrelated normal controls (n=3), all performed in duplicates using Affymetrix GeneChip Human Exon 1.0 ST arrays, to identify genes and pathways that are significantly dysregulated in affected individuals. Moreover, we have performed global transcriptional profiling in whole blood from patients (n=2) and healthy controls (n=13) using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Global transcriptional profiling of affected individuals and controls revealed significant alterations of genes and pathways in a pattern consistent with previous microarray studies of autism spectrum disorder patients.

Microarray analysis was used to compare the gene expression profiles of Deaf-1-transduced mouse mammary epithelial cells (MECs) relative to Deaf-1-deficient MECs.

Resveratrol supplementation does not improve metabolic function in non-obese women with normal glucose tolerance

Resveratrol has been reported to improve metabolic function in metabolically-abnormal rodents and humans, but has not been studied in non-obese people with normal glucose tolerance.  We conducted a randomized, double-blind, placebo-controlled trial to evaluate the metabolic effects of 12 weeks of resveratrol supplementation (75 mg/day) in non-obese, postmenopausal women with normal glucose tolerance.  Although resveratrol supplementation was well-tolerated and increased plasma resveratrol concentration without adverse effects, it did not change body composition, resting metabolic rate, plasma lipids, or inflammatory markers.  A two-stage hyperinsulinemic-euglycemic clamp procedure, in conjunction with stable isotopically-labeled tracer infusions, demonstrated that resveratrol did not increase liver, skeletal muscle, or adipose tissue insulin sensitivity.  Consistent with the absence of in vivo metabolic effects, resveratrol did not affect its putative molecular targets, including AMPK, Sirt1, Nampt, and Pgc-1α, in either skeletal muscle or adipose tissue.  These findings demonstrate that resveratrol supplementation does not have metabolic effects in non-obese women.

Inhibitor of NFκB ζ (IκBζ) is a transcriptional key regulator of   monocyte chemoattractant protein-1 (MCP-1/CCL2)

Monocyte chemoattractant protein 1 (MCP-1/CCL2) is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive MCP-1 secretion has been linked to many (auto)inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that the atypical inhibitor of NF-κB ζ (IκBζ), a transcriptional co-activator required for the selective expression of a subset of NF-κB target genes, is a key activator of the Ccl2 gene. IκBζ-deficient macrophages exhibited impaired secretion of MCP-1 when challenged with diverse inflammatory stimuli, such as lipopolysaccharide or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of IκBζ. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that IκBζ is directly recruited to the proximal promoter region of the Ccl2 gene and required for histone H3K9 trimethylation. Finally, IκBζ-deficient mice showed significantly impaired MCP-1 secretion and monocyte infiltration in an experimental model of peritonitis. Together, these findings suggest a distinguished role of IκBζ in mediating the targeted recruitment of monocytes in response to local inflammatory events.

Chronic tendon injuries, also known as tendinopathy, are common among professional and recreational athletes.  These injuries result in a significant amount of morbidity and health care expenditure and yet little is known about the molecular mechanism leading to tendinopathy.  We have used histological evaluation and molecular profiling to determine the gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy.  Diseased tendons have altered extracellular matrix, fiber disorientation, increased cellular content and vasculature and the absence of inflammatory cells.  Global gene expression profiling identified 1783 transcripts with significant different expression patterns in the diseased tendons.  Global pathway analysis further suggests altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response.  We have identified pathways and genes regulated in tendinopathy samples that will help contribute to the understanding of the disease towards the development of novel therapeutics.

Gene expression analysis of 12  B-cell Chronic Lymphocytic Leukemia samples and 5 CD19+ control samples

Elevated levels of microRNA miR-155 represent a candidate pathogenic factor in chronic B-lymphocytic leukemia (B-CLL). In this study, we present evidence that MYB (v-myb myeloblastosis viral oncogene homolog) is overexpressed in a subset of B-CLL patients. MYB physically associates with the promoter of MIR155 host gene (MIR155HG, also known as BIC, B-cell integration cluster) and stimulates its transcription. This coincides with the hypermethylated histone H3K4 residue and spread hyperacetylation of H3K9 at MIR155HG promoter. Our data provide evidence of oncogenic activities of MYB in B-CLL that include its stimulatory role in MIR155HG transcription.
We have studied differentially expressed genes in the whole genome and also in the preselected groups of  MYB target genes and miR-155 microRNA predicted targets.

Early and late passage endothelial cells with or without PDCD10 downregulation

Primary cells deficient for PDCD10/CCM3 do not enter senescence as control cells. Microarray analysis was performed in cells transduced with non-targeting shRNA and CCM3 shRNA at passage 7 (early passage) and passage 11 (late passage), when control cells are already senescent.

HDAC6 and HSP90 control the functions of Foxp3+ T regulatory cells

Foxp3+ T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection. Foxp3+ Tregs express histone/protein deacetylases (HDACs) that regulate chromatin remodeling, gene expression and protein function. Pan-HDAC inhibitors developed for oncology enhance Treg production and suppression but have limited non-oncologic applications given their broad effects. We show, using HDAC6-deficient mice and WT mice treated with HDAC6-specific inhibitors, that HDAC6 inhibition promotes Treg suppressive activity in models of inflammation and autoimmunity, including multiple forms of experimental colitis and fully MHC-incompatible cardiac allograft rejection. Many of the beneficial effects of HDAC6 targeting are also achieved by inhibition of the HDAC6-regulated protein, HSP90. Hence, selective targeting of a single HDAC isoform, HDAC6, or its downstream target, HSP90, can promote Treg-dependent suppression of autoimmunity and transplant rejection.

Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells.

To examine the impact of tumors on the immune system, we compared global gene expression profiles of peripheral blood T cells from previously untreated patients with B cell chronic lymphocytic leukemia (CLL) with those from age-matched healthy donors. Although the cells analyzed were not part of the malignant clone, analysis revealed differentially expressed genes, mainly involved in cell differentiation in CD4 cells and defects in cytoskeleton formation, vesicle trafficking, and cytotoxicity in CD8 cells of the CLL patients. In coculture experiments using CLL cells and T cells from healthy allogeneic donors, similar defects developed in both CD4 and CD8 cells. These changes were induced only with direct contact and were not cytokine mediated. Identification of the specific pathways perturbed in the T cells of cancer-bearing patients will allow us to assess steps to repair these defects, which will likely be required to enhance antitumor immunity.
Gene expression profiling was performed to determine whether CLL cells induce changes in T cells in patients with CLL.

Gene expression profiling of mouse prostate over-expressing the Notch1 intracellular domain.

Molecular signaling that regulates differentiation, survival and proliferation of the prostate luminal epithelial cells has not been thoroughly understood. Herein, we show that increased canonical Notch1 activity suppresses terminal differentiation of prostate luminal epithelial cells but is insufficient to transform. Augmented Notch1 activity delays anoikis of luminal epithelial cells by augmenting NF-κB activity independent of Hes-1, stimulates luminal cell proliferation by potentiating the PI3K-AKT signaling, and rescues the capacities of a fraction of prostate luminal epithelial cells for unipotent differentiation in vivo and short-term self-renewal in vitro. Epithelial cell-autonomous AR signaling is dispensable for the Notch-mediated cellular survival and proliferation. This study reveals a previously unappreciated role of Notch in prostatic luminal epithelial cell differentiation, supports the presence of a lineage hierarchy within the prostate luminal epithelial cells, and implies a pro-metastatic function of Notch signaling during prostate cancer progression.

purified CD34+ cells from bone marrow of imatinib-treated patients were compared to those of healthy donors
Keywords = CML

In vivo function of NR2E3 in establishing photoreceptor identity during mammalian retinal development

Rod and cone photoreceptors in mammalian retina are generated from common pool(s) of neuroepithelial progenitors.  NRL, CRX and NR2E3 are key transcriptional regulators that control photoreceptor differentiation.  Mutations in NR2E3, a rod-specific orphan nuclear receptor, lead to loss of rods, increased density of S-cones, and supernormal S-cone-mediated vision in humans.  To better understand its in vivo function, NR2E3 was expressed ectopically in the Nrl-/- retina, where post-mitotic precursors fated to be rods develop into functional S-cones similar to the human NR2E3 disease.  Expression of NR2E3 in the Nrl-/- retina completely suppressed cone differentiation and resulted in morphologically rod-like photoreceptors, which were not functional.  Gene profiling of FACS-purified photoreceptors confirmed the role of NR2E3 as a strong suppressor of cone genes and an activator of a subset of rod genes (including rhodopsin) in vivo.  Ectopic expression of NR2E3 in cone precursors and differentiating S-cones of wild type retina also generates rod-like cells.  The dual regulatory function of NR2E3 is not dependent upon the presence of NRL and/or CRX, but on the timing and level of its expression.  Our studies reveal a critical role of NR2E3 in establishing functional specificity of post-mitotic photoreceptor precursors during retinal neurogenesis.

Transgenic (Probasin driven Myr-AKT)or wild-type littermates were treated with RAD001 or placebo and sacrificed at 12 and 48 hours following the beginning of treatment
Keywords = AKT Prostate RAD001 mTOR PIN

Imatinib has become the current standard therapy for patients with chronic myelogenous leukaemia (CML). For a better understanding of the Imatinib-related molecular effects in vivo, we assessed gene expression profiles of Philadelphia Chromosome positive (Ph+) CD34+ cells from peripheral blood of 6 patients with de novo CML in chronic phase. After 7 days of treatment with Imatinib the Ph+ CD34+ cells were reassessed to look for changes in the transcriptome. The expression level of 303 genes was significantly different comparing the transcriptome of the Ph+ CD34+ cells before and after 7 days of Imatinib therapy (183 down-regulated, 120 up-regulated, lower bound ≥1.2-fold). For a substantial number of genes governing cell cycle and DNA replication, the level of expression significantly decreased (CDC2, RRM2, PCNA, MCM4). On the other hand, therapy with Imatinib was associated with an increase of genes related to adhesive interactions, such as L-selectin or CD44. A group of 8 genes with differential expression levels were confirmed using a gene specific quantitative real-time PCR. Thus, during the first week of treatment, Imatinib is preferentially counteracting the bcr-abl induced effects related to a disturbed cell cycle and defective adhesion of leukemic Ph+ CD34+ cells.

Analysis of SW480 cells following knockdown of Ezrin using RNAi.  Ezrin is a protein that regulate the organization of cytoskeleton.  Ezrin KD SW480 was used to study the role of ezrin in colon cancer.
Keywords: ezrin, colon cancer, migration

MEFs were grown from WT and PDPN-deficient mouse embryos and gene expression was analyzed. The aim was to determine whether a lack of PDPN caused significant global changes in gene expression in primary fibroblasts.
Here, we performed a microarray on WT and PDPN-deficient mouse embryonic fibroblasts to determine whether there were signficiant underlying changes in gene expression.

Expression data from osteoblastic lineage cells isolated from normal and leukemic mice

Multipotent stromal cells (MSC) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. During myeloproliferative neoplasm (MPN) development, MSCs are stimulated to overproduce functtionally altered OBCs, which accumulate in the BM cavity as myelofibrotic cells. These MPN-expanded OBCs, in turn, impair the maintenance of normal HSCs but not of leukemic stem cells.
We used microarrays to detail the global gene expression changes in OBCs during BCR/ABL-induced MPN development, and understand the molecular deregulations contributing to their functional changes.

Expression profiles in WT and MLL1-KO MEF at two different circadian time point

We found that a H3K4 specific histone methyltransferase MLL1, a mammalian homologue of Drosophila trithorax, is essential for circadian transcription. MLL1 is in a complex with CLOCK:BMAL1 and contributes to their rhythmic recruitment to circadian promoters and cyclic H3K4 tri-metylation. To analyze the function of MLL1 on circadian gene regulation, we performed comparative microarray analysis of global gene expression levels in WT and MLL1-deficient MEF, at two different circadian time points (CT18 and CT30). This analysis identified several genes whose expression levels were remarkably changed between CT18 and CT30 in WT and MLL1-KO MEF. Typical clock-regulated genes such as Per2, Per3, Bmal1, or Dbp were found to be changing in WT but not in MLL1-KO MEFs.

Addiction, Drugs of abuse, Time-course, Immediate Early Genes, Glucocorticoid receptor dependent genes, Cocaine, Heroin, Nicotine, Ethanol, Morphine, Methamphetamine

Transcriptional networks regulated by drugs of abuse in mouse striatum

In summary, we characterized genomic signatures of response to drugs of abuse and we found positive correlations between the drug-induced expression and various behavioral effects. These signatures are formed by two dynamically inducible transcriptional networks: (1) CREB/SRF-dependent gene pattern that appears to be related to drug-induced neuronal activity, (2) the pattern of genes controlled at least in part via release of glucocorticoids and androgens that are associated with rewarding and harmful drug effects. The discovery of co-expressed networks of genes allowed for the identification of master-switch controlling factors involved in molecular response to the drugs. Finally, using the pharmacological tools we were able to dissect and inhibit particular gene expression patterns from genomic profile.
Type: Drug response, Time-course, Gene expression profiling with Illumina Microarrays

Expression profiling of tumor and adjacent non-tumorous tissues of Hepatocellular Carcinoma (HCC) patients. Illumina HumanHT-12 V4.0 expression beadchip was used to obtain expression profiles across more than 31,000 annotated genes. Samples included 59 tumors and 59 adjacent non-tumorous samples.

A functional liaison between E2F and the aberrant ETS oncogene EWS/FLI1 in Ewing's sarcoma

Translocations of ETS transcription factors are driver mutations in diverse cancers. We investigated the genomic network of the ETS fusion EWS/FLI1 in Ewing's sarcoma (ESFT) as a model of ETS-driven tumorigenesis. ChIP-Seq and transcriptional analysis identified E2F3 as a principle co-factor of EWSFLI1 defining functionally distinct gene sets. While EWS/FLI1 binding independent of E2F3 predominantly associated with repressed differentiation genes, significant co-localization with E2F3 was discovered at proximal promoters of activated growth-related genes. Thus, EWS/FLI1 promotes oncogenesis by simultaneously perturbing differentiation state and augmenting the expression of genes co-regulated by E2F3. Integration of additional E2F3 and ERG localization data from prostate cancer containing TMPRSS2/ERG verified that the ETS-E2F module is also found in prostate cancer and may be of general relevance to ETS driven cancers.

Analysis of E14 embryonic stem cells lacking Trip12 ubiquitin ligase activity.
We used microarrays to detail the global gene expression profiles affected by Trip12 ubiquitin ligase activity in mouse embryonic stem cell.

Knockdown of transactive response DNA-binding protein TDP-43 downregulates histone deacetylase 6

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and  mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementias  and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear  localization is observed in disease. To identify relevant targets of TDP-43, we performed  an expression profiling study. Thereby, histone deacetylase 6 (HDAC6) downregulation  was discovered upon TDP-43 silencing on mRNA and protein level in human embryonic  kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation  of the major HDAC6 substrate, acetyl-tubulin. Expression of wild-type but neither  RNA-binding- nor nuclear-localization-deficient TDP-43 restored HDAC6 expression.  Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional  interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster  also showed HDAC6 mRNA decrease. HDAC6 is necessary for protein aggregate  formation and degradation. Indeed, downregulation of HDAC6 reduced aggregate  formation and increased cytotoxicity of expanded poly-glutamine ataxin-3 in TDP-43  silenced cells. This was completely restored by co-transfection with HDAC6. In  conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby  contribute to pathogenesis.

Pancreatic cancer circulating tumor cells express a cell motility gene signature that predicts survival after surgery

Most cancer deaths are caused by metastases, which are the end-results of circulating tumor cells (CTC) that detach from the cancer primary and succeed to survive in distant organs. The aim of the present study was to develop a gene signature of CTC and to assess its prognostic relevance after surgery for pancreatic ductaladenocarcinoma (PDAC).
A negative depletion fluorescence activated cell sorting (FACS) procedure was developed and validated with spiking experiments using cancer cell lines in whole human blood samples. This FACS-based method was used to enrich for CTC from the blood of 10 patients who underwent surgery for PDAC. Total RNA was isolated from 4 subgroup samples, i.e. CTC, haematological cells (G), original tumor (T), and non-tumoral pancreatic control tissue (P). After RNA quality control, samples of 6 patients were eligible for further analysis. Whole genome microarray analysis was performed after double linear amplification of RNA. The ‘Ingenuity Pathway Analysis’ software and AmiGO were used for functional data analyses. A CTC gene signature was developed and validated with the nCounter system on expression data of 78 primary PDAC using Cox regression analysis for disease-free (DFS) and overall survival (OS).
Using stringent statistical analysis, we finally retained 8,152 genes to compare expression profiles of CTC vs. other subgroups, and found 1,059 genes to be differentially expressed. The pathway with the highest expression ratio in CTC was p38 mitogen-activated protein kinase (p38 MAPK) signaling, known to be involved in cancer cell migration. In the p38 MAPK pathway TGF-β1, cPLA2, and MAX were significantly upregulated. In addition, 9 other genes associated with both p38 MAPK signaling and cell motility were over-expressed in CTC. High co-expression of TGF-1 and our cell motility panel (≥ 4 out of 9 genes for DFS and ≥ 4 out of 9 genes for OS) in primary PDAC was identified as an independent predictor of DFS (p=0.041, HR (95% CI) = 1.885 (1.025 – 3.559)) and OS (p=0.047, HR (95% CI) = 1.366 (1.004 – 1.861)).
Pancreatic CTC isolated from blood samples using a FACS-based negative depletion method,express a cell motility gene signature. The expression of this newly defined cell motility gene signature in the primary tumor is able to predict survival of patients who undergo surgical resection for pancreatic cancer.

The role of mtDNA mutations in age-related hearing loss in mice carrying a mutator DNA polymerase gamma

Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related disorders. Previously, we created mice expressing a proofreading-deficient version of the mtDNA polymerase gamma (Polg) which accumulate age-related mtDNA mutations and display premature aging. Here we performed microarray gene expression profiling to identify mtDNA mutation-responsive genes in the cochlea of aged mitochondrial mutator mice. Age-related accumulation of mtDNA mutations was associated with transcriptional alternations consistent with reduced inner ear function, mitochondrial dysfunction, neurodegeneration, and reduced cell structural modulation. Hearing assessment and histopathological results confirmed that aged PolgD257A/D257A (D257A) mice exhibited moderate hearing loss and severe cochlear degenerations. Age-related accumulation of mtDNA mutations also resulted in alternations in gene expression consistent with induction of apoptosis, proteolysis, stress response, and reduced DNA repair. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay confirmed that the cochleae from aged D257A mice showed significantly more TUNEL positive cells compared to wild-type (WT) mice. The levels of cleaved caspase-3 were also found to increase in the cochleae of aged D257A mice. These observations provide evidence that age-related accumulation of mtDNA mutations is associated with apoptotic cell death in aged cochlea. Our results provide the first global view of molecular events associated with mtDNA mutations in postmitotic tissue, and suggest that apoptosis is the major mechanism of mtDNA mediated cell death in the development of age-related hearing disorder.

Data expression in alveolar macrophages induced by lipopolysaccharide in humans

Rationale: Lipopolysaccharide (LPS) is ubiquitous in the environment. Inhalation of LPS has been implicated in the pathogenesis and/or severity of several lung diseases, including pneumonia, chronic obstructive pulmonary disease and asthma. Alveolar macrophages are the main resident leukocytes exposed to inhaled antigens.   Objectives: To obtain insight into which innate immune pathways become activated within human alveolar macrophages upon exposure to LPS in vivo.

Profiling of MCF-7 cell lines stably overexpressing (ca)Raf-1, (ca)MEK, (ca)erbB-2, or ligand-activatable EGFR.

Profiling of MCF-7 cell lines stably overexpressing constitutively active Raf-1, constitutively active MEK, constitutively active c-erbB-2, or ligand-activatable EGFR as models of overexpressed growth factor signaling, as well as control vector transfected cells (coMCF-7) and control vector transfected cells long-term adapted for estrogen-independent growth (coMCF-7/lt-E2).
Keywords: Cell Line Comparison

Comparison of wildtype versus transgenic pineal gland gene expression

Pineal gland gene expression of 129 Wt P0 mice versus NeuroD1 pineal gene expression at P0

This study determines pineal gland gene expression levels in the NeuroD1 knockout mouse at postnatal day zero.
Comparison was performed against pineal gland gene expression levels in 129 wildtype mice also disected at P0.

As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D).
Microarrays were used to measure the changes in gene expression induced by 1,25D treatment of H37Rv-infected THP-1 cells

Transcriptional profiling of WAT comparing wild-type control with Ahnak Knockout mice fed regular chow and high fat diet

Isolation and enrichment of cancer stem cells in colorectal carcinoma to study role of epithelial-mesenchymal transition regilators in tumor malignancy.

Gene expression profiles of whole blood incubated with live meningococci

Blood from two donors was incubated for timepoints up to 24 hours in the presence and absence of live meningococci. The donors are labelled NP and SW. 'SWB' refers to blood that was incubated with meningococci; 'Unstim' refers to control blood incubated in the absence of meningococci.  Duplicate arrays were prepared for the majority of timepoints ('a' and 'b'), except where the array of of poor quality and not used in the analysis.
We infected heparinised whole blood from two healthy donors (adult volunteers) with 108 live meningococci, serogroup B, type MC58, as previously described.8 Blood was incubated at 37°C for 0, 1, 3, 6, 12, and 24 h. At every timepoint we removed a sample and harvested the plasma by centrifugation (1200 g, 10 min). We gathered the remaining blood into RNA stabilisation fluid. Uninfected control blood was obtained for every timepoint.
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

The transcriptional activities of c-Myb and its oncogenic variant v-Myb were compared by expressing them in human MCF7 cells using recombinant adenovirus vectors. A hybrid construct, 3Mutc, which is a variant of c-Myb harboring three v-Myb-derived DNA binding domain mutations was also analyzed.  All the samples were compared to cells infected with a control adenovirus.  The results showed that v-Myb, which differs from c-Myb only by N- and C-terminal deletions and eleven amino acid substitutions, has a qualitatively different transcriptional activity.
Keywords: repeat

Time series of diabetes and exercise training induced expression changes in cardiac muscle of mice

We investigated the effects of diabetes, physical training, and their combination on the gene expression of cardiac muscle. Mice were divided to control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed 1, 3, or 5 weeks of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 hours after the last training session. Gene expression of cardiac muscles were analyzed using Affymetrix Gene chip MG U74Av2 (Affymetrix , Inc., Santa Clara, CA).

We studied the genomic locations of three key regulatory proteins (OCT4, NANOG and CTCF) in human and mouse embryonic stem (ES) cells [see Series GSE20650]. To identify the conserved and unique human OCT4 targets, we performed an OCT4 RNAi knock-down experiment. We find that species-specific transposable elements have profoundly altered the transcriptional circuitry of pluripotent stem cells.

Novel Estrogen Receptor-{alpha} Binding Sites and Estradiol Target Genes Identified by ChIP Cloning in Breast Cancer.

Estrogen receptor-{alpha} (ER{alpha}) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ER{alpha} was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ER{alpha} target loci in breast cancer cells. The ER{alpha}-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ER{alpha}-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ER{alpha} responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ER{alpha} that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer.

Exposure to high levels of arsenic in drinking water is associated with several types of cancers including lung, bladder and skin, as well as vascular disease and diabetes. Drinking water standards are based primarily on epidemiology and extrapolation from higher dose experiments, rather than measurements of phenotypic changes associated with chronic exposure to levels of arsenic similar to the current standard of 10ppb, and little is known about the difference between arsenic in food as opposed to arsenic in water. Measurement of phenotypic changes at low doses may be confounded by the effect of laboratory diet, in part because of trace amounts of arsenic in standard laboratory chows, but also because of broad metabolic changes in response to the chow itself. Finally, this series contrasts 8hr, 1mg/kg injected arsenic with the various chronic exposures, and also contrasts the acute effects of arsenic, dexamethasone or their combination. Male C57BL/6 mice were fed on two commercially available laboratory diets (LRD-5001 and AIN-76A) were chronically exposed, through drinking water or food, to environmentally relevant concentrations of sodium arsenite, or acutely exposed to dexamethasone.

Gene expression profiles of Lkb1 WT and KO hematopoietic stem cells (HSCs)

LKB1 encodes a Ser/Thr kinase and acts as an evolutionarily conserved sensor of cellular energy status in eukaryotic cells. LKB1 functions as the major upstream kinase to phosphorylate AMPK and 12 other AMPK-related kinases, which is required for their activation in many cellular contexts. Once activated, AMPK and AMPK-related kinases phosphorylate a diverse array of downstream effectors to switch on ATP-generating catabolic processes and switch off ATP-consuming anabolic processes, thus restoring energy balance during periods of energetic stress. To study the role and mechanisms of Lkb1 in the regulation of hematopoietic stem cell (HSC) biology, we performed transcriptome analysis of sorted LSK (Lin-, Sca-1+, c-Kit+) cells from Lkb1 WT and KO bone marrows at 1 day post-completing tamoxifen injection (DPI). To identify more proximal molecular effects, we chose 1 DPI due to the modest phenotypes in Lkb1 KO mice, yet documentation of efficient Lkb1 deletion in LSK cells at this very early time point.

To identify molecular pathological alterations in AD brains, we performed interspecies comparative microarray analyses using RNAs prepared from postmortem human brain tissues donated for the Hisayama study and hippocampal RNAs from the triple-transgenic mouse model of AD (3xTg-AD)
Three-way ANOVA of microarray data from frontal cortex, temporal cortex and hippocampus with presence/absence of AD and vascular dementia, and sex, as factors revealed that the gene expression profile is most significantly altered in the hippocampi of AD brains. Comparative analyses of the brains of AD patients and a mouse model of AD showed that genes involved in non-insulin dependent DM and obesity were significantly altered in both, as were genes related to psychiatric disorders and Alzheimer’s disease.

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. Notably, some cellular interferon-inducible genes were upregulated six hours after exposure to virions and LPS, but not after exposure to VLPVP40-GP. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response.

Expression data from murine hearts exposed to ischemic preconditioning comparing A2BAR null and wildtype BL6/C57 mice

Genetically targeted mice with deficiency for the A2BAR show increased susceptibility to acute myocardial ischemia and are not protected by IP, a powerful strategy for cardioprotection, where short and repeated episodes of ischemia and reperfusion prior to myocardial infarction result in attenuation of infarct size.
The purpose of this study was to identify genes that are potentially under the control of the cardiac A2B adenosine receptor

Expression profiling in proliferative thyroid lesions of different histologies and normal thyroid tissue

Experiment a) Establishment of expression profiles in conventional papillary thyroid carcinomas (PTCs) with a BRAF mutation vs. PTCs without a BRAF mutation and using normal thyroid (TN) specimens as reference.
Experiment b) Establishment of expression profiles in follicular adenomas (FAs) of the thyroid and follicular variant of papillary thyroid carcinomas (FVPTCs).

mouse embryonic fibroblasts derived from FHL2-/- and WT mouse embryos (14.5 days)

The LIM-only protein FHL2 acts as a transcriptional modulator that positively or negatively regulates multiple signaling pathways. We recently reported that FHL2 cooperates with CBP/p300 in the activation of ß-catenin/TCF target gene cyclin D1. In this paper, we demonstrate that FHL2 is associated with the cyclin D1 promoter at the TCF/CRE site, providing evidence that cyclin D1 is a direct target of FHL2. We show that deficiency of FHL2 greatly reduces the proliferative capacity of spontaneously immortalized mouse fibroblasts which is associated with decreased expression of cyclin D1 and p16INK4a, and hypophosphorylation of Rb. Reexpression of FHL2 in FHL2-null fibroblasts efficiently restores cyclin D1 levels and cell proliferative capacity, indicating that FHL2 is critical for cyclin D1 activation and cell growth. Moreover, ectopic cyclin D1 expression is sufficient to override growth inhibition of immortalized FHL2-null fibroblasts. Gene expression profiling revealed that FHL2 deficiency triggers a broad change of the cell cycle program that is associated with downregulation of several G1/S and G2/M cyclins, E2F transcription factors and DNA replication machinery, thus correlating with reduced cell proliferation. This change also involves downregulation of the negative cell cycle regulators, particularly INK4 inhibitors, which could counteract the decreased expression of cyclins, allowing cells to grow. Our study illustrates that FHL2 can act on different aspects of the cell cycle program to finely regulate cell proliferation.
Keywords: mouse embryonic fibroblasts derived from FHL2-/- and WT mouse embryos (14.5 days)

Depletion of Jak2V617F MPN-propagating stem cells by interferon-alpha in a murine model of polycythemia vera

Interferon alpha (IFNa) is an effective treatment for patients with myeloproliferative neoplasms (MPN). In addition to inducing hematological responses in most MPN patients, IFNa reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNa on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNa on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNa treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNa treatment induces cell-cycle activation of Jak2V617F mutant long-term hematopoietic stem cells (LT-HSC) and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNa on Jak2V617F mutant and normal hematopoiesis and suggest that IFNa achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN, by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNa and targeting the proliferating downstream progeny with JAK2-inhibitors or cytotoxic chemotherapy.

Expression profile of HeLa THOC1-depleted  stable cell lines (HeTH-4)

THO/TREX is an eukaryotic conserved complex with a role at the interface between transcription and mRNP biogenesis/export that in yeast has been shown to play an important role in preventing transcription-associated genome instability. However, whereas a role of mammalian THO/TREX in mRNA processing and export seems clear, a role on either transcription or genome stability is still being argued. In this work we show, by microarray analysis of gene expression, that THO depletion in human cell lines has a global effect on transcription, with a significant impact on genes involved in transcription and DNA metabolism.
We performed a microarray analysis of gene expression on  THOC1-depleted cells in order to know if there was a change in the genome-wide expression profile. Our data data suggest that the absence of THOC1 has a global effect on transcription process

The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A1.  Progerin is also sporadically expressed in wild type cells and has been linked to physiological aging.  HGPS cells exhibit extensive nuclear defects including abnormal chromatin structure and increased DNA damage.  At the organismal level, HGPS affects several tissues particularly of mesenchymal origin.  How the cellular defects of HGPS cells lead to the organismal defects has been unclear.  To begin to unravel how progerin leads to disease phenotypes, we analyzed time-dependent changes in transcriptional profiles in response to progerin expression in a hTERT-immortalized skin fibroblast cell line expressing either GFP-progerin or the  GFP-wt lamin A control
Keywords: time course, cell line comparison

Genomic profiles of Macaca fascicularis exposed to Venezuelan equine encephalitis

Gene expression analysis of PBMC exposed to VEE
Keywords: VEEV challenge in Monkey

Regulatory T cells require the phosphatase PTEN to restrain type 1 and follicular helper T-cell responses

The interplay between effector and regulatory T (Treg) cells is crucial for adaptive immunity, but how Treg control effector cell flexibility is elusive. We found that the phosphatase PTEN links Treg stability to the repression of TH1 and TFH (follicular helper) responses. Depletion of PTEN in Treg resulted in excessive TFH and germinal center responses and spontaneous inflammatory disease. These defects are considerably blocked by deletion of Interferon-γ, indicating coordinated control of TH1 and TFH responses. Mechanistically, PTEN maintains Treg stability and proper metabolic balance between glycolysis and mitochondrial fitness. Moreover, PTEN deficiency markedly upregulates mTORC2-Akt activity, and loss of this activity restores PTEN-deficient Treg function. Our studies establish a PTEN-mTORC2 axis that actively maintains Treg stability and coordinates Treg-mediated control of effector cell flexibility.

Identification of genes that are linked with optineurin expression

This study aimed to identify genes that are linked with optineurin expression using a combined siRNA-microarray approach
Keywords: siRNA treatment study

X-linked inhibitor of apoptosis (XIAP) is the most potent endogenous caspase inhibitor preventing cell death via caspase-9, -7 and -3 (initiator and executioner caspase pathways). Using short hairpin RNA (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones have been developed. XIAP mRNA levels were established by RT-PCR, the four X (XIAP knockdown) clonal cell lines show 82-93% reduction in XIAP mRNA when compared to the four L (luciferase control) cell lines. Immunoblot analysis showed a 67-89% reduction in XIAP protein in X cell lines compared to L. RNA was analysed by microarray and XIAP knockdown was confirmed in 7 probe sets, there was no significant compensation of other IAP family members. XIAP knockdown induced a 2-fold increase in the basal level of apoptosis without modification of caspase 3/7 activity. Finally, XIAP knockdown sensitises cells to radiotherapy by 20%, to recombinant TRAIL by a 3-fold factor, and to paclitaxel and docetaxel by >2 fold factor. Future work should focus on targeted agents such as rhTRAIL in combination with strategies to down regulate XIAP. XIAP antisense is now in clinical development in oncology.

Comparison of genes regulated in P0 Klhl40 WT and KO skeletal muscle

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, and their role in the disease remains unresolved. Here, we demonstrate that loss of a muscle-specific protein, Klhl40, results in a nemaline-like myopathy in mice that closely phenocopies the muscle abnormalities observed KLHL40 deficient patients. We show that Klhl40 dynamically localizes to the sarcomere I-band and A-band and binds to Nebulin (Neb), a protein frequently implicated in NM, as well as a putative thin filament protein, Lmod3. Klhl40 belongs to the BTB-BACK-Kelch (BBK) family of proteins, some of which have been previously shown to promote degradation of their substrates.  In contrast, we find that Klhl40 promotes stability of Neb and Lmod3 and blocks Lmod3 ubiquitination.  Accordingly, loss of Klhl40 reduces Neb and Lmod3 protein in skeletal muscle of mice and KLHL40 deficient patients. Because loss of sarcomere thin filament proteins is a frequent cause of NM, our data establishes a possible molecular basis for NM in KLHL40 deficient patients by establishing a novel pro-stability function of Klhl40 for Neb and Lmod3.

Missense mutations in the gene for the ubiquitously expressed superoxide dismutase-1 (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (ALS), the most common adult onset motor neuron disease in humans killing selectively large motor neurons. Mice and rats overexpressing mutant SOD1 develop an adult onset neurodegenerative disease with hindlimb-paralysis and subsequent death similar to the human condition. In order to analyze the effects of mutant SOD1 expression onto the most affected cell-type in ALS, a small subpopulation of spinal cord cells, we propose to use laser microdissection to isolate mouse lumbar motor neurons and to assess the changes onto the mRNA expression profile using Affymetrix GeneChips compared to control animals. While two studies applying a genomic approach on the ALS mouse models used the entire spinal cord, contributions of changes to motor neurons were masked by the inflammatory effects of mutant SOD1 and the much larger population of non-motor neuronal cells. What is therefore needed is a cell-type specific expression profile that could reveal dysregulations in the transcriptome of the affected motor neurons.
In order to find and analyze disease relevant gene expression profile changes in the mutant vs. the wildtype overexpressing SOD1 mice, we propose to use two different mutant SOD1 lines. Both lines develop the same ALS-like motor neuron disease, however, line 1 (with a G37R mutation) retains the full activity of the SOD1 enzyme while line 2 (with a G85R mutation) has almost no SOD1 enzymatic activity. We believe that by using these two most extreme variants of ALS-inducing mutant SOD1 forms, candidate genes that will be similarly dysregulated in both mutant lines will have great potential to be a disease relevant hit. We will collect from both mutant SOD1 mouse lines lumbar motor neurons from three presymptomatic timepoints, equally distributed during their adult life and before obvious hindlimb-weakness or paralysis signs and compare them to two control lines, negative littermates and wildtype SOD1 overexpressing mice. Furthermore, to determine if their are already mutant SOD1-induced changes since the formation of the affected cells, we will also isolate embryonic spinal cord motor neurons and compare them between mutant SOD1 and wildtype SOD1 overexpressing animals.
Both, mice deleted in wildtype SOD1 and mice overexpressing mutant human SOD1 clearly established that the toxic property of mutant SOD1 is based upon a gain-of-toxic function phenomenon rather than a loss-of-function effect, since the SOD1 knock-out mice are overall normal. Furthermore, mice overexpressing wildtype SOD1 are healthy and serve as ideal control animals. Therefore, one of the most important question in ALS is to determine what slowly acting mechanism, or build-up of toxic products is responsible for ultimately killing more than 50-70% of the large lumbar spinal cord motor neurons. We hypothezise that the toxic property of mutant SOD1 must already induce changes onto the transcriptome of the most at-risk cell-type, the large lumbar spinal cord motor neuron, very early on in the presymptomatic phase of the disease course, when the animal is still without any paralysis signs and moving normally around the cage.
We will choose 3 timepoints before phenotypic disease-onset that is at 22 weeks for the SOD1G37R line (L42) and at 11 months for the SOD1G85R line (L148). The timepoints are 8, 15 and 18 weeks for SOD1G37R line and 4, 7.5 and 9 months for the SOD1G85R line all compared to age-matched control animals (wildtype SOD1 overexpressing mice (L76) for the SOD1G37R line (high mutant SOD1 levels) and negative littermates for the SOD1G85R line (low mutant SOD1 levels)). Using the Leica lasermicrodissection system on fresh frozen spinal cord sections, we process a single spinal cord sample in 3 days resulting in approximately 4000 ventral horn motor neurons collected from 150 consecutive 20 um sections of the most affected lumbar spinal cord region (L4-L6). To visualize the motor neurons, we have established a fast Nissel-staining protocol that is optimized for speed to preserve the integrity of the RNA. Before lasermicrodissection slides containing 5 spinal cord sections are dessicated for 1 hour and samples are collected directly into RNA-preserving lysis-buffer. Using Stratagene's NanoPrep RNA-extraction columns a yield of around 150 ng of total RNA was obtained (Ribogreen-quantification), that is enough for both the 2-round linear RNA amplification and for future candidate confirmation studies using real-time PCR analysis. We are using the Affymetrix Two-Cycle 3'-Amplification Kit, starting with 100 ng of total RNA and will hybridize on Affymetrix Mouse 430 2.0 GeneChips. Embryonic motor neurons are isolated from e14 mutant SOD1G93A overexpressing rat embryos and compared to wildtype SOD1 overexpressing embryos using a metrazimide gradient followed by a p75-antibody purification in combination with magnetic beads. Cells from one litter of embryos were directly collected into lysis-buffer, yielding at least 200-500 ng of total RNA, enough for one Rat 230 2.0 GeneChip (2-round amplification using 100 ng total RNA).

In the present study, we investigated whether, and to what extent, P2Rs and their ligands are involved in the regulation of AML cells. Our findings show that AML blasts express several receptors belonging to the P2X and P2Y family. Although different samples respond differently to ATP and UTP stimulation (reflecting the variability intrinsic to the group of acute myeloid leukemias), all the tested samples appear to be responsive to purinergic signalling, as demonstrated by intracellular calcium mobilization.
GEP analysis demonstrated that ATP induced the expression of cell cycle inhibitors and negative modulators of cell motility, such as inhibitors of GTPase activity. On the contrary, ATP inhibits the expression cell-cycle related genes (cyclins and CDKs), activators of cell motility (Rho GTPases regulators, matrix degradation enzyme and cytoskeleton proteins) and adhesion molecules involved in homing and engraftment.

Genomic Microarray of Rat Alveolar Type 2 cells Following Chronic Ethanol Ingestion

Chronic alcohol ingestion changes the alveolar landscape.  We used microarrays to characterize the change in mRNA expression following chronic alcohol ingestion in male Sprague Dawley rates (EtOH 36% of calories)

Pathway and single gene analysis of Caco-2 cell differentiation by ascorbate-stabilized quercetin

The aim was to investigate mechanisms contributing to quercetin’s previously described effects on cell-proliferation and -differentiation, which contradicted its proposed anti-carcinogenic potency. In a 10-day experiment, 40 µM quercetin stabilized by 1mM ascorbate reduced Caco-2 differentiation up to 50% (P<0.001). Caco-2 RNA from days 5 and 10, hybridized on HG-U133A2.0 Affymetrix® GeneChips®, showed 1,743 affected genes on both days (P<0.01). All 14 Caco-2 differentiation-associated genes showed decreased expression (P<0.01), including intestinal alkaline phosphatase that was confirmed technically (qRT-PCR) and functionally (enzyme-activity).
The 1,743 genes contributed to 27 affected pathways (P<0.05) categorized under 6 gene ontology (GO) processes, including apoptosis and cell-cycle. Genes within these GO-processes showed fold changes that suggest increased cell-survival and -proliferation. Furthermore, quercetin downregulated expression of genes involved in tumor-suppression and phase II metabolism, and upregulated oncogenes.
Gene expression changes mediated by ascorbate-stabilized quercetin were concordant with those occurring in human colorectal carcinogenesis (≈ 80-90%), but were opposite to those previously described for Caco-2 cells exposed to quercetin in the absence of ascorbate (≈ 75-90%).
In conclusion, gene expression among Caco-2 cells exposed to ascorbate-stabilized quercetin showed mechanisms contrary to what is expected for a cancer-preventive agent. Whether this unexpected in vitro effect is relevant in vivo, remains to be elucidated.

Expression data from GNMT and MAT1A knockout models that develop all the stages of non-alcoholic fatty liver disease including hepatocellular carcinoma [GNMT_MAT1A_3&8_months]

Liver global gene expression patterns of 9 GNMT-knockout mice histopathologically determined to have non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) together with 10 MAT1A-knockout mice histopathologically determined to have steatosis and NASH. All these have their respective wild type patterns. These were analyzed to define signatures to study the pathogenesis of NAFLD-derived HCC, explore which subtypes of cancers can be investigated using mouse models and define a signature of HCC differential survival that can be used to characterize HCC subtypes of different survival derived from mixed etiologies.

Lim1 mutant, kidney development, gene expression profile comparison

Laser capture-microarray analysis of Lim1 mutant kidney development.

The Lim1 gene has essential functions during several stages of kidney development. In particular, a tissue specific knockout in the early metanephric mesenchyme results in the formation of the earliest nephron precursor, the renal vesicle, but failure of this structure to progress to the next stage, the comma shaped body. To better understand the molecular nature of this developmental arrest we used a laser capture microdissection-microarray strategy to examine the perturbed gene expression pattern of the mutant renal vesicles. Among the genes found differently expressed were Chrdl2, an inhibitor of BMP signaling, the pro-apoptotic factor Bmf, as well as myob5, an atypical myosin which modulates chemokine and transferring signaling, and pdgfr1, which is important in epithelial folding. Of particular interest, the microarray data indicated that the Dkk1 gene, which encodes an inhibitor of Wnt signaling, was downregulated  nine fold in mutants. This was confirmed by in situ hybridizations. It is interesting to note that Lim1 and Dkk1 mutant mice have striking similarities in phenotype.  These results suggest that the Dkk1 gene might be a key downstream effector of Lim1 function.
Keywords: Lim1 mutant, kidney development, gene expression profile comparison

Exploring the gene expression associated with Pten deficiency in the developing inner ear

In our recent study, we reported the function of phosphatase and tensin homolog (PTEN) during inner ear development. PTEN is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. To better understand the genes and signaling networks related to auditory neuron maintenance, we examined the profiles of differentially expressed genes (DEGs) using microarray analysis in Pten-deficient mice at E14.5. We identified 46 statistically significant DEGs using Significant Analysis of Microarrays (SAM) analysis with a false discovery rate (FDR) equal to zero. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time polymerase chain reaction (RT-PCR) and in situ hybridization, respectively.    Ingenuity pathway analysis (IPA) with DEGs identified significant signaling networks associated with cellular movement and axon guidance. Significant networks revealed that Spp1-mediated cellular movement and G-protein signaling 4 (RGS4)-Akt are related with axon guidance. This result was consistent with the phenotypic defects of spiral ganglion in Pten conditional knockout (cKO) mice (e.g., abnormal migration of spiral ganglion and irregular formation of neuritis). From this study, we suggest two key regulatory signaling networks mediated by Spp1 and RGS4, which may play potential roles in neuronal differentiation of developing auditory neurons.

Gene expression in T1alpha null mutant lungs compared to wild type at dpc 18.5 and term

We compared gene expression in T1alpha (-/-) lungs vs wild type at dpc 18.5 when the phenotype is moderate and at term when the phenotype is more severe. Lungs present narrow and irregular alveolar spaces, thicker mesenchyme, reduced number of attenuated type I cells, normal numbers of type II cells and secreted surfactant. At term, but not a dpc 18.5, there is increased proliferation of distal cells.

We compared gene expression of T1alpha null mutant lungs to wild type lungs at dpc 18.5. The mutant phenotype at this developmental stage is moderate but the alveolar sacs are narrower than normal.

Comparison of post-mortem tissue from brain BA10 region between schizophrenic and control patients.

Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function.
Schizophrenia is a severe psychiatric disorder with a world-wide prevalence of 1%. The pathophysiology of the illness is not understood, but is thought to have a strong genetic component with some environmental influences on aetiology. To gain further insight into disease mechanism, we used microarray technology to determine the expression of over 30 000 mRNA transcripts in post-mortem tissue from a brain region associated with the pathophysiology of the disease (Brodmann area 10: anterior prefrontal cortex) in 28 schizophrenic and 23 control patients.

The Acute Respiratory Distress Syndrome (ARDS)/Acute Lung Injury (ALI) was described 30 years ago, yet the interaction between specific sets of genes involved in this syndrome remains incompletely understood.

Transscriptomics, proteomics, quercetin-exposed and control rats

Transcriptomics and proteomics in colon mucosa from rats fed quercetin

Quercetin has been shown to act as an anti-carcinogen in experimental colorectal cancer (CRC). The aim of the present study was to characterise transcriptome and proteome changes occurring in the distal colon mucosa of rats supplemented with 10 g quercetin/kg diet for 11 weeks. Transcriptome data analysed with Gene Set Enrichment Analysis showed that quercetin significantly downregulated the potentially oncogenic mitogen-activated protein kinase (Mapk) pathway. In addition, quercetin enhanced expression of tumor suppressor genes, including Pten, Tp53 and Msh2, and of cell cycle inhibitors, including Mutyh. Furthermore, dietary quercetin enhanced genes involved in phase I and II metabolism, including Fmo5, Ephx1, Ephx2 and Gpx2. Quercetin increased PPARα target genes, and concomitantly enhanced expression genes in volved in of mitochondrial fatty acid degradation. Proteomics performed in the same samples revealed 33 affected proteins, of which 4 glycolysis enzymes and 3 heatshock proteins were decreased. A proteome-transcriptome comparison showed a low correlation, but both pointed out towards altered energy metabolism.

In conclusion, transcriptomics combined with proteomics showed that dietary quercetin evoked changes contrary to those found in colorectal carcinogenesis. These tumor-protective mechanisms were associated with a shift in energy production pathways, pointing at decreased glycolysis in the cytoplasm towards increased fatty acid degradation in the mitochondria.

Jurkat T cell line was transfected with SIV Nef controlled by an unducible promoter system. Nef was induced by addition of 10 micromolar pronasterone A and gene expression values were determined after 24 hrs. Control Jurkat cells were untransfected and treated with 10 micromolar pronasterone A and gene expressio values were determined after 24 hrs.
Keywords: SIV Nef, Jurkat, pronasterone A

Parsing the roles of transcription factors Gata4 and Gata6 in adult cardiac hypertrophic responses

Cardiac hypertrophy is regulated by the zinc finger-containing DNA binding factors Gata4 and Gata6, both of which are required to mount a productive growth response of the adult heart.  To determine if Gata4 and Gata6 are redundant or have non-overlapping roles, we performed cardiomyocyte-specific conditional gene deletions for Gata4 and Gata6 in conjunction with reciprocal replacement with a transgene encoding either Gata4 or Gata6, during the pressure overload response.  We determined that Gata4 and Gata6 play a redundant and dosage-sensitive role in programming the hypertrophic growth response itself following pressure overload stimulation. However, non-redundant functions were identified as functional decompensation induced by either Gata4 or Gata6 deletion was not rescued by the reciprocal transgene, and only Gata4 heart-specific deletion produced a reduction in capillary density after pressure overload.  Gene expression profiling from hearts of these gene-deleted mice showed both overlapping and unique transcriptional codes, with Gata4 exhibiting the strongest impact. These results indicate that Gata4 and Gata6 play a dosage-dependent and semi-redundant role in programming cardiac hypertrophy, but that each has a unique role in maintaining cardiac homeostasis and adaptation to injury that cannot be compensated by the other.
Microarray-bassed gene expression profiling identified overlapping, distinct, and quantitatively/differentially regulated classes of Gata4 or Gata6 regulated genes.

Total RNA was extracted from mouse liver samples using an Ambion RNaqueous RNA extraction kit. Targets were produced using modified Affymetrix procedure from 5ug of total RNA. 3 biological replicates were analysed per each group of animals.
Keywords = growth hormone

Expression data from Control, Uninfected and BRAF infected cells

Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or N-RAS oncogene and express markers of senescence, including p16. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 20-30% of melanoma are thought to arise in association with a pre-existing nevus. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. We set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as cyclin D1 and c-myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, cyclin D1 and c-myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signalling in nevi correlated inversely with nevus maturation, an established dermatopathological correlate of clinical benignancy. Single cell analyses of lone epidermal melanocytes and nevus melanocytes showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16-positive non-malignant melanocytes. We conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of clinical benignancy, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi.
We used microarrays to detail the global programme of gene expression after lentiviral infection of BRAF in 3 replicates.

The endogenous peptide Apelin is crucial for maintaining heart function in pressure overload and aging
Keywords: knockout

genetic modification, wilde type (wt) vs. knock out (k.o.) liver samples

Impaired liver regeneration in Nrf2 knockout mice caused by ROS-mediated insulin/IGF-1 resistance

The liver is frequently challenged by surgery-induced metabolic overload, viruses, or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signalling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress. As a consequence, tissue damage was aggravated, and liver regeneration after partial hepatectomy was delayed.
Using in vitro and in vivo studies we identified oxidative stress-induced insulin/insulin-like growth factor resistance as the underlying mechanism. This deficiency impaired the activation of p38 mitogen-activated kinase, Akt kinase, and downstream targets after hepatectomy, resulting in enhanced death and delayed proliferation of hepatocytes. Our results reveal novel roles of Nrf2 in the regulation of growth factor signalling and in tissue repair. In addition, they provide new insight into the mechanisms underlying oxidative stress-induced defects in liver regeneration and thus offer new avenues to improve regeneration in patients with acute or chronic liver damage.

Identification of genes controlled by LMX1B in the developing mouse hindlimb bud

A control vs. genetic knockout experiment aimed at determining what RNAs are upregulated or downregulated in e11.5 mouse proximal limb tissue lacking the Lmx1b gene.  Because Lmx1b is required for dorsal-ventral patterning of the limb, this screen gives insight into what putative downstream targets of Lmx1b contribute to dorsal-ventral patterning.
Keywords: Genetic allele comparison

Glomerular abnormalities have been demonstrated in kidney biopsies of patient after orthotopic liver transplantation (OLT). We hypothesize that these changes exist prior to OLT and may play a role in the development of renal failure after OLT. We use gene expression microarrays to investigate the mechanism of kidney disease in patients listed for OLT.
Gene expression profiles of biopsies of cirrhotic patients were compared with pre-implantation living donor biopsies. Glomerular abnormalities were seen in 92% of the biopsies, the most common being increase in mesangial matrix. Electron microscope showed effacement of podocytes (93%) and duplication (35%) and widening of glomerular basement membrane (45%). Gene Ontology analysis revealed significant up-regulation of genes implicated in immune response, including T-cell, leucocyte and platelet activation and differentiation. Pathogenesis-based transcripts analysis revealed significantly increased expression of cytotoxic T-cell, macrophage, B-cell, natural killer cell, and endothelial cell associated transcripts, indicating an ongoing inflammatory immune response.

Transcriptome profiling of Transforming Growth Factor B1-Induced Mesangial Gene Expression

TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood.  In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease.

Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs.

Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 °C for 5 min, equilibrated at 45 °C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice.

Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david).

Abdominal Aortic Aneurysm in the ApoE Knockout Mouse: Time Course of Control vs. Angiotensin II

Transcriptional profiling of suprarenal aorta from ApoE-/- mice  (12-14 weeks old, C57BL/6J background)  treated by subcutaneous pump with angiotensin II or saline for 7d, 14d and 28d. Includes Ang II-treated samples at 7d found to have dissected aneurysms.
Goal was to examine gene expression in developing AAA in this model over time.

Cardiac hypertrophy, Tropomyosin, Mutation, Transgenic mouse, Microarray

Microarray analysis of gene expression during early stages of mild and severe cardiac hypertrophy

Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function.  We previously developed two transgenic mouse models that carry FHC associated mutations in  alpha-tropomyosin (TM):  FHC  alpha-TM175 mice show patchy areas of mild ventricular disorganization and limited hypertrophy; whereas FHC  alpha-TM180 mice exhibit severe hypertrophy and fibrosis and die within 6 months.  To obtain a better understanding of the molecular mechanisms associated with the early onset of cardiac hypertrophy, we conducted a detailed comparative analysis of gene expression in 2.5-month-old control and FHC  alpha-TM175 and  alpha-TM180 ventricular tissue.  Results show that 754 genes (from a total of 22,600) were differentially expressed between the NTG and the FHC hearts.  There are 178 differentially regulated genes between NTG and the FHC  alpha-TM175 hearts, 388 genes are differentially expressed between NTG and FHC  alpha-TM180 hearts, and 266 genes are differentially expressed between FHC  alpha-TM175 and FHC  alpha-TM180 hearts.  Genes that exhibit the largest increase in expression belong to the "secreted/extracellular matrix" category, and those with the most significant decrease in expression are associated with "metabolic enzymes."
Keywords: Cardiac hypertrophy, Tropomyosin, Mutation, Transgenic mouse, Microarray

Insulin and IGF-1 promote adipocyte differentiation via complex and overlapping signalling networks.  Here we used microarray analysis of brown preadipocytes derived from wild-type and insulin receptor substrate (IRS) knockout (KO) animals, which exhibited progressively impaired differentiation, to define the set of genes that predict adipogenic potential in these cells.  374 genes/ESTs were identified whose expression in preadipocytes correlated with their ultimate ability to differentiate.  Many of these genes were related to early adipogenic events, including genes involved in extracellular matrix, cytoskeletal organization, growth arrest, post-mitotic clonal expansion, and inhibitors of adipogenesis, including preadipocyte factor-1 and multiple members of the Wnt-signalling pathway.  Reconstitution of IRS-1 KO cells with IRS-1 reversed these changes and restored the ability to differentiate.  Several of these genes showed concordant changes in brown adipose tissue in vivo.  Necdin was markedly increased in IRS-1 KO cells that could not differentiate, and knockdown of necdin restored brown adipogenesis with down-regulation of Pref-1 and Wnt10a expression.  We demonstrated a necdin-E2F4 interaction repressing PPARg transcription.  IRS proteins regulated necdin via a CREB dependent pathway, defining a signalling network involved in brown preadipocyte determination.
Keywords = brown fat

Transcriptome Profiling of High Glucose and Glucosamine-Induced Mouse Mesangial Cells

The renal mesangial cells play an important role in the development of diabetic glomerulosclerosis and renal failure. We have previously demonstrated some of the effects of high glucose are mediated via the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 chips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on the transcriptomes of a mouse mesangial cell line (MES-13). Of the 34,000 genes on the chip, ~55-60% genes are detected in MES-13 cells. HG induces the expression of ~369 genes at >2-fold where 263 genes are up and 106 genes down regulated. Similarly, GlcN increases the expression of 120 genes and decreases 94 genes. Seventy-two genes are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The differential expressions of several genes found in the microarray are also confirmed by quantitative PCR. Significant pathways co-modulated by HG and GlcN are the thioredoxin system (a 20-fold increase in thioredoxin interacting protein expression), endoplasmic reticulum (ER) stress, extracellular matrix and interferon-inducible genes. Furthermore, HG and GlcN target various intracellular pathways including the mitogen-activated protein kinase, TOLL-like receptor, fructose and mannose metabolism, and the biosynthesis of steroids and N-glycans. We conclude from this microarray data and other experimental results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes ER stress, oxidative stress and the interferon-inducible gene expression to cause cell cycle arrest, ECM gene expression and apoptosis.

Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with the desired concentrations of glucose and GlcN for 48h (LG, 5.5 mM; HG, 25 mM and GlcN, 1.5 mM + LG).

Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix U430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA (from LG, HG and GlcN treated MES-13 cells) was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled-cRNAs were purified in RNeasy spin columns (Qiagen, Valencia, CA). Fifteen mg of each cRNA was fragmented at 94 °C for 35 min in a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). These cRNA fragments were then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100). The solution was equilibrated at 45 °C for 5 min and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the mater mix) were hybridized to GeneChip Mouse Genome U430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm. After the overnight hybridization, the chips were washed with a non-stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by a stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween-20) at 50 °C. The GeneChips were then stained with streptavidin-phycoerythrin (Molecular Probe), washed with 6x saline/sodium phosphate/EDTA, incubated with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin. Finally, a third wash with 6x saline/sodium phosphate/EDTA was performed using the GeneChip Fluidics Station 450. The arrays were then scanned in a GeneChip Scanner 3000 (Affymetrix).  Each experiment was repeated twice.

Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (http://appls.niaid.nih.gov/david).

Analysis of target genes induced by the Amyloid Precursor Protein Intracellular Domain

The amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer’s disease (AD). Processing of APP by β- and γ-secretase activities results in the production of ß-amyloid (Aß), the main constituent of Alzheimer plaques, but also in the generation of the APP intracellular domain (AICD). Recently, it has been demonstrated that AICD has transactivation potential, however, the targets of AICD dependent gene regulation and hence the physiological role of AICD remain largely unknown. In this work we analysed transcriptome changes during AICD dependent gene regulation using a human neural cell culture system inducible for expression of AICD, its co-activator Fe65, or the combination of both. Induction of AICD was associated with increased expression of genes with known function in the organization and dynamics of the actin cytoskeleton as well as genes involved in the regulation of apoptosis.
Keywords: (Tet-Off)

AIRE-deficient CD8+CD28low regulatory T lymphocytes fail to control experimental colitis

Mutations in the gene encoding the transcription factor AutoImmune REgulator (AIRE) are responsible for the ‘Autoimmune PolyEndocrinopathy Candidiasis Ecodermal Dystrophy’ syndrome. AIRE directs expression of tissue restricted antigens in the thymic medulla and in lymph node stromal cells and thereby substantially contributes to induction of immunological tolerance to self-antigens. Data from experimental mouse models showed that AIRE-deficiency leads to impaired deletion of autospecific T cell precursors. However, a potential role for AIRE in the function of regulatory T cell populations, which are known to play a central role in prevention of immunopathology, has remained elusive. Regulatory T cells of CD8+CD28low phenotype efficiently control immune responses in experimental autoimmune and colitis models in mice. We here show that CD8+CD28low Treg from AIRE-deficient mice are transcriptionally and phenotypically normal, exert efficient suppression of in vitro immune responses, but completely fail to prevent experimental colitis in vivo. Our data therefore demonstrate that AIRE plays an important role in the in vivo function of a naturally occurring regulatory T cell population.

Mechanism of the anti-inflammatory effect of colchicine in rheumatic diseases

Objective. Colchicine is an alkaloid that is used to alleviate acute gout and to prevent acute attacks of familial Mediterranean fever (FMF). However, it is not beneficial when given during the occurrence of an acute episode of FMF. It is believed that colchicine exerts its anti-inflammatory effect through direct interaction with microtubules. We aim to study the molecular basis of colchicine action by analysing the effect of this drug on global gene expression of HUVEC (human umbilical vein endothelial cell line) cells. Methods. HUVEC cells were exposed to various concentrations of colchicine and were harvested at different time points. Ribonucleic acid was extracted, amplified, reverse transcribed and hybridized to complementary deoxyribonucleic acid microarrrays containing more than 40,000 probes to human expressed sequence tags. This approach enabled us to have a global look at the transcriptional response induced by colchicine treatment. Results. Colchicine changed the expression of many genes in HUVEC cells following exposure to a concentration of 100 ng/ml or higher. Following short exposure (30 or 120 min), colchicine affected genes known to be involved in the cell cycle and its regulation. However, change in expression of genes involved in neutrophil migration or other inflammatory processes were observed mainly after 12 to 24 h. Conclusions. The anti-inflammatory effect of colchicine may be mediated not only through direct interaction with microtubules but also through changes at the transcriptional level. This latter effect apparently requires a higher concentration and a longer time to occur. This can explain the observation that colchicine does not have an immediate effect when given during an acute attack of FMF.
A dose response design type examines the relationship between the size of the administered dose and the extent of the response of the organism(s).

Expression data from normal human fibroblasts expressing prelamin A or progerin, untreated or treated with farnesyltransferase inhibitor (FTI)

We compared the transcriptomes of isogenic diploid fibroblasts expressing progerin or elevated levels of wild-type prelamin A with that of wild-type fibroblasts. We subsequently used the reversion towards normal of two phenotypes, reduced cell growth and dismorphic nuclei, by treatment with farnesyltransferase inhibitor (FTI) or overexpression of ZMPSTE24, as a filtering strategy to identify genes linked to the onset of these two phenotypes.

Expression data from RUNX1(41-214)-expressing and Runx1-knockout mice KSL cells

Mutations of RUNX1 are detected in patients with myelodysplastic syndrome (MDS). In particular, C-terminal truncation mutations lack a transcription regulatory domain and have increased DNA binding through the runt homology domain (RHD). The expression of the RHD, RUNX1(41-214), in mouse hematopoietic cells induced progression to MDS and acute myeloid leukemia (AML). Analysis of pre-myelodysplastic animals revealed expansion of c-Kit+Sca-1+Lin- (KSL) cells and skewed differentiation to myeloid at the expense of the lymphoid lineage. These abnormalities correlate with the phenotype of Runx1-deficient animals, as expected given the reported dominant-negative role of C-terminal mutations over the full-length RUNX1. However, MDS is not observed in Runx1-deficient animals. Gene expression profiling revealed that RUNX1(41-214) KSLs have an overlapping yet distinct gene expression profile from Runx1-deficient animals. Moreover, an unexpected parallel was observed between the hematopoietic phenotype of RUNX1(41-214) and aged animals. Genes deregulated in RUNX1(41-214), but not in Runx1-deficient animals, were inversely correlated with the aging gene signature of hematopoietic stem cells (HSC), suggesting that disruption of the expression of genes related to normal aging by RUNX1 mutations contributes to development of MDS. The data presented here provide insights into the mechanisms of development of MDS in HSCs by C-terminal mutations of RUNX1.

Synthetic Lethal Interaction Between Oncogenic KRAS Dependency and Suppression of STK33 in Human Cancer Cells

Many human oncogenes are challenging therapeutic targets. An alternative to direct targeting of oncogenes is to perform “synthetic lethality” screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of the broad spectrum of mutant KRAS-driven cancers, and demonstrate the potential of RNAi screens for discovering critical functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers associated with “undruggable” genetic alterations.

Anti-TNF-alpha therapy has made a significant impact on the treatment of psoriasis. Despite being designed to neutralize TNF-alpha activity, the mechanism of action of these agents in the resolution of psoriasis remains unclear. The aim of this study was to better understand the mechanism of action of etanercept by examining very early changes in the lesional skin of psoriasis patients. 20 chronic plaque psoriasis patients were enrolled and received 50mg etanercept twice weekly. Skin biopsies were obtained before treatment and on days 1, 3, 7 and 14 post-treatment. Skin mRNA expression was analysed by microarray.

Huntington’s Disease (HD) is an inherited neurodegenerative disease caused by a glutamine repeat expansion in huntingtin protein.  Transcriptional deregulation and altered energy metabolism have been implicated in HD pathogenesis. We report here that mutant huntingtin causes disruption of mitochondrial function by inhibiting expression of PGC-1a, a transcriptional coactivator that regulates several metabolic processes including mitochondrial biogenesis and respiration.  Mutant huntingtin represses PGC-1a  gene transcription by associating with the promoter and interfering with the CREB/TAF4-dependent transcriptional pathway critical for the regulation of PGC-1a gene expression.  Crossbreeding of PGC-1a knockout mice with HD knock-in mice leads to increased neurodegeneration of striatal neurons and motor abnormalities in the HD mice.  Importantly, expression of PGC-1a partially reverses the toxic effects of mutant huntingtin in cultured striatal neurons.  Moreover, lentiviral-mediated delivery of PGC-1a in the striatum provides neuroprotection in the transgenic HD mice.  These studies suggest a key role for PGC-1a in the control of energy metabolism in the early stages of HD pathogenesis.
Keywords: PGC-1a, striatum

Expression data from T-cell prolymphocytic leukemia (TPLL) and normal T cells

T-cell prolymphocytic leukemina (T-PLL) is an agressive lymphoma derived from mature T-cells, which is in most cases characterized by the presence of an inv(14)(q11q32) and a characteristic pattern of secondary chromosomal abberations.
We used microarrays to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor derived peripheral blood cells with five highly purified inv(14)-positive T-PLL blood samples.

Adult and Neonatal dorsal horn gene expression 7 day post sciatic nerve SNI injury

As rats do not develop neuropathic pain like hypersensitivity as neonates post nerve injury but do as adults we have used these arrays to help define the processes involved in this process.
Rat spinal cord (ipsilateral dorsal horn) was assayed 7 days post SNI injury to the sciatic nerve relative to sham injury. Two age groups of animals were tested Neonates (P10) and Adult (8-12wks).

Gene expression profiles induced by knockdown and overexpression of p63 variants in MCF-10A mammary epithelial cell line

p63 is critical for epithelial development yet little is known about the transcriptional programmes it regulates. The p63 transactivating (TA) isoforms contain an amino-terminal exon that encodes a p53-like transactivation domain, whereas ΔN-isoforms lack this domain but contain the common DNA binding domain (DBD), suggesting that TAp63 and ΔNp63 isoforms may have opposing functions.  By characterising transcriptional changes and cellular effects following modulation of p63 expression, we have defined a vital role for p63 in cellular adhesion. Knockdown of p63 expression caused downregulation of cell adhesion-associated genes, cell detachment and anoikis in mammary epithelial cells and keratinocytes. Conversely, overexpression of the TAp63γ or ΔNp63α isoforms of p63 upregulated cell adhesion molecules, increased cellular adhesion and conferred resistance to anoikis.

Gfi1 regulates survival and lineage commitment of hematopoietic precursors and prevents myeloproliferative diseases

Hematopoietic stem cells (HSCs) and lymphoid-primed multi-potential progenitors (LMPPs) are able to initiate both lymphoid and myeloid differentiation. We show here that the transcriptional repressor Gfi1 (growth factor independence 1) implements a specific gene expression program in HSCs and LMPPs that is critical for their survival and lymphoid differentiation potential. We present evidence that Gfi1 is required to maintain expression of genes involved in lymphoid development such as Flt-3, IL7R, Ebf1, Rag1, CCR9 and Notch1 and controls myeloid lineage commitment  by regulating expression of genes such as Hoxa9 or M-CSFR. Gfi1 also inhibits apoptosis in HSCs by repressing pro-apoptotic genes such as Bax or Bak. As a consequence, Gfi1-/- mice show defects in self renewal, survival and both myeloid and lymphoid development of HSCs and LMPPs. Co-expression of a Bcl-2 transgene can partially restore the function of HSCs in Gfi1-/- mice, but not the defects in early lymphoid development. Of interest, Gfi1-/- x Bcl-2 transgenic mice show an accelerated expansion of myeloid cells and succumb to a fatal myeloproliferative disease resembling chronic myelomonocytic leukemia (CMML). Our data show that Gfi1 protects HSCs against apoptosis, ensures the proper development of LMPPs and plays a role in the development of myeloid leukemia.
We used microarrays to detail the global gene expression changes following knockout of Gfi1 in mouse LSK cells

Protocadherin 12 (Pcdh12) is a transmembrane adhesive protein with homophilic adhesive properties and expressed in endothelial cells, the glycogen trophoblast cells of the placenta, and the mesangial cells of kidney glomeruli. Pcdh12-deficient mice are alive although they show alterations in placenta development.
To reveal Pcdh12-associated pathways, 45,000 - Affymetrix probe arrays were used on Pcdh12 knockout and control placentas.

HD;Huntingtin;cognitive disorder;R6/2;transgenic mice;gene expression;microarray;ordered

Huntington’s disease is a genetic disease caused by a single mutation. It is characterised by progressive movement, emotional and cognitive deficits. R6/2 transgenic mice carrying the Huntington’s disease mutation have a progressive neurological phenotype, including deterioration in cognitive function. The mechanism underlying the cognitive deficits in R6/2 mice is unknown, but dysregulated gene expression, reduced neurotransmitter levels and abnormal synaptic function are present before the cognitive decline becomes pronounced. Our goal here was to ameliorate the cognitive phenotype in R6/2 mice using a combination drug therapy (tacrine, moclobemide and creatine) aimed boosting neurotransmitter levels in the brain. Treatment from 5 weeks of age prevented deterioration in two different cognitive tasks until at least 12 weeks. However, motor deterioration continued unabated. Microarray analysis of global gene expression revealed that many genes significantly up- or down-regulated in untreated R6/2 mice had returned towards normal levels after treatment. Thus dysregulated gene expression was reversed by the combination treatment in the R6/2 mice and probably underlies the observed improvements in cognitive function. Our study shows that cognitive decline caused by a genetic mutation can be slowed by a combination drug treatment, and gives hope that cognitive symptoms in HD can be treated.
Keywords = HD

We performed global scale microarray analysis to identify detailed mechanisms by which sodium butyrate (SB) induce cell growth arrest and differentiation of colonic epithelial cells by using an Affymetrix GeneChip system. Colonic epithelial MCE301 cells used in this study were derived from transgenic mice harboring a tsSV40 large T-antigen. Arrested cell growth and a differentiated phenotype accompanying elevations of alkaline phosphatase activity and histone acetylation were observed in the cells treated with 2 mM SB. Of the 22,690 probe sets analyzed, approximately 2,000 genes were down- and up-regulated by a factor of 2.0 or greater in the cells treated with SB.
Keywords: sodium butyrate, gene expression, colonic epithelial cell

Comparison of the genetic extinction of NRAS to pharmacological MEK inhibition in an inducible mouse model of melanoma

Since direct pharmacological inhibition of RAS has thus far been unsuccessful, we explored system biology approaches to identify synergistic drug combination(s) that can mimic direct RAS inhibition. Leveraging an inducible mouse model of NRAS-mutant melanoma, we compare pharmacological MEK inhibition to complete NRAS-Q61K extinction in vivo. NRAS-Q61K extinction leads to a complete and durable tumor regression by enhancing both apoptosis and cell cycle arrest. By contrast, MEK inhibition only produces tumor stasis at best and we find that it robustly activates apoptosis but does not significantly impede proliferation.
We used microarrays to determine which transcripts were affected by NRAS-Q61K extinction but insufficiently by MEK inhibition. We selected a  single comparative timepoint, 4 days post-treatment. Downstream analyses included GSEA and TRAP algorithms, leading to the identification of a differentially affected CDK4-driven proliferation network. Immune genes were also identified as significant, but control experiments determined these to be largely the off-target effects of doxycycline and not of NRAS-Q61K extinction.

Molecular analysis of circadian rhythm in mice. Liver tissue of wildtype, Clock mutant and Cry deficient C57BL/6  8- to 10-week-old male mice examined.
Keywords = circadian rhythm

Comparison of  Wildtype and FSH-Receptor Knock out Ovaries at 8 months of age.

To understand consequences of loss of FSH receptor, we used genetically altered mouse mutants with deletion of this receptor. Follitropin Receptor Knockout (FORKO) mice present various developmental and age-dependent abnormalities such as infertility, senescence and increased tumor incidences in addition to other phenotypes. To explore why the atrophic ovaries of FORKO mice develop ovarian anomalies later in life, we used expression profiling studies to gain a more comprehensive view of genes that are misregulated.
Keywords: Comparison of  Wildtype and FSH-Receptor Knock out Ovaries at 8 months of age.

Age-related transcriptional changes and the effect of dietary supplementation of vitamin E in the mouse heart

To investigate the global effects of vitamin E supplementation on heart aging, we used high-density oligonucleotide arrays to measure transcriptional alterations in 30-month-old B6C3F1 mice supplemented with α- and γ-tocopherol since middle age (15 months).
Keywords: expression profiling

DNA methylation status of myelinating Schwann cells during development and in diabetic neuropathy [Gene Expression Array: GNMT mice]

DNA methylation is a key epigenetic regulator of mammalian embryogenesis and somatic cell differentiation. Using high-resolution genome-scale maps of methylation patterns, we show that the formation of myelin in the peripheral nervous system, proceeds with progressive DNA demethylation, which coincides with an upregulation of critical genes of the myelination process. More importantly, we found that, in addition to expression of DNA methyltransferases and demethylases, the levels of S-adenosylmethionine (SAMe), the principal biological methyl donor, could also play a critical role in regulating DNA methylation during myelination and in the pathogenesis of diabetic neuropathy. In summary, this study provides compelling evidence that SAMe levels need to be tightly controlled to prevent aberrant DNA methylation patterns, and together with recently published studies on the influence of SAMe on histone methylation in cancer and embryonic stem cell differentiation show that in diverse biological processes, the methylome, and consequently gene expression patterns, are critically dependent on levels of SAMe.
Axonal myelination by Schwann cells in the peripheral nervous system is essential for rapid saltatory impulse conduction, and malformation or destruction of myelin sheaths can lead to severe motor and sensory disabilities (peripheral neuropathies). Using high-resolution genome-scale methylome maps, we found that DNA methylation could play a critical role in the generation of myelinated Schwann cells. This process was accompanied by a global DNA demethylation at most genomic elements. Notably, demethylation at gene-regulatory regions was associated with activation of critical myelination-specific genes. Furthermore, we found an aberrant DNA methylation pattern in a mouse model of diabetic neuropathy, which could be involved in the pathogenesis of the disease. Importantly, we found that these methylation patterns in both situations could be regulated by levels of S-adenosylmethionine (SAMe), the principal biological methyl donor. Together with recent studies on the influence of SAMe on histone methylation in diverse biological processes, we conclude that the methylation landscape of cells could be critically dependent on levels of SAMe. These provide a mechanistic link between metabolism and gene regulatory networks in normal and pathological situations.

Expression profiling the response to inhibition of DNA methylation and histone deacetylation.
Comparison of expression in HepG2 cells treated with 5-aza-dC, Trichostatin A, both, or none (control) to change methylation and acetylation status.
Background:DNA methylation and histone deacetylation are epigenetic mechanisms that play major roles in eukaryotic gene regulation. We hypothesize that many genes in the human hepatoma cell line HepG2 are regulated by DNA methylation and histone deacetylation. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) to inhibit DNA methylation with and/or Trichostatin A (TSA) to inhibit histone deacetylation should allow us to identify genes that are regulated epigenetically in hepatoma cells.
Results:5-aza-dC had a much larger effect on gene expression in HepG2 cells than did TSA, as measured using Affymetrix® HG-U133A Plus 2.0 microarrays. The expression of 1504 probe sets was affected by 5-aza-dC (at p < 0.01), 535 probe sets by TSA, and 1929 probe sets by the combination of 5-aza-dC and TSA. 5-aza-dC treatment turned on the expression of 211 probe sets that were not detectably expressed in its absence. Expression of imprinted genes regulated by DNA methylation, such as H19 and NNAT, was turned on or greatly increased in response to 5-aza-dC. Genes involved in liver processes such as xenobiotic metabolism (CYP3A4, CYP3A5, and CYP3A7) and steroid biosynthesis (CYP17A1 and CYP19A1), and CCAAT element-binding proteins (CEBPA, CEBPB,  and CEBPG) were affected by 5-aza-dC or the combination. Many of the genes that fall within these groups are also expressed in the developing fetal liver. Quantitative real-time RT-PCR assays confirmed selected gene expression changes seen in microarray analyses.
Conclusions:Epigenetics play a role in regulating the expression of several genes involved in essential liver processes such as xenobiotic metabolism and steroid biosynthesis in HepG2 cells. Many genes whose expression is normally silenced in these hepatoma cells were re-expressed by 5-aza-dC treatment. Many genes that are expressed in the fetal liver are up-regulated by demethylation, indicating that DNA methylation is a major factor in restricting the expression of fetal genes during liver development.

STAT1 independent control of a neurotropic measles virus challenge in primary neurons and infected mice

The objective of this study was to compare the ability of mice that lack STAT1 to resolve a neurotropic viral challenge, and to assess the ability of neurons obtained from these mice to be effectively cleared of virus by interferon gamma

Pit-1 overexpression in the human breast adenocarcinoma MCF-7 cell line

The aim of the study is to evaluate Pit-1-induced genes in the MCF-7 cell line
The Pit-1 transcription factor (also known as POU1F1) plays a critical role in cell differentiation during organogenesis of the anterior pituitary in mammals and is a transcriptional activator for pituitary gene transcription. Increased expression of Pit-1 has been reported in human tumorigenic breast cells. Here, we found that Pit-1 overexpression or knockdown in human breast cancer cell lines induced profound phenotypic changes in the expression of proteins involved in cell proliferation, apoptosis, and invasion. In immunodeficient mice, Pit-1 overexpression induced tumoral growth and promoted metastasis in lung. In patients with invasive ductal carcinoma of the breast and node-positive tumors elevated expression of Pit-1 was significantly and independently associated with the occurrence of distant metastasis. These findings suggest that Pit-1 could help to make a more accurate prognosis in patients with node positive breast cancer and may represent a new therapeutic target (Journal of Clinical Investigation 2010, 120:4289-4302)

Abstract.
Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematological malignancies enhanced NF-kB exerts prosurvival functions. Here we investigated the role of NF-kB in mouse and human c-MYC-transformed lymphomas. The NF-kB-pathway is extinguished in murine lymphoma cells and extrinsic stimuli typically inducing NF-kB activity fail to activate this pathway. Genetic activation of the NF-kB pathway induces apoptosis in these cells, while inhibition of NF-kB by an IkBa superrepressor provides a selective advantage in vivo. Furthermore, in human Burkitt´s lymphoma cells we find that NF-kB activation induces apoptosis. NF-kB upregulates Fas and predisposes to Fas-induced cell death, which is caspase 8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kB-induced apoptosis and persistent inacvtivity of NF-kB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC driven lymphoma cells is reversed by activation of NF-kB. Our observations provide a molecular explanation for the described absence of the NF-kB signaling in Burkitt´s lymphoma and question the applicability of NF-kB inhibitors as candidates for treatment of this cancer.

Trovafloxacin-Induced Gene Expression Changes: Comparison of Primary Human Hepatocytes to HepG2 Cells

Primary human hepatocytes (PHH) are a main instrument in drug metabolism research and in the prediction of drug-induced phase I/II enzyme induction in humans.  The HepG2 liver-derived cell line is commonly used as a surrogate for human hepatocytes, but their use in ADME and toxicity studies can be limited because of lowered basal levels of metabolizing enzymes.  Despite their widespread use, the transcriptome of HepG2 cells compared to PHH is not well characterized.  In this study, microarray analysis was conducted to ascertain the differences and similarities in mRNA expression between HepG2 cells and human hepatocytes before and after exposure to a panel of fluoroquinolone compounds. Comparison of the naïve HepG2 cell and PHH transcriptomes revealed a substantial number of basal gene expression differences.  When HepG2 cells were dosed with a series of fluoroquinolones, trovafloxacin, which has been associated with human idiosyncratic hepatotoxicity, induced substantially more gene expression changes than the other quinolones, similar to previous observations with PHH. While TVX-treatment resulted in many gene expression differences between HepG2 cells and PHH, there were also a number of TVX-induced commonalities, including genes involved in RNA processing and mitochondrial function.   Taken together, these results provide insight for interpretation of results from drug metabolism and toxicity studies conducted with HepG2 cells in lieu of PHH, and could provide further insight into the mechanistic evaluation of TVX-induced hepatotoxicity.
Keywords: Cell Type Comparison

Genome-wide comparative analysis of gene expression in  wild type (WT3) and TRPM7-/- (K09) mouse embryonic stem cells

TRPM7 is a ubiquitous ion channel and kinase, a unique ‘chanzyme’, required for proper early embryonic development. In order to assess the effects of TRPM7 activity on cellular gene expression, mouse embryonic stem cells with TRPM7 gene deletion (TRPM7-/- mESC) were established. By using microarray analysis, we identified genes with transcription significantly different in TRPM7-deficient mESC.

Expression profiling of cyclin D1 splice variants cyclin D1a and D1b in mouse 3T3 cells.

Cyclin D1 is an important cell cycle regulator but in cancer its overexpression also increases cellular migration mediated by p27KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N-terminus as the canonical cyclin D1a isoform but a distinct C-terminus. Analysis was performed of mouse cyclin D1 knockout 3T3 cells infected with splice variants of cyclin D1. 3T3 cells transduced with retroviral vectors expressing each cyclin D1 isoform were processed for expression analysis.
Keywords: Cancer associated risk factor

Gene expression profiling of mouse uterine tissue from progesterone receptor-Cre knockout of Dicer

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNAs (miRNA) have been implicated in several reproductive processes, the specific role of Dicer and miRNA in uterine development is not known. To address the roles of miRNA in the regulation of these key uterine pathways, we generated a conditional knockout (cKO) of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor (PR)-Cre. These Dicer cKO are sterile with small uteri, which demonstrate significant defects including absence of glandular epithelium and enhanced stromal apoptosis, beginning at postnatal day 15 with expression of Cre and deletion of Dicer. Although these mice had normal serum steroid hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/Beta-catenin canonical pathway, were dysregulated at the mRNA level.

Influenza virus infection-induced gene expression changes of regional B cells are mediated in part through type I IFN

Influenza virus infection-induced gene expression changes of regional B cells are mediated at least in part through type I Interferon:
Our objective is to determine whether the influenza virus-infection induced gene expression changes in regional lymph node B cells are facilitated at least in part through type I interferon.  Our specific aim is to compare the gene expression profile of highly FACS-purified B cells in the regional lymph nodes of wildtype and IFNR-/- mice prior to and 48h following infection with influenza virus infection and to contrast this expression profile with that of FACS-purified wildtype B cells activated in vitro with IFN-beta +/- anti-CD86 for 12h.

In this experiments different treatments were applied to lung cancer cell lines

1)H720 cell line suspension was treated by 1 micromole of 5-lipoxygenase activating protein inhibitor MK886 serum-free in TIS medium for 24 h.
2) A549 cell line was treated as H720 by MK886
3) A549 was treated by 100 nM of silencing cocktail (siRNA) prepared against the 4-th exon of the gene and generated by RNASEIII digestion of double stranded RNA. The cocktail was prepared by Ambion technology, see the manufacturer's protocol. The observed  silencing was >90%
4) Based on Oligoengine vectors, silencing constructs were created against HRPA2B1 transcript(HNRPA2B1 is a subunit of spliceosome). Stable transfectants were raised harboring the construct.

Study muscle regeneration in Emerin and Lamin AC KO mice. Gastrocnemius muscles were injected with cardiotoxin. Muscles were isolated at different time points.
Keywords: Time-Series

Knockout mice reveal key roles for claudin-18 in alveolar fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJ), where they serve as regulators of paracellular permeability to ions and solutes. Claudin-18, a member of the large claudin family, is highly expressed in lung epithelium. To elucidate the role of claudin-18 in alveolar epithelial barrier function and fluid homeostasis, we generated claudin-18 knockout (C18 KO) mice. Increased alveolar fluid clearance (AFC) observed in C18 KO mice may have accounted for absence of lung edema despite increased alveolar solute permeability compared to wild type (WT) controls. Higher AFC in C18 KO mice was associated with higher Na-K-ATPase activity and increased expression of the Na-K-ATPase β1 subunit compared to WT controls. Consistent with in vivo findings, alveolar epithelial cell (AEC) monolayers derived from C18 KO mice exhibited lower transepithelial electrical resistance (RT) accompanied by increased solute and ion permeability without changes in ion selectivity. Expression of claudin-3 and claudin-4 was markedly increased in whole lung and in freshly isolated AEC from C18 KO mice, while claudin-5 was unchanged. In contrast, occludin, another major component of the TJ complex, was significantly decreased in C18 KO lung. Further analysis revealed rearrangements in the F-actin cytoskeleton in C18 KO MAECM. These findings demonstrate a crucial non-redundant role for claudin-18 in regulation of alveolar epithelial tight junction composition and permeability to ions and solutes.  Importantly, increased AFC in C18 KO mice identifies additional roles for claudin-18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties of the alveolar epithelium.

Expression data from microdissected glomeruli to examine the role of hypoxia in glomerulosclerosis of human Nephrosclerosis (NSC).

Comparative analysis of gene expression  WT and Nrf2-/- mice Type II cells

We hypothesize that gene expression in the Type II cells of Nrf2+/+ and Nrf2-/- mice are divergent thus contributing the cell growth. More specifically, type II cells from Nrf2-/- mice have increased reactive oxygen species that cause the impaired cell growth.  In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between Nrf2+/+ and Nrf2-/- cells.
Keywords: comparative expression profiling

Gene expression is differently affected by pimecrolimus and betamethasone in lesional skin of atopic dermatitis.

Topical corticosteroids and calcineurin inhibitors are well known treatments of atopic dermatitis (AD), but differ in their efficacy and side effects. A study in AD patients has demonstrated that betamethasone valerate (BM) though clinically more efficient impaired skin barrier repair in contrast to pimecrolimus. Objective: The present study elucidates the mode of action of topical BM and pimecrolimus cream in AD.
Methods: These two components were subjected to gene expression profile analysis in lesional AD skin after topical treatment.
Results: BM resulted in a significant reduction of mRNA levels of genes encoding for markers for dendritic cells, T cells, cytokines, chemokines and serine proteases, whereas pimecrolimus exerted minor effects only. This corroborates the clinical finding that BM reduces inflammation more effectively than pimecrolimus. Genes encoding molecules important for skin barrier function were differently affected. Both BM and pimecrolimus normalized the expression of filaggrin and loricrin. BM, but not pimecrolimus, significantly reduced the expression of rate-limiting enzymes for lipid synthesis and the expression of involucrin and small proline-rich proteins, which covalently bind ceramides. This may explain the lack of restoration of functional stratum corneum layers observed after BM treatment.
Conclusion: The gene expression profiles are consistent with previous findings that corticosteroids may exert a more potent anti-inflammatory effect but may impair the restoration of the skin barrier. Hence, this study confirms the hypothesis at the molecular level that corticosteroids are a suitable treatment for acutely exacerbated AD, but that pimecrolimus is preferable for long-term treatment and stabilization.

Global gene expression is altered in heart failure. This syndrome can be caused by cardiovascular diseases, including dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), hypertrophic cardiomyopathy, viral or toxic myocarditis, hypertension, and valvular diseases.
We used microarrays to evaluate the impact of heart failure on human nucleocytoplasmic transport-related genes examining simultaneoulsly both dilated and ischemic human cardiomyopathies compared to normal hearts.

Integration-Free Induced Pluripotent Stem Cells Model Genetic and Neural Developmental Features of Down Syndrome Etiology

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of iPSCs to model DS phenotypes, as a prototypical complex human disease, we generated bona-fide DS and wild-type (WT) non-viral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency, and had remarkably similar lineage potency, differentiation kinetics, proliferation and axon extension at early time points. However, at later time points DS cultures showed a two-fold bias towards glial lineages.
Moreover, DS neural cultures were up to two times more sensitive to oxidative stress induced apoptosis, and this could be prevented by the anti-oxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy-21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Non-viral DS iPSCs can therefore recapitulate features of complex human disease in vitro, and provide a renewable and ethically unencumbered discovery platform.

Comparison of gene expression pattern between Wild-type and Trib1-deficient mice

Comparison of gene expression pattern between Wild-type and Trib1-deficient mice (Gene chip data for JEM 20070183)

The purpose of this experiment was to compare the gene expression pattern between wild-type and Trib1-deficient macrophages in response to LPS.
Keywords: Comparison of gene expression pattern between Wild-type and Trib1-deficient mice

Adam10, a cell surface protease, cleaving many proteins including TNF-alpha and E-cadherin. Here we investigate the genome wide effects of Adam10 knock out on the transcriptome.
Commercial microarrays (Affymetrix Mouse Gene ST 1.0) were used to generate genome wide mRNA profiles.

Idiosyncratic drug reactions (IDRs) cause significant morbidity and mortality. In an animal model of IDRs, 50-80% of Brown Norway rats exposed to D-penicillamine develop an autoimmune syndrome after several weeks of treatment. The symptoms of the IDR are similar to that observed in humans who take D-penicillamine. The mechanism of this reaction is unknown, and no effective biomarkers have been identified to predict susceptibility. We postulate that cell stress caused by drugs is required to initiate the response. We used a highthroughput approach to identify factors that might represent danger signals by profiling hepatic gene expression 6 h after dosing with D-penicillamine (150 mg/kg). Our results show that the drug-treated animals cluster into two distinct groups. One group exhibits substantial expression changes relative to control animals. The most significantly altered transcripts have a role in stress, energy metabolism, acute phase response, and inflammation. We used quantitative reverse transcriptase polymerase chain reaction to measure transcript levels in liver biopsies of 33 rats and found that resistant animals cluster together. This 'resistant' cluster of animals contains 87.5% (7/8) resistant animals but only 48% (12/25) 'sensitive' animals. This separation is statistically significant at the p 0.01 level.
Keywords: Response to Drug

Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (KO) and immunocompetent CBA mice, were used in microarrays containing more than 12,000 genes and expressed sequence tags (ESTs). We found 4515 transcripts expressed in duplicate experiments in all three strains. Out of these, 38 were differentially expressed genes (21 up-regulated >2 fold and 17 down-regulated <-2 fold) between CBA and Btk defective mice. Ten out of these genes were selected and quantitative Real-Time PCR was conducted for validation and further investigation. Real-Time experiments correlated nicely with the microarray data.

No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes, whose expression differed (>2 fold) between the two strains. Moreover, when the 38 genes, which differed between immunocompetent CBA and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant negative form.

Genes regulated by AGR2 in pancreatic cancer cell lines FA6 and MiaPaCa2

We and others have shown that AGR2 is frequently upregulated during the development of pancreatic cancer.
We used microarray to look at the target genes regulated by AGR2 in pancreatic cancer cell lines FA6 and MiaPaCa2.

Fibrogenic and redox-related but not proinflammatory genes are upregulated in lewis rat model of chronic silicosis

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNAs from controls or chronically silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of seven genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, qPCR, ELISA, Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after chronic silica exposure and compared with 14 d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, CCL2, and CCL7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis; however, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis; however, genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.

retinoblastoma, fetal liver, erythroblast, macrophage, cell death

Rb null embryos exhibit defective fetal liver erythropoiesis. We used microarrays to compare  Wt and Rb null fetal livers and to analyse gene expression differences which accompany and may underlie Rb null fetal liver degeneration, erythroid failure, and erythropoietic island dissolution.
We used microarrays to compare  Wt and Rb null fetal livers and analyse gene expression changes which accompany and may underlie fetal liver.

genetic modification, strain comparison, cell stimulation, time course, inflammatory response

response to IL-1b of WT and IRAK4 kinase dead mouse embryonic fibroblasts

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Though regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. In order to investigate the role of IRAK-4 kinase function in vivo, ‘knock-in’ mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase deficient IRAK-4 protein (IRAK-4 KD). Analysis of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice with a number of experimental techniques demonstrated that they greatly lack responsiveness to stimulation with IL-1b or a Toll-like receptor 7 (TLR7) agonist. One of the techniques used, microarray analysis, identified IRAK-4 kinase-dependent IL-1b response genes in mouse embryonic fibroblasts and revealed that the induction of IL-1b-responsive mRNAs was largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1R/TLR7-mediated induction of inflammatory responses.
Keywords: genetic modification, strain comparison, cell stimulation, time course, inflammatory response

Comparison of embryonic stem cell genomes between wild type and calreticulin knockouts

Expression data from wild type and calreticulin deficient murine embryonic stem cells

Primordial genomic challenge compromises embryonic development and survival, and surveillance of deployed transcriptional programs may provide an early opportunity to forecast phenotype abnormalities. Here, comparisons between wild-type and calreticulin-ablated embryonic stem cells revealed transcriptome shifts precipitated by calreticulin loss. Bioinformatic analysis identified down and up-regulation in 1187 and 418 genes, respectively. Cardiovascular development precedes other organogenic programs, and examination of cardiogenic genes revealed a map of calreticulin-calibrated expression profiles that encompass the developmental regulators, Ccnd1, Ccnd2 and Notch1. Interrogation of primary function in the resolved network forecasted abnormalities during myocardial development. Whole embryo magnetic resonance imaging, verified by pathoanatomical analysis, diagnosed prominent ventricular septal defect. Correlation clustering and network resolution of probesets associated with protein folding/chaperoning and calcium handling demonstrated 14 and 19 genes, respectively, modulated by calreticulin deficiency. Calreticulin deletion provoked ontological re-prioritization of gene expression, molecular transport and protein trafficking that translated into multiple subcellular functional outcomes. Individual stem cell-derived cardiomyocytes lacking calreticulin demonstrated a disorganized contractile apparatus with mitochondrial paucity and architectural aberrations. Thus, bioinformatic deconvolution of primordial embryonic stem cell transcriptomes enables predictive phenotyping of defective developmental networks that coalesce from complex systems biology hierarchies.

Effect of FTO overexpression and knockdown on transcriptome of HEK293 cells

To gain insight into FTO function, we knocked down and overexpressed FTO in HEK293 cells.Genetrail analyses of expression profiles pointed to the RNA splicing and processing machinery. Intriguingly, using immunocytochemistry and confocal laser scanning microscopy, we observed strong enrichment of FTO in nuclear speckles and - to a lesser extent - in nucleoli, but not in other known nuclear bodies. We also studied RNA samples of Fto knockout and wild type mice with regard to content of methylated and unmethylated nucleosidesand observed that ratios of modified and unmodified uracil and adenine were different depending on the presence of FTO. Taken together, our data suggest that FTO is involved in RNA processing and modification.
We used microarrays to investigate global gene expression changes depending on the level of FTO

Comparison of granulosa cells from WT and Inha KO mice following gonadotropin stimulation

Inhibin α knockout (Inha-/-) female mice develop sex cord-stromal ovarian cancer with complete penetrance and previous studies demonstrate that the pituitary gonadotropins [follicle stimulating hormone (FSH) and luteinizing hormone (LH)] are influential modifiers of granulosa cell tumor development and progression in inhibin-deficient females. Recent studies have demonstrated that Inha-/- ovarian follicles develop precociously to the early antral stage in prepubertal mice without any increase in serum FSH and these studies suggested that in the absence of inhibins, granulosa cells differentiate abnormally, and thus at sexual maturity may undergo an abnormal response to gonadotropin signaling. To test this hypothesis, we stimulated immature WT and Inha-/- female mice prior to gross tumor formation with gonadotropin analogs, and subsequently examined post-gonadotropin induced ovarian follicle development, as well as preovulatory and hCG-induced gene expression changes in granulosa cells.  We find that at three weeks of age, inhibin-deficient ovaries do not show further antral development nor undergo cumulus expansion.  Widespread alterations in the transcriptome of gonadotropin-treated Inha-/- granulosa cells suggest that gonadotropins initiate an improper program of cell differentiation in Inha-/- cells. Overall, our experiments reveal that inhibins are essential for the normal gonadotropin-dependent response of granulosa cells.

Transcriptional response of normal human lung WI-38 fibroblasts to benzo[a]pyrene diol epoxide: a dose-response study

Cellular responses to carcinogens are typically studied in transformed cell lines, which do not reflect the physiological status of normal tissues. To address this question, we have characterized the transcriptional program and cellular responses of normal human lung WI-38 fibroblasts upon exposure to the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE). Exposure to BPDE induces a strong inflammatory response in WI-38 primary fibroblasts. Whole-genome microarray analysis shows induction of several genes related to the production of inflammatory factors, including those that encode interleukins (ILs), growth factors, and enzymes related to prostaglandin synthesis and signaling. This is the first demonstration that a strong inflammatory response is triggered in primary fibroblasts in response to a reactive diol epoxide derived from a polycyclic aromatic hydrocarbon.

Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here, we report the identification and characterization of a novel heterochromatinization factor in vertebrates, Bromo Adjacent Homology Domain-containing protein 1 (BAHD1). BAHD1 interacts with HP1, MBD1, HDAC5 and with several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes formation of large heterochromatic domains, which lack acetyl histone H3 and are enriched in H3 trimethylated at lysine 27. Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic X inactive chromosome. As highlighted by whole genome microarray analysis of BAHD1 knock down cells, BAHD1 represses several proliferation and survival genes and in particular, the insulin-like growth factor II gene (IGF2). BAHD1 specifically binds the CpG-rich P3 promoter of IGF2. This region contains DNA binding sequences for the transcription factor SP1, with which BAHD1 co-immunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting proteins that coordinate heterochromatin assembly at specific sites in the genome.
We used microarrays to identify BAHD1 gene targets. We compared the transcriptome profile of BAHD1 depleted cells with siRNA to that of cells treated with control siRNA.

Characterization of whole blood gene expression profiles in sickle-cell disease patients using globin mRNA reduction

Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation .  In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations.  Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable.

Expression levels in immortalized B cells from unrelated individuals and twins undergoing ER stress

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called “ER stress” which induces the unfolded protein response (UPR) which is a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B-cells and then measured gene expression at 10 time-points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in ER stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE which showed significant increase in expression following ER stress in B-cells and
in primary fibroblasts. To study the links between unfolded protein response and disease susceptibility, we identified ER stress responsive genes that are associated with human diseases and assessed individual differences in ER stress response. Many of the UPR genes are associated with Mendelian disorders such as Wolfram syndrome and complex
human diseases including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility.

BCR-ABL1 and a dominant negative isoform of Ikaros cooperate to induce human acute myeloid leukemia

Analysis of lineage depleted human cord blood cells sequentially transduced with retro- (BCR-ABL1) and lentiviral (Ik6) vectors and the corresponding controls. Results provide important informations on the collaboration of BCR-ABL1 and Ik6 in human hematopoietic cells.

BT-20 breast cancer cells were exposed to a mock transfection, GFP siRNA, or FoxM1 siRNA.  Each condition was performed in triplicate, and RNA was collected after 48hrs.

Gene expression profiling in 50 glial brain tumors and 4 normal brains using 42,000-feature cDNA microarrays (from total RNA). Tumors: 50 fresh-frozen glioma specimens subjected to standard WHO classification. Specimens included astrocytic [2 juvenile pilocytic astrocytomas, 1 low-grade astrocytic glioma, 1 anaplastic astrocytomas, 31 glioblastomas (of these 2 secondary glioblastomas and 2 gliosarcomas)], oligodendroglial [5 oligodendrogliomas, 3 anaplastic oligodendrogliomas], and 6 anaplastic oligoastrocytomas tumors. One tumor had been classified as glioneuronal neoplasm. Normal brain was purchased from Stratagene. Stratagene Universal Common Reference was used as reference RNA. The results provide insights into molecular mechanisms and pathways associated with gliomagenesis.
type: Logical Set

HSC (Sca+ SP) were isolated from 8-12 week C57B6 mice at various time points after treatment with 5-Fluorouracil.  RNA was isolated from 50,000-100,000 FACS sorted cells and subjected to two rounds of T7 based linear amplification using Ambion's Message Amp kit.  Two replicates from each time point were analyzed.

http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371%2Fjournal.pbio.0020301

Regulation of Liver Regeneration and Hepatocarcinogenesis by Suppressor of Cytokine Signaling 3

Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration. We developed hepatocyte-specific Socs3 knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after 2/3 partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after 2/3 PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic kinases extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor (EGF) or interleukin 6 (IL-6) stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes after PH, which fall into diverse physiologic categories. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma (HCC) at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiologic and neoplastic proliferative processes in the liver, and may act as a tumor suppressor.
Keywords: SOCS3, liver regeneration

muscle biopsies of normal and dermatomyositis patients

Microarray analysis was designed to study the effect of PIAS1 on interferon (IFN) signaling pathway by comparing the gene activation profiles of wild type and Pias1 null cells in response to IFN treatments. Microarray analysis was performed essentially following the manufacturer’s instructions (Affymetrix). Briefly, bone marrow-derived macrophages (BMMs) from wile type or Pias1 null littermates were either untreated, or treated with IFN-beta (500 U/ml) or IFN-gamma (10 ng/ml) for 2h or 4h. Total RNA was prepared with RNA-STAT60 (TEL-TEST) and purified with the RNeasy kit (Qiagen). Double stranded complementary DNA was synthesized from 20 ug of total RNA according to Affymetrix methodology, and purified with Phase Lock Gels (Eppendorf). Biotin-labeled RNA was synthesized with the BioArray High Yield RNA Transcript Labeling Kit (Enzo). Samples were cleaned, fragmentated and hybridized to murine genome (MGU74Av2) Genechips (Affymetrix) as instructed. GeneChips were stained with phycoerythrin-streptavidin (Molecular Probes) and scanned with a GeneChip scanner (Affymetrix).

OBF1, also known as Bob.1 or OCA-B, is a B lymphocyte-specific transcription factor which coactivates Oct1 and Oct2 on B cell specific promoters. So far, the function of OBF1 has been mainly identified in late stage B cell populations. The central defect of OBF1 deficient mice is a severely reduced immune response to T cell-dependent antigens and a lack of germinal center formation in the spleen. Relatively little is known about a potential function of OBF1 in developing B cells. Here we have generated transgenic mice overexpressing OBF1 in B cells under the control of the immunoglobulin heavy chain promoter and enhancer. Surprisingly, these mice have greatly reduced numbers of follicular B cells in the periphery and have a compromised immune response. Furthermore, B cell differentiation is impaired at an early stage in the bone marrow. A first block is observed during B cell commitment and a second differentiation block is seen at the large preB2 cell stage. The cells that succeed to escape the block and to differentiate into mature B cells have post-translationally downregulated the expression of transgene, indicating that expression of OBF1 beyond the normal level early in B cell development is deleterious. Indeed ID3, which is a negative regulator of B cell differentiation, is upregulated in the EPLM and preB cells of the transgenic mice. Furthermore ID3 promoter contains an octamer site suggesting that it is a potential OBF-1 direct target gene. These results provide evidence that OBF1 expression has to be tightly regulated in early B cells to allow efficient B lymphocyte differentiation.

Analysis of Aiolos and OBF-1 deletions in bone marrow from 7 week old mice

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes l5l5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the l5l5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

OBF-1 or Aiolos or both were deleted and the gene expression profiles for these animals investigated using Affymetrix arrays

Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n=40), normal lung specimens (n=4), normal pleura specimens (n=5), and MPM and SV40-immortalized mesothelial cell lines (n=5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma which correlate loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (i.e. software) to create and explore complex physiological pathways that may be relevant in mesothelioma tumorigenesis, pathobiology, or both.

Tissues and cell lines profiled using microarrays. Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Boston's Brigham and Women's Hospital (BWH) between October 1998 and August 2000. All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed. All normal specimens were obtained from patients who were never diagnosed with MPM. Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH. The JMN1B MPM cell line19,20 has been described previously. The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection. Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM. All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used). The microscopic slides from the patients' resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases. Linked clinical and pathological data were obtained for all patients who contributed tumor specimens. Specimens and data were rendered anonymous to protect patient confidentiality. Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH.

Array profiling of dystrophin-deficient mice with a secondary glycosylation defect

A deletion in the CMAH gene in humans occurred approximately 3.5 million years ago. This resulted in the inactivation of the CMP-Neu5Ac hydroxylase enzyme, and hence, in the specific deficiency in N-glycolylneuraminic acid (Neu5Gc), a form of sialic acid, in all modern humans. Although there is evidence that this molecular milestone in the origin of humans may have led to the evolution of human-specific pathogens, how deficiency in Neu5Gc might alter progression of non-infectious human diseases remains unanswered.  Here, we have investigated cardiac and skeletal muscle gene expression changes in mdx mice, a model of Duchenne muscular dystrophy (DMD), that do or do not carry the human-like inactivating mutation in the mouse Cmah gene.  We have evidence that Neu5Gc-deficiency in humans might explain some of the discrepancies in the disease phenotype between mdx mice and DMD patients.

Pulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations.
However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.

macrophages;VDR;activated vitamin D3;IFNgamma;KO mice;dose response

IFNgamma and 1a,25(OH)2D3 dependent gene expression in bone marrow derived macrophages

Gene expression profiling of macrophages derived from WT and Vdr deficient mice after stimulation with IFNgamma and/or 1alpha,25(OH)2D3
Keywords = macrophages

NOD2 and desease associated variant NOD2-L1007fsinsC dependent genomewide transcriptional regulation in stable Flp-In HEK cells

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP) and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs e.g. Crohn disease, asthma and atopic eczema. It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined.  To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2wt or NOD2L1007fsinsC to stimulation with MDP. Importantly, a clear loss-of-function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, while the NOD2wt cells showed differential regulation of growth factors, chemokines and several antagonists of NF-κB, e.g. TNFAIP3 (A20) and IER3.

Quercetin effect on gene expression in HepG2 hepatocellular cancer cells

Quercetin is a flavonol modifying numerous cell processes with potent antiproliferative effects on cancer cell-lines. The aim of this study was to explore by gene-array analysis the effect of quercetin on cancer-related gene expression in HepG2 cells, followed by verification with RT-PCR and analysis of the expected phenotypic changes (migration, cell cycle, cell proliferation). Quercetin induces significant changes on cell-adhesion related genes, leading to reduced migratory capacity and disorganization of the actin cytoskeleton. Several genes related to DNA functions, cellular metabolism and signal-transducer activities were also modified, while an early effect on G–protein related cascades possibly via protease-activated receptor 2 and phospholipase C-γ1 was identified. Cyclin-D associated events in G1 and ubiquitin-dependent degradation of cyclin-D1 were also affected, resulting in cell-cycle arrest without activation of apoptosis pathways. In conclusion quercetin (3μM) exerts its cellular effects by modifying numerous genes related to mechanisms involved in cancer initiation and promotion.

Genome wide expression data from discordant twins (ulcerative colitis, primary mucosal tissue)

Background and aims. The etiopathology of inflammatory bowel diseases is still poorly understood. To date, only few little data are available on the microbiota composition in ulcerative colitis (UC), representing a major subform of inflammatory bowel diseases. Currently, one of the main challenges is to unravel the interactions between genetics and environmental factors in the onset or during the progression and maintenance of the disease. The aim of the present study was to analyse twin pairs discordant for UC for both gut microbiota dysbiosis and host expression profiles at a mucosal level and to get insight into the functional genomic crosstalk between microbiota and mucosal epithelium in vivo.   Methods. Biopsies were sampled from the sigmoid colon of both healthy and diseased siblings from UC discordant twin pairs but also from healthy twins. Microbiota profiles were assessed by 16S rDNA libraries while mRNA expression profiles were analysed from the same volunteers using Affymetrix microarrays.
Results. UC patients showed a dysbiotic microbiota with lower diversity and more species belonging to Actinobacteria and Proteobacteria phyla. On the contrary, their healthy siblings’ microbiota contained more bacteria from the Lachnospiracea and Ruminococcaceae family than did healthy individuals . Sixty-three host transcripts significantly correlated with bacterial genera in healthy individuals whereas only 43 and 32 correlated with bacteria in healthy and UC siblings from discordant pairs, respectively. Several transcripts related to oxidative and immune responses were differentially expressed between unaffected and UC siblings.  Conclusion. A loss of crosstalk between gut microbiota and host was highlighted in UC patients. This defect was also striking in healthy siblings from discordant pairs, as was the lower biodiversity within the microbiota. Our results suggest disease-relevant interactions between host transcriptome and microbiota. Moreover, unusual aerobic bacteria were noticed in UC mucosal microbiota, whereas healthy siblings from discordant pairs had higher percentages of potentially beneficialusual commensal  bacterial species.

Expression data from E12.5 embryonic kidneys isolated from wild-type and Ret-deficient mice

To better understand the signaling and transcriptional events involved in the GDNF-independent emergence of the ureteric bud from the Wolffian duct, microarray expression analysis was performed on embryonic kidneys from wild-type and Ret-deficient mice.  Microarray data was used to identify genes and gene networks involved in the GDNF-independent outgrowth of the ureteric bud.

Analysis of GPR120 which play roles for the fatty acid sensor in adipose tissue. Results provide insight into the transcriptional effects caused by the loss of the GPR120 proteins and provide further insight into their functions.

Identification of IL-22 regulated genes in the ileum after infection with Toxoplasma gondii

IL-22 acts on epithelial cells and has been shown to induce tissue protective and wound healing responses in these cells. But it has recently been decribed that IL-22 exacerbates ileatis after infection with T. gondii.
The goal of the study is to identify the IL-22-dependent genes during T. gondii infection in order to understand why IL-22 is pathogenic in this context.

Genome-wide identification of genes differentially expressed between wild-type and Prdm14-deficient ES cells grown in 2i culture conditions.

Analysis of Prdm14 function in mouse embryonic stem cells. Prdm14 null and overexpressing ES cells were generated and analyzed by microarray, immunoflurescence, flow cytometry, ELISA, qPCR in different culture conditions.

Gene expression profiles were recorded from rectal suction specimens of Cystic Fibrosis (CF) patients, carrying the CF-specific D508 mutated CFTR-allele.  These profiles were compared with gene expression profiles from rectal suction specimens of non-CF subjects (control).

Claudin 1 mediates TNFα-induced gene expression and cell migration in human lung carcinoma cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFß1 and TNFa were used to induce EMT in human lung carcinoma A549 cells, we were surprised to find an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFa and not the TGFß1 that induced the fibroblast-like morphology changes. TNFa also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFa-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFa-induced molecular functional networks related to inflammation and cell movement. Wound-healing assay showed that Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFa-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.

Barrett's esophagus, Barrett's-associated adenocarcinomas and normal esophageal epithelium

Samples were obtained from 8 patients with Barrett's associated adenocarcinomas after transhiatal esophagectomy. Samples representative of the normal esophageal epithelium (N), Barrett’s esophagus (B) and esophageal adenocarcinomas (ADC) were obtained from every patient by experienced GI pathologists. RNA were extracted and samples were profiled for detection of genes differentially expressed in B and ADC relative to N and in ADC relative to B.

Modifications of the Rat Airway Explant Transcriptome by Cigarette Smoke

Although a number of animal model studies have addressed changes in gene expression in the parenchyma and their relationship to emphysema, much less is known about the pathogenesis of cigarette smoke-induced small airway remodeling. In this study, we exposed rat tracheal explants to whole smoke for 15 minutes, and then cultured the explants in air. The airway transcriptome was evaluated using RAE 230_2 GeneChips. By 2 hours after starting smoke exposure, expression levels of 502 genes were changed up or down by more than 1.5 times (p values <0.01 or less), and by 24 hours, 1870 genes were significantly changed up or down. These included genes involved in anti-oxidant protection, epithelial defense and remodeling, inflammatory mediators and transcription factors, and a number of unexpected genes including the MMP-12 inducer, tachykinin-1 (substance P). Pre-treatment of the explants with 1 x 10-7 M dexamethasone reduced the number of significantly changed genes by approximately 47% at 2 hr and 68% at 24 hours, and in almost all instances, reduced the magnitude of the smoke-induced changes. We conclude that even a very brief exposure to cigarette smoke can lead to rapid changes in the expression of a large number of genes in rat tracheal explants, and that these effects are directly mediated by smoke, without a need for exogenous inflammatory cells.  Steroids, contrary to the usual belief, are able to ameliorate many of these changes, at least in this very acute model.

Microarray gene expression profiling of aorta genes of APOE-deficient mice receiving the ACE inhibitor captopril

Microarray gene expression profiling of aorta genes of APOE-deficient mice receiving atherosclerosis treatment with the ACE inhibitor captopril.
Hypercholesterolemic APOE-deficient mice were used as a standard model of atherosclerosis to study gene expression changes during atherosclerosis treatment with the ACE inhibitor captopril. Microarray analysis was performed of whole aortas isolated from captopril-treated APOE-deficient mice relative to untreated APOE-deficient mice with overt atherosclerosis, and nontransgenic control mice. Microarray gene expression profiling revealed that captopril-mediated atherosclerosis prevention involved inhibition of aorta-infiltrating immune cells such as pro-atherogenic T lymphocytes and macrophages.

Amniotic fluid RNA gene expression profiling provides insights into the phenotype of Turner syndrome

Background: Turner syndrome, a common sex chromosome aneuploidy, has characteristics and malformations associated with the phenotype.  Fetal amniotic fluid is a complex biological material that could contribute to the understanding Turner syndrome pathogenesis.  Global gene expression analysis of Turner syndrome fetal amniotic fluid supernatant was utilized to identify organ systems and specific genes that may play a role in the pathophysiologic changes that are seen in individuals with Turner syndrome.
Methods: Global gene expression analysis was performed utilizing cell-free RNA from five midtrimester fetuses with Turner syndrome matched with five euploid female fetuses.  Total RNA was extracted, amplified, hybridized onto GeneChip® Human Genome U133 Plus 2.0 arrays.  Network and pathway analysis of differentially expressed genes were completed.  Chromosomal distribution of gene expression differences, differential expression by pathway and organ system (a “Turner syndrome core transcriptome”), and candidate genes that could play a pathological role were identified.
Results: There were 470 differentially expressed genes identified in the Turner syndrome transcriptome.   The differentially expressed genes were distributed randomly across different chromosomes.  Among genes on the X chromosome, XIST was down-regulated, and SHOX not differentially expressed.  The most highly represented organ systems were hematologic/immune and neurologic.  Increased representation of differentially expressed genes in the hematologic/immune system distinguishes the Turner syndrome transcriptome from the euploid, trisomy 18 and trisomy 21 transcriptomes previously studied in our laboratory.  Manual curation of the differentially expressed gene list identified genes including NFATC3, IGFBP5, and LDLR that warrant further study.

Expression data from MTDH knockdown in the Hec50co endometrial cancer cell line

Understanding the molecular underpinnings of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH has recently been implicated in poor clinical outcome in breast cancer, neroblastoma, hepatocellular carcinoma and prostate cancer. In this present study, we focused on the therapeutic benefit of MTDH depletion to restore sensitivity to cell death mediated by a combinatorial therapy of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL), which promotes death of cancerous cells of the human reproductive tract, and histone deacetylase (HDAC) inhibitors, which have been shown to increase sensitivity of cancer cells to TRAIL-induced apoptosis. Our data indicate that depletion of MTDH in endometrial cancer cells results in sensitization of cells that were previously resistant to cell death mediated by combinatorial treatment with TRAIL and HDAC inhibitor LBH589. MTDH was found to be involved in G2/M checkpoint regulation in response to LBH589 alone or LBH589 in combination with TRAIL, suggesting that MTDH functions at the cell cycle checkpoint to accomplish resistance.Using microarray technology, we identified 57 downstream target genes of MTDH, including Calbindin 1 and Galectin 1, which may contribute to MTDH-mediated resistance to combinatorial TRAIL and HDAC inhibitor targeted therapy. Inhibition of PDK1,AKT phosphorylation and increase Bim expression and XIAP degradation may result in sensitivity to cell death induction in MTDH depleted Hec50co cells by TRAIL and LBH 589 combination treatment. These findings indicate that depletion of MTDH is a potentially novel avenue for effective cancer therapy.

Heart;Mycardial infarction;1 day post-MI;7 day post-MI;parallel sample

Genomewide analysis of gene expression associated with Tcof1 in mouse neuroblastoma.  NB N1E-115 cells with wildtype, overexpression, knockdown of Tcof1.

Gene expression profiling of spleen marginal zone B cells and spleen follicular B cells in IRF8 conditional KO mice

Conditional IRF8 KO mice (mice with a conditional allele of Irf8 crossed with CD19-Cre mice) showed increased numbers of both Gene expression data spleen marginal zone (MZ) and Gene expression data spleen follicular (FO) B cells compared to control mice.
To evaluate gene expression patterns that distinguished FO or MZ B cells derived from conditional KO and control mice, we used Affymetrix GeneChip® Mouse gene 1.0 ST Array.

Conditional keratinocyte-specific ADAM17 knockout in mice alters skin homeostasis

This study investigates the role of ADAM17 (a disintegrin and metalloproteinase 17) in skin homeostasis. Here, we show that mice lacking ADAM17 in keratinocytes have a normal epidermal barrier and skin architecture at birth, but develop pronounced defects in epidermal barrier integrity soon after birth and chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 days after birth, followed by transepidermal water loss and inflammatory immune cell infiltration. Our results identify a previously unappreciated critical role of the ADAM17/EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier.

Illumina array was employed to analyze the genes whose expression are altered when ARID1A gene is downregulated by shRNA in normal ovarian surface epithelial cells OSE4 and IOSE80pc.  This leads to discovery of p53-regulated genes such as p21 and SMAD3.

Analysis of Gsk-3-Deficient Embryonic Stem Cells Reveals Large-Scale Alterations in Gene Expression

The two vertebrate Gsk-3 isoforms, Gsk-3a and Gsk-3b, are encoded by distinct genetic loci and exhibit mostly redundant function in murine embryonic stem cells (ESCs). Here we report that deletion of both Gsk-3a and Gsk-3b in mouse ESCs results in significant changes in gene expression. In contrast, deletion of either Gsk-3a or Gsk-3b individually had little effect on gene expression. These data support the notion that Gsk-3 isoforms are functionally redundant in embryonic stem cells. In addition, we did not find the expected upregulation of known Wnt target genes. Our data suggests that Gsk-3-meidated regulation of gene expression in embryonic stem cells is complex, and likely involves affects on numerous signaling pathways.

Role of the Cytoskeleton in muscle transcriptional response to altered use

Desmin is a cytoskeletal protein in muscle involved in integrating cellular space and transmitting forces.  In this study we sought to determine the combinatory effects of desmin deletion, aging and eccentric exercise on skeletal muscle at the transcriptional level across many pathways of muscle physiology.

Accumulating evidence suggests that mitochondrial dysfunction underlies the pathophysiology of bipolar disorder (BD) and schizophrenia (SZ). We performed large-scale DNA microarray analysis of postmortem brains of patients with BD or SZ, and examined expression patterns of mitochondria-related genes. We found a global down-regulation of mitochondrial genes, such as those encoding respiratory chain components, in BD and SZ samples, even after the effect of sample pH was controlled. However, this was likely due to the effects of medication. Medication-free patients with BD showed tendency of up-regulation of subset of mitochondrial genes. Our findings support the mitochondrial dysfunction hypothesis of BD and SZ pathologies. However, it may be the expression changes of a small fraction of mitochondrial genes rather than the global down-regulation of mitochondrial genes. Our findings warrant further study of the molecular mechanisms underlying mitochondrial dysfunction in BD and SZ.

Mice lacking the function of the PcG protein CBX2 (also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes.
To clarify the molecular basis of the defects, RNA prepared from both wild type and KO gonads were subjected to microarray analyses.

Effect of imatinib on philadelphia chromosome positive acute lymphoblastic leukemia

The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 tyrosine kinase, which defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. In this study, the tyrosine kinase inhibitor imatinib was used for pharmacological inhibition of BCR-ABL1. Gene expression profiles of Ph+ ALL cell lines were analyzed in response to imatinib treatment.

Cell culture, genetic, oxidative phosphorylation, microarray, skeletal muscle, type 2 diabetes

Transcription profiling of myotubes from patients with type 2 diabetes

Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independent of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin-responsiveness in primary human muscle cells from patients with type 2 diabetes.
Results: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using both approaches for global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis, PPARGC1A and NRF-1, or in mitochondrial mass between diabetic and control myotubes.

Gene expression in immortalized B-lymphocytes derived from patients with multiple sclerosis

This study aims to determine if global gene expression and transcription factor networks in B-lympocytes of siblings with MS were different from healthy siblings.
Keywords: Multiple sclerosis, sibling comparisons

Transcriptome analysis of Brg1 deficient small intestinal epithelium in the context of normal and aberrant Wnt signalling

Brg1 has been reported to act as a trans-activator for the Wnt pathway by interacting with beta-catenin. Given this interaction and the crucial role Wnt signalling plays in the intestinal homeostasis, we aimed to investigate the effect of Brg1 loss on gene expression in normal and Wnt activated small intestinal epithelium.

Expression of HOXB genes is significantly different in acute myeloid leukemia with a partial tandem duplication of MLL vs. a MLL translocation: a cross-laboratory study

In acute myeloid leukemia (AML), the mixed lineage leukemia (MLL) gene may be rearranged to generate a partial tandem duplication (PTD), or fused to partner genes through a chromosomal translocation (tMLL). In this study, we first explored the differentially expressed genes between MLL-PTD and tMLL using gene expression profiling of our cohort (15 MLL-PTD and 10 tMLL) and one published data set. The top 250 probes were chosen from each set, resulting in 29 common probes (21 unique genes) to both sets. The selected genes include four HOXB genes, HOXB2, B3, B5, and B6. The expression values of these HOXB genes significantly differ between MLL-PTD and tMLL cases. Clustering and classification analyses were thoroughly conducted to support our gene selection results. Second, as MLL-PTD, FLT3-ITD, and NPM1 mutations are identified in AML with normal karyotypes, we briefly studied their impact on the HOXB genes. Another contribution of this study is to demonstrate that using public data from other studies enriches samples for analysis and yields more conclusive results.

Compared gene enrichment in Ago2-RNA immunopreciptant between wildtype and Mir140-null chondrocytes
Identified Mir140 targets that are most exclusively regulated by Mir140

Gastrointestinal Stromal Tumor frequently harbor mutations in the KIT receptor tyrosine kinase and depend on its activity for growth. This underlies the efficacy of imatinib, a inhibitor of KIT activity, in GIST management. GIST882 is a patient derived GIST cell line that harbor a K640E exon 13 KIT mutation and is sensitive to imatinib treatment. To analyze the downstream effect of KIT inhibition, GIST882 cells were treated for 8 hours with  1μM Imatinib.

Evi1 is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly, high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. Improvement of the therapeutic outcome of leukemia with activated Evi1 is one of the most challenging issues. However, mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here we show that Evi1 directly represses PTEN transcription in the murine bone marrow, which leads to activation of AKT/mTOR signaling. In a murine bone marrow transplantation model, Evi1 leukemia showed remarkable sensitivity to an mTOR inihibitor rapamycin. Furthermore, we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN downregulation, which reveals a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and chromatin immunoprecipitation assays using human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1.

Expression data for human epithelium from subjects with atopic dermatitis, psoriasis and nonatopic controls

The expression and function of tight junction genes, in particular claudin-1, was studied in order to further understanding of the biology underlying atopic dermatitis.

Liver of rat offsprings exposed perinatally to low doses of 2,2',4,4'-tetrabromodiphenyl ether

Background: Polybrominated diphenyl ethers are a group of flame-retardant chemicals appearing increasingly in the environment. Their health effects and mechanisms of toxicity are poorly understood.Objectives: We screened for the sensitive effects and mechanisms of toxicity of 2,2 ,4,4 -tetra-bromodiphenyl ether (BDE-47) by analyzing the gene expression profile in rats exposed to doses comparable to human exposure.Methods: Wistar dams were exposed to vehicle or BDE-47 (0.002 and 0.2 mg/kg body weight) every fifth day from gestation day 15 to postnatal day 20 by injections to caudal vein. Total RNA was extracted from the livers of pups and hybridized to the whole-genome RNA expression micro-arrays. The list of genes 2-fold differentially expressed was exported to PANTHER and Ingenuity Systems for analysis of enriched ontology groups and molecular pathways.Results: Oxidoreductase and transferase protein families were enriched in exposed rats as were these biological process categories: carbohydrate metabolism; electron transport; and lipid, fatty acid, and steroid metabolism. Four signaling pathways (cascades of activation of drug-metabolizing enzymes) and 10 metabolic pathways were significantly enriched. Drug-metabolizing enzymes appear to be regulated by BDE-47 through an aryl hydrocarbon receptorindependent mechanism. Direct interaction with retinoid X receptor or its upstream cascade may be involved. The main metabolic effects consisted of activation of metabolic pathways: alpha- and omega-oxidation of fatty acids, glycolysis, and starch hydrolysis.Conclusions: Altered expression of genes involved in metabolic and signaling pathways and functions of the organism occurs after perinatal exposure of rat offspring to BDE-47 at doses relevant for the general population.

Genome-wide analysis of c-Myb-responsive gene expression in prostate cancer cells

Analysis of c-Myb-regulation of  gene expression. The hypothesis tested in the present study was that c-Myb influences the expression of specific sets of genes that are involved in cell cycle, DNA replication, recombination and repair. Results provide important information on c-Myb-responsive genes that may be crucial to the cell survival and the progression of prostate cancer.

GORD;Barrett's Oesophagus;Oesopageal Adenocarcinoma.;time-course

Differential gene expression analysis of oesophageal cells stimulated with a low pH environment. Study designed to identify pathways involved in progression of gastro-oesophageal reflux disease through Barrett's oesophagus to adenocarcinoma. Identified many subsets of genes with involvement in pathogenesis.
Keywords = GORD

Background and Aims: Although the zinc finger transcription factor GATA4 has been implicated in regulating jejunal gene expression, the contribution of GATA4 in controlling jejunal physiology has not been addressed. Methods: We generated mice in which the Gata4 gene was specifically deleted in the small intestinal epithelium.  Measurements of plasma cholesterol and phospholipids, intestinal absorption of dietary fat and cholesterol, and gene expression were performed on these animals. Results: Mice lacking GATA4 in the intestine displayed a dramatic block in their ability to absorb cholesterol and dietary fat. Comparison of the global gene expression profiles of control jejunum, control ileum, and GATA4 null jejunum by gene array analysis demonstrated that GATA4 null jejunum lost expression of 53% of the jejunal-specific gene set and gained expression of 47% of the set of genes unique to the ileum. These alterations in gene expression included a decrease in mRNAs encoding lipid and cholesterol transporters as well as an increase in mRNAs encoding proteins involved in bile acid absorption. Conclusion: Our data demonstrate that GATA4 is essential for jejunal function including fat and cholesterol absorption and confirm that GATA4 plays a pivotal role in determining jejunal versus ileal identity.

knockout, mouse embryo development, angiogenesis, yolk sac, E9.0, E10.5

Comparison of gene expression pattern between Wild-type and Hypb knockout embryo yolk sacs

This study aimed at exploring the physiological function of mammalian HYPB by means of knockout mouse model. Homogenous disruption of mouse Hypb gene leads to embryonic lethality at E10.5-E11.5. Severe vascular defects were observed in the Hypb-/- embryos, yolk sac and placenta.In the mutant embryo and yolk sac, disorganized and abnormally dilated capillaries cannot be remodeled into large blood vessels or intricate networks. Thus, our results suggest that the mammalian HYPB HMT plays an important role in embryonic vascularization.
Keywords: knockout, mouse embryo development, angiogenesis, yolk sac, E9.0, E10.5

Effect of NPAP1/C15orf2 overexpression on transcriptome of HEK293 cells

To gain insight into the function of Nuclear pore associated protein 1 (NPAP1, formerly C15orf2), we overexpressed NPAP1 in HEK293 cells. We detected no significant difference between NPAP1-expression of induced and uninduced cells in three technical replicates, exept for an approximately 10-fold increase in the NPAP1 transcript itself. This indicates that overexpression of NPAP1 does not change mRNA expression profiles of HEK293 cells. We used microarrays to investigate global gene expression changes depending on the level of NPAP1/C15orf2

CHD8 is a putative chromatin remodeling ATPase of the SNF2 family. We found that depletion of CHD8 impairs cell proliferation. In order to identify CHD8 target genes, we performed a transcriptomic analysis of CHD8-depleted cells. CHD8 was knockdown by doxycyclin-dependent expression of a shRNA that target the CHD8 mRNA.
Keywords: CHD8 knockdown analysis

Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of lineage genes [Expression profiling]

Polycomb group (PcG) proteins play important roles in repressing lineage-specific genes and maintaining the undifferentiated state of mouse embryonic stem cells (mESCs). However, the mechanisms by which PcG proteins are recruited to their targets are largely unknown. Here, we show that the histone demethylase Kdm2b is highly expressed in mESCs and regulated by the pluripotent factors Oct4/Sox2 directly. Depletion of Kdm2b in mESCs causes de-repression of lineage-specific genes and induces early differentiation. The function of Kdm2b depends on its CXXC-ZF domain, which mediates Kdm2b’s genome-wide binding to CpG islands (CGIs). Kdm2b interacts with the core components of the Polycomb repressive complex 1 (PRC1) and recruits the complex to the CGIs of early lineage-specific genes. Thus, our study not only reveals a novel Oct4/Sox2-Kdm2b-PRC1-CGI regulatory axis and its function in maintaining undifferentiated state of mESCs, but also demonstrates a critical function of Kdm2b in recruiting PRC1 to the CGIs of lineage-specific genes to repress their expression.
In this dataset, we include the expression data for control and Kdm2b knockdown mouse embryonic stem cells.

BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers.

Pten deficiency cooperates with KrasG12D to activate NFkB pathway promoting the development of malignant pancreatic ductal adenocarcinoma

Almost all human pancreatic ductal adenocarcinomas (PDACs) are driven by oncogenic Kras and the progression of the disease is characterized by the serial appearance of certain genetic lesions. Mouse models have convincingly shown that Kras mutation induces classical PanIN lesions that can progress to PDAC in the appropriate tumor suppressor background. However, the cooperative mechanism between mutant Kras-dependent signaling surrogates and other oncogenic pathways remains to be fully elucidated in order to devise better therapeutic strategy. Mounting evidence PTEN/PI3K perturbation on PDAC tumorigenesis, we observed frequent PTEN inactivation at both genomic and histopathological levels in primary human PDAC samples. The importance of PTEN/PI3K pathway during the development of PDAC was further supported by genetic studies demonstrating that Pten deficiency in cooperation with Kras activation accelerated the formation of invasive PDAC. Mechanistically, combined Kras mutation and Pten inactivation leads to NFkB activation and subsequent induction of cytokine pathways, accompanied with strong stromal activation and immune cell infiltration. Therefore, PTEN/PI3K pathway dictates the activity of NFkB network and serves as a major surrogate during Kras-mediated pancreatic tumorigenesis.

TRANSCRIPTOMIC PROFILING OF ASTROCYTES TREATED WITH THE RHO KINASE INHIBITOR FASUDIL REVEALS CYTOSKELETAL AND PRO-SURVIVAL RESPONSESTRANSFORMATION INDUCED BY THE RHO KINASE INHIBITOR FASUDIL.

Analysis of genome wide gene expression changes over a time course of 24 hours induced by the Rho kinase inhibitor Fasudil in primary mouse astrocyte cultures.

Gene expression profiles of adult peripheral and cord blood mononuclear cells altered by lipopolysaccharide

Neonatal gram-negative sepsis is often characterized by a fulminat clinical course compared to adults resulting in higher morbidity and mortality. Genome-wide gene expression analysis can provide insights into the molecular alterations in sepsis. To evaluate in vitro activation of the neonatal and adult immune system, gene expression patterns were compared in mononuclear cells from cord (CBMNC) and adult peripheral blood (APBMNC). To better understand the influence of early molecular signals on the effects of sepsis, Affymetrix gene profiling (8475 genes) was done on RNA isolated from CBMNC and APBMNC without and after incubation with 100 ng/ml lipopolysaccharide (LPS). We demonstrated significant alterations in the expression of 108 CBMNC and APBMNC genes compared with basal levels, 188 significant changes in CBMNC, and 97 in APBMNC, including cytokines, chemokines, and immunoregulatory genes. Furthermore, we found five genes showing a significant interaction effect between cell-type and LPS-stimulation, including tumor necrosis factor receptor superfamily, member 6 (FAS), absent in melanoma 2 (AIM2), malic enzyme 1 (ME1), hemoglobin epsilon 1 (HBE1), and trans-prenyltransferase (TPRT). These results provide further support for a marked difference in the pathogenesis of neonatal and adult sepsis and may stimulate additional studies to investigate some of the altered genes as potential new targets for diagnostic tools and therapeutic strategies.
Keywords: LPS response

The effect of Neuroserpin expression upon HeLa cell gene expression

HeLa cells were stably transfected using the Tet-On® Advanced Cell Line system with Wildtype, G392E and Delta Neuroserpin with neuroserpin expression induced with doxycycline.  Gene expression was examined in the absence or presence of 2 ug/ml doxycycline.

Transcriptome of murine testis from wild type mice and mice lacking telomerase for three generations (G3-Terc), Ku86 or  both telomerase and Ku86.

Proteasome inhibition blocks estrogen-dependent gene transcription by decreasing histone H2B monoubiquitination in human breast cancer cells

The estrogen receptor-alpha (ERα) determines breast cancer cell phenotype and is a prognostic indicator. A better understanding of the mechanisms controlling ERα function may uncover improved strategies for the treatment of breast cancer. Proteasome inhibition was previously reported to regulate estrogen-induced transcription but the mechanisms by which it influences ERα function remain controversial. In this study we investigated the transcriptome-wide effects of the proteasome inhibitor Velcade on estrogen-regulated transcription in MCF7 human breast cancer cells and demonstrate a specific global decrease in estrogen-induced transcription.

Background:  Prostate cancer is the second leading cause of cancer mortality among US men.  Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1α,25 dihydroxyvitamin D3 (1,25(OH)2D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown.
Results: We examined the effect of 1,25(OH)2D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D (Affymetrix U133 Plus 2.0, n=4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60 % up), 48 h, 3566 (38 % up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements.  Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling.  Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways).  There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h.
Conclusions: Our data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention.  In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.

Expression data of cardiac and gastrocnemius muscles of tumor bearing C26 mouse

We surveyed the transcriptomes of the whole heart and whole gastrocnemius muscle taken from two different types of Balb/c-DBAj hybrid mice (10-11 weeks old). The colon cancer bearing mice are called C26. The NTB are the non-tumor bearing mice.
This dataset includes Affymetrix expression data collected from the biological triplicates of heart and gastrocnemius from both C26 and NTB mice.

Transcriptomic analysis of the effect of CT16 (PAGE5) silencing by RNAi in A2058 melanoma cells

The cellular gene expression profiles were investigated after CT16 (PAGE5) silencing in CT16 positive A2058 melanoma cells. Control siRNA treated A2058 cells was used as a negative control.

Global gene expression from SOX7 and SOX17 over-expressing human embryonic stem cells (CA1 and CA2 lines)

This study aimed to understand the transcriptional networks regulating endoderm specification from HESC and therefore explored the phenotype of CA1 and CA2 HESC constitutively over-expressing SOX7 or SOX17. Cell lines were created using an inducible construct whereby clonal populations containing transgene integration are selected by Neomycin resistance without expressing of the gene of interest (NoCre controls). Transgene expression is induced via Cre-mediated recombination and selected for puromycin resistance (SOX O/E). The phenotype of the resulting cells suggests that SOX7 expressing HESC represent stable extraembryonic endoderm progenitors, while SOX17 expressing HESC represent early definitive endoderm progenitors. Both in vitro and in vivo SOX7 expressing HESC are restricted to the extraembryonic endoderm lineage, while SOX17 expressing HESC demonstrate mesendodermal specificity.  In vitro, SOX17 expressing HESC efficiently produce mature definitive endoderm derivatives.
The molecular phenotype of the resulting SOX7 and SOX17 expressing HESC was characterized by microarray analysis

Alpha2-6-linked Sialic Acids on N-Glycans Modulate Carcinoma Differentiation In Vivo

Sialic acids on vertebrate cell surfaces mediate many biological roles. Altered expression of certain sialic acid types or their linkages can have prognostic significance in human cancer. A classic but unexplained example is enhanced α2-6-sialylation on N-glycans, resulting from over-expression of the Golgi enzyme β-galactoside:α2-6-sialyltransferase (ST6Gal-I). Previous data supporting a role for the resulting Siaα2-3Galβ1-4GlcNAc (Sia6LacNAc) structure in tumor biology were based on in vitro studies in transfected carcinoma cells, in which increased Sia6LacNAc on β1-integrins enhanced their binding to ligands, and stimulated cell motility. Here we examine for the first time the in vivo role of the ST6Gal-I enzyme in the growth and differentiation of spontaneous mammary cancers in mice transgenic for an MMTV-promoter-driven polyoma-middle-T antigen, a tumor in which beta1-integrin function is important for tumorigenesis, and in maintaining the proliferative state of tumor cells. Tumors induced in St6gal1 null animals were more differentiated in comparison to those in the wild-type background, both by histological analysis and by protein expression profiles. Furthermore, we show the St6gal1 null tumors have selectively altered expression of genes associated with focal adhesion signaling, and have decreased phosphorylation of FAK, a downstream target of β1-integrins.  This first in vivo evidence for a role of ST6Gal-I in tumor progression was confirmed using a novel approach, which conditionally restored St6gal1 in cell lines derived from the null tumors. These findings indicate a role for ST6Gal-I as a mediator of tumor progression, with its expression causing a less differentiated phenotype, via enhanced β1-integrin function.
Keywords: sialic acid, genetic modification, mouse mammary carcinoma

Geminin-regulated genes during neural fate acquisition of mouse embryonic stem cells

Formation of the complex vertebrate nervous system begins when pluripotent cells of the early embryo are directed to acquire a neural fate.  Although cell intrinsic controls play an important role in this process, the molecular nature of this regulation is not well defined.  Here we assessed the role for Geminin, a nuclear protein expressed in embryonic cells, in neural fate acquisition from mouse embryonic stem (ES) cells. While Geminin knockdown does not affect the ability of ES cells to maintain or exit pluripotency, we found that it significantly impairs their ability to acquire a neural fate. Conversely, Geminin overexpression promotes neural gene expression, even in the presence of growth factor signaling that antagonizes neural transcriptional responses. These data demonstrate that Geminin’s activity contributes to mammalian neural cell fate acquisition. We investigated the mechanistic basis of this phenomenon and found that Geminin maintains a hyperacetylated and open chromatin conformation at neural genes. Interestingly, recombinant Geminin protein also rapidly alters chromatin acetylation and accessibility even when Geminin is combined with nuclear extract and chromatin in vitro. These findings define a novel activity for Geminin in regulation of chromatin structure. Together, these data support a role for Geminin as a cell intrinsic regulator of neural fate acquisition that promotes expression of neural genes by regulating chromatin accessibility and histone acetylation.

Genomewide analysis of PTEN deletion in mouse lung epithelial cells

Triple transgenic mice harboring a conditional Pten allele were developed  and Pten was conditionally deleted in vivo from the lung epithelial cells using the doxycycline dependent Cre/LoxP approach. Microarray analysis of lung RNA isolated from Pten-deleted lung epithelial cells (PtenΔ/Δ) and control mice was performed to identify potential PTEN responsive genes.

Sex differences during  acute myocarditis against coxsackievirus B3 (CVB3)-induced myocarditis (10 dpi) and  chronic myocarditis/dilated cardiomyopathy (DCM) against CVB3-induced myocarditis (90 dpi)

Men are at an increased risk of dying from heart failure caused by inflammatory heart diseases such as atherosclerosis, myocarditis and dilated cardiomyopathy (DCM). We previously showed that immune responses in the heart are phenotypically distinct in male compared to female mice 10 days after infection resulting in severe DCM in males.

The aim of this study was to identify new biomarkers and to investigate pathways involved in the progression of human carotid atheroma.

Cadherin-11 depletion in aggressive MDA-MB-231 breast cancer cells.

Cadherin-11 expression is associated with tumor progression and metastasis in various cancers, including basal-like breast carcinoma and advanced prostate cancer, and invasive cell lines, yet is absent in normal epithelium.  We now show cadherin-11 attenuation in aggressive breast and prostate cancer cells results in marked decreases in proliferation, migration, and invasion.  Cadherin-11 depletion in MDA-231 cells prevents tumor growth in mice and alters gene expression associated with poor prognosis malignancies.  Additionally, a novel small molecule inhibitor targeting its unique adhesive interface significantly inhibits the growth and migration of cadherin-11 positive cells.  Cadherin-11 is essential for malignant progression of MDA-231 basal-type cells, and may serve as both an aggressive tumor marker and viable therapeutic target for poor prognosis carcinomas expressing it.

Carbonyl chloride (phosgene) is a toxic industrial compound (TIC) widely used in industry for the production of synthetic products, such as polyfoam rubber, plastics, and dyes.  Exposure to phosgene results in a latent (1-24 hr), potentially life-threatening pulmonary edema and irreversible acute lung injury.  A genomic approach was utilized to investigate the molecular mechanism of phosgene-induced lung injury.  CD-1 male mice were exposed whole-body to either air or a concentration x time (c x t) amount of 32 mg/m3 (8 ppm) phosgene for 20 min (640 mg x min/m3).  Lung tissue was collected from air- or phosgene-exposed mice at 0.5, 1, 4, 8, 12, 24, 48, and 72 hr post-exposure.  RNA was extracted from the lung and used as starting material for the probing of oligonucleotide microarrays to determine changes in gene expression following phosgene exposure.  The data were analyzed using principal component analysis (PCA) to determine the greatest sources of data variability.  A three-way analysis of variance (ANOVA) based on exposure, time, and sample was performed to identify the genes most significantly changed as a result of phosgene exposure.  These genes were rank ordered by p-values and categorized based on molecular function and biological process.  Some of the most significant changes in gene expression reflect changes in glutathione synthesis and redox regulation of the cell, including upregulation of glutathione S-transferase alpha-2, glutathione peroxidase 2, and glutamate-cysteine ligase, catalytic subunit (also known as γ-glutamyl cysteine synthetase).  This is in agreement with previous observations describing changes in redox enzyme activity after phosgene exposure.  We are also investigating other pathways that are responsive to phosgene exposure to identify mechanisms of toxicity and potential therapeutic targets.

Male CD-1 mice were exposed to air or 32 mg per cubic meter phosgene for 20 min and tissue collected at 30 min, 1 hr, 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, or 72 hr before collection and analysis of lung tissue using oligonucleotide microarrays.
Keyword=phosgene
Keyword=mouse
Keyword=lung
Keyword=carbonyl chloride

Gene profile of glioblastoma cells treated with y15 and temozolomide

The gene expression profiles were identified in glioblastoma cells treated with FAK inhibitor Y15, temozolomide alone or with combination of Y15 and Temozolomide

Expression profiling of the developing and mature Nrl -/- mouse retina (Yoshida et al. 2004, HMG)

The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed.
Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation.

Three male 8-week-old Wistar rats were used. The peroneal nerve of the animals was transected and sutured to the biceps femoris muscle. Four weeks later, the proximal stump was sutured to the distal stump. The animals survived for 8 weeks.
All experiments were performed under Ketanest (ketamine hydrochloride, Essex Pharma GmbH, Munich, Germany; 100 mg/kg) and Rompun (xylazine hydrochloride, Bayer, Leverkusen, Germany; 10 mg/kg) anesthesia. Both substances were injected intraperitoneally. RNA was extracted from the ipsilateral and contralateral tibialis anterior muscles of each animal. The gene array analysis compared the ipsilateral muscle with the contralateral muscle of each animal.

Gene expression in skeletal muscle in AMPK gamma3 knock-out mice and wild type littermates

Analysis of AMPK gamma3-dependent transcriptional responses by analyzing global gene expression in the white portion of the gastrocnemius muscle in AMPK gamma3 knock-out mice and corresponding wild type littermates.

Gene expression profiling of lung cancer cells transfected with scrambled siRNA and siRNA targeting the ETS2 gene

ETS2 is a canonical transcriptional factor and member of the ETS family of genes. ETS2 binds to consensus ERE binding sites in a broad spectrum of genes thus affecting many intracellular molecular functions. However, the role of ETS2 in the biology and pathogenesis of lung cancers is still not known.
We have found that ETS2 is down-regulated in lung tumors compared to normal lung tissue and the expression of the coding protein of the gene was a significant independent predictor of favorable outcome in NSCLC patients pinpointing to a potential tumor suppressor role for this gene. To better understand its molecular function in NSCLC, we compared and contrasted the transcriptome of lung cancer cells transfected with control siRNA and siRNA targeting ETS2.

Erbb2 regulates inflammation and proliferation in the skin after ultraviolet irradiation.

Exposure to ultraviolet (UV) irradiation is the major cause of nonmelanoma skin cancer, the most common form of cancer in the United States. UV irradiation has a variety of effects on the skin associated with carcinogenesis, including DNA damage and effects on signal transduction. The alterations in signaling caused by UV regulate inflammation, cell proliferation, and apoptosis. UV also activates the orphan receptor tyrosine kinase and proto-oncogene Erbb2 (HER2/neu). In this study, we demonstrate that the UV-induced activation of Erbb2 regulates the response of the skin to UV. Inhibition or knockdown of Erbb2 before UV irradiation suppressed cell proliferation, cell survival, and inflammation after UV. In addition, Erbb2 was necessary for the UV-induced expression of numerous proinflammatory genes that are regulated by the transcription factors nuclear factor-kappaB and Comp1, including interleukin-1beta, prostaglandin-endoperoxidase synthase 2 (Cyclooxygenase-2), and multiple chemokines. These results reveal the influence of Erbb2 on the UV response and suggest a role for Erbb2 in UV-induced pathologies such as skin cancer.
Keywords: time course, ultraviolet irradiation, UV, erbB2, mouse skin

AMPK gamma3 knock-out and mutant (R225Q) transgenic mice compared to corresponding wild type littermates

Note: GSE4063 and GSE4065 are not directly comparable.

Gene expression in skeletal muscle in AMPK gamma3 mutant (R225Q) transgenic mice and wild type littermates.

Analysis of AMPK gamma3-dependent transcriptional responses by analyzing global gene expression in the white portion of the gastrocnemius muscle in AMPK gamma3 mutant (R225Q) transgenic mice and corresponding wild type littermates.

To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics, and employed it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed 3~5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced 1.5~2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, TGF-? and SDF-1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, dependent on the E2 concentration especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions. (paper abstract)
Keywords: Dose Response, Time Course

FGF Receptor and Wnt-1 Signaling Cooperate in Mammary Tumorigenesis

The fibroblast growth factor pathway is known to cooperate with the highly oncogenic Wnt/beta-catenin pathway in mouse models of breast cancer.  To investigate the mechanisms involved in this cooperativity, we utilized MMTV-driven transgenic mouse lines expressing a drug-inducible model for FGF Receptor signaling (iFGFR) crossed with the previously characterized MMTV-Wnt-1 mouse model.  In these bigenic mice, both iFGFR1 and iFGFR2 activation resulted in a dramatic enhancement of mammary tumorigenesis.  Tumor microarray analysis identified no transcriptional enhancement of Wnt/beta-catenin targets, however, identified a protein translational gene signature that also correlated with elevated FGFR1 and FGFR2 expression in several human breast cancer data sets.  Additionally, iFGFR1 activation resulted in enhanced polysome recruitment and a marked increase in protein expression of several different Wnt/beta-catenin target oncogenes.  Rapid FGFR-induced ERK activation and phosphorylation of key translation regulators was observed both in vivo in the transgenic mouse model, and in human breast cancer cell lines treated with exogenous FGF.  These studies suggest that translational regulation is a key rate-limiting step required for oncogenic cooperativity between the Wnt and FGF pathways.

The whole-genome expression analysis in MIF knockdown cells and the control cells

siRNA-mediated knockdown of MIF expression in HEK293 cells resulted in  inhibition of cell proliferation and cell cycle progress.   The microarray study of MIF KD cells reveald knockdown of MIF would lead to extensive changes in gene expression profiles which may elucidate the molecular mechanism of MIF siRNA-mediated inhibition of cell cycle and cell proliferation.
Keywords: The whole-genome expression analysis in MIF knockdown cells and the control cells

Nf-kB activity is associated with the key pathological features of chronic respiratory diseases including epithelial remodelling, excess mucous production, and submucosal gland hyperplasia.  However, the role of Nf-kB activity in airway epithelial differentiation remains controversial.  In the present study we demonstrate that Nf-kB adaptor protein Myd88 deficiency promotes increased airway submucosal gland abundance and abnormal epithelial differentiation in proximal adult airways.  Abnormal airway differentiation was not developmentally determined, became exacerbated following acute lung injury, and did not involve altered epithelial proliferation or apoptosis.  Instead, we demonstrate that tracheal Myd88 deficiency promotes upregulation of a unique gene expression profile that includes activation of alternate, Myd88-independent Nf-kB signalling.  Finally, we show that these effects are not intrinsically maintained in vitro using an air-liquid interface epithelial culture.  This finding indicates that Myd88 deficiency promotes adult airway remodelling by regulating non-epithelial, non-cell autonomous Nf-kB activity.

Effects of Sirt1 manipulation on gene expression in human Caco-2 cell line

To compare gene expression in human intestinal cell line after Sirt1 manipulation by overexpression or targetted siRNA knockdown

To identify the target genes of Evi-1 in hematopoietic stem cells (HSCs), we carried out genome-wide transcriptional analysis using wild-type and Evi-1-deleted HSCs.

Surviving Sepsis: bcl-2 Overexpression Modulates Splenocyte Transcriptional Responses in vivo

Background: We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the affect of bcl-2 on improving survival during sepsis.

Methods: Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2.  Whole spleens were obtained 6 hours after septic injury.  DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements.  Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene.

Results:  A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied.  At 6 hours, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., IL-6).  These sepsis-induced alterations were blocked by expression of the bcl-2 transgene.  Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1.  Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1.

Conclusion:  These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks.  In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses.

Time course effect of the glucocorticoid mometasone furoate on the proliferation of primary human lung fibroblasts

The effect of mometasone furoate on the proliferation of 2 patients of human lung fibroblasts was investigated at 8 different time points

Stem cell factor Sox2 regulates the tumorigenic potential in human gastric cancer cells

Gastric cancer is still one of the most common causes of cancer-related death worldwide, which is mainly attributable to late diagnosis and poor treatment options. Infection with H. pylori, different environmental factors and genetic alterations are known to influence the risk of developing gastric tumors. However, the molecular mechanisms involved in gastric carcinogenesis are still not fully understood, making it difficult to design targeted therapeutic approaches.
Aberrant expression of the specific gastric differentiation marker Sox2 (sry-related HMG box 2) has been observed in stomach cancer. However, the role of Sox2 in gastric tumors has not been well established to date. To elucidate the role of Sox2 in gastric tumorigenesis, Sox2 transcriptional activity was blocked in AZ521 cells. Interestingly, inhibition of Sox2 reduced cell proliferation and migration, increased apoptosis and induced changes in cell cycle. Blocking of Sox2 also reduced the tumorigenic potential of AZ521 cells in vivo. In addition, correlation of Sox2 expression and proliferation was observed in a subset of human gastric tumours. Finally, target genes of Sox2 were for the first time identified by RNA microarray in gastric cancer cells.
Taken together, the results presented here indicate that Sox2 controls several aspects related to gastric cancer development and progression by regulating the expression of members of important signalling pathways. These findings could provide new therapeutic options for a subset of gastric cancers exhibiting Sox2 deregulation.

Tyrosine phosphorylation is a hallmark for activation of Signal Transducer and Activator of Transcription (STAT) proteins, but their transcriptional activity also depends on other secondary modifications. Type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the Sin3a complex as an important mediator of this STAT3 transcriptional repression. Sin3a directly interacts with the DNA-binding domain of STAT3 and alters its acetylation status. SIN3A silencing enhances recruitment of STAT3 and enhanceosome components to the SOCS3 promoter, resulting in histone hyperacetylation and enhanced transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against Influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent STAT1/3 transcriptional switch.

Elk1 Directs a Critical Component of Growth Signaling by the Androgen Receptor in Prostate Cancer

Elk1 directs selective gene induction that is a substantial and critical component of growth signaling by AR in PC cells.

Liver adapts mitochondrial function to insulin-resistant and diabetic states in mice

Objective: To study if diabetic and insulin-resistant states lead to mitochondrial dysfunction in the liver, or alternatively, if there is adaption of mitochondrial function to these states in the long-term range.

Results: High-fat diet (HFD) caused insulin resistance and severe hepatic lipid accumulation, but respiratory chain parameters were unchanged. Livers from insulin-resistant IR/IRS-1+/- mice had normal lipid contents and normal respiratory chain parameters, however showed mitochondrial uncoupling. Livers from severely hyperglycemic and hypoinsulimic, streptozotocin (STZ)-treated mice had massively depleted lipid levels, but respiratory chain abundance was unchanged. However, their mitochondria showed increased abundance and activity of the respiratory chain, which was better coupled compared to controls.

Conclusions: Insulin resistance, either induced by obesity or by genetic manipulation, does not cause mitochondrial dysfunction in the liver of mice. However, severe insulin deficiency and high blood glucose levels in mice cause an enhanced performance of the respiratory chain, probably in order to maintain the high energy requirement of the unsuppressed gluconeogenesis.

Microarray was used to identify differential gene expression pattern in Barrett's esophagus (BE), compared to the normal adjacent epithelia gastric cardia (GC) and normal squamous esophagus (NE)

Control rat lacrimal gland samples representing a normal unoperated, a sham, and paired contralateral controls. Experimental LG samples representing paired and unpaired contralateral controls.  Paired contralateral control LGs represent LG from the same animal, one side was operated and the other side unoperated.

Microarray of Dexamethasone-treated primary chondrocytes identifies downstream targets of glucocorticoid signalling

Background: Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures.  Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated.
Results:  This study systematically identifies a spectrum of GC target genes in embryonic growth plate chondrocytes treated with synthetic GR agonist, dexamethasone (DEX), at 6 and 24 hrs. Conventional analysis of this data set and gene set enrichment analysis (GSEA) was performed. Transcripts associated with metabolism were enriched in the DEX condition along with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template.
Conclusions: Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes monolayers. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides novel insights into the effects of pharmacological GC on chondrocyte gene transcription and establishes the basis for subsequent functional studies.

Experimental autoimmune encephalomyelitis;multiple sclerosis;1;25(OH)2D3;repeat sample

B10.PL mice with severe (stage 2.5-3) experimental autoimmune encephalomyelitis were treated with placebo or 200 ng 1,25(OH)2D3.  Six hours later, the spinal cords were harvested and mRNA was extracted for microarray analysis.  Naive mice serve as controls.  Individual samples were hybridized to individual microarrays.
Keywords = Experimental autoimmune encephalomyelitis

A time course of orotic acid induced fatty liver disease. Kyoto and Wistar strain rats were exposed to orotic acid for days 1, 3 and 14. Controls are also included.
Keywords = Wistar

To determine the role of NOTCH3 in human esophageal epitheila homeostasis/squamous cell differentiation
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial to mesenchymal transition (EMT), senescence and cancer stem cell maintenance through mutual negative regulation of the microRNA (miR)-200 family members. However, little is known as to how ZEB expressing tumor cells may emerge during invasive growth. We find that canonical Notch signaling prevents expansion of a unique subset of cells expressing ZEBs through NOTCH3 (N3). In primary esophageal squamous cell carcinoma (ESCC), ZEB1 is induced in tumor cells displaying EMT-like dedifferentiation at the invasive front of tumor nests with reciprocal downregulation of the miR-200. ZEB expression was associated with the lack of cellular capability of undergoing squamous differentiation through dysfunction of N3, implicated at the onset of normal esophageal squamous differentiation. Dominant-negative Mastermind-like1 (DNMAML1), a genetic pan-notch inhibitor, prevented CSL-dependent transcription, resulting in suppression of N3 expression and squamous differentiation while enriching EMT competent cells with robust upregulation of ZEBs and downregulation of the miR-200. Such a cell population demonstrated enhanced anchorage independent growth as well as tumor formation in nude mice. RNA interference (RNAi) experiments documented the requirement of ZEBs in TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition in organotypic 3D culture, a form of human tissue engineering. Finally, RNAi experiments revealed N3 as a key factor limiting the expansion of the ZEB expressing cells, providing novel mechanistic insights into the role of Notch signaling in ESCC cell fate regulation and disease progression.

Age-related transcriptional changes and the effect of dietary supplementation of vitamin E in the mouse brain

We established the transcriptional profile of brain aging and examine the global effects of vitamin E supplementation on age-related alterations in expression in the aged mouse brain.

PCOS, microarray, global pathway analysis, insulin resistance, pioglitazone, protein metabolism, mitochondrial oxidative metabolism

Gene expression profiling in skeletal muscle of PCOS after pioglitazone therapy

Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZD) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZD in PCOS is in part mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity.
Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy control women. Treatment with pioglitazone improved insulin-stimulated total, oxidative and non-oxidative glucose metabolism, and reduced fasting serum insulin (all p < 0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes involved in mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome complexes in PCOS patients after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1α expression (p < 0.05). Expression of genes involved in ribosomal proteins and OXPHOS was down-regulated in PCOS patients before treatment compared to matched healthy women using GenMAPP 2.1 and GSEA 2.1.
These data indicate that pioglitazone therapy restores insulin sensitivity in part by a coordinated upregulation of genes involved in mitochondrial oxidative metabolism and protein biosynthesis in skeletal muscle of PCOS. These transcriptional effects of pioglitazone therapy may contribute to prevent the onset of type 2 diabetes in these women.

Mouse surgical model of acute lymphedema induction. We performed three sets of microarrays with three replicates each for a total of 9 arrays.  Each array was run using pooled RNA from three animals.  The three conditions were Normal tail skin (no intervention), Lymphedema tail skin(due to surgical lymphatic vessel blockage), and Surgical Sham control tail skin(surgical incision with no lymphatic vessel blockage).  15ug of test and reference (e17.5 mouse whole embryo) RNA was used for labeling.

Expression data from Parkinson's iPSCs with four copies of SNCA, and equivalent cell lines from an unaffected first degree relative

A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding α-synuclein. α-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double α-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of α-synuclein, and for mechanistic experiments to study PD pathogenesis.
This gene expression microarray study was carried out as part of the validation process for demonstrating that the generated iPSC lines are pluripotent.

Gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression

Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in KIT or PDGFRA of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations.

Expression data from pre-iPSCs with a control, histone methyltransferase or Cbx3 (HP1g) knockdown

Transition from  a partially reprogrammed pre-iPSC state to iPSC state can be achieved by modulating levels of histone modifying enzymes or proteins that can bind to histone modifications
We used microarrays to determine the gene expression profile of pre-iPSCs depleted for either 3 histone methyltransferases together or the HP1gamma protein

Expression and regulation of corticotropin-releasing factor receptor type 2β in developing and mature mouse skeletal muscle.

The purpose of the study was to further understand the molecular mechanisms mediating the effect of Crhr2 signaling on insulin sensitivity in skeletal muscle. To that end we compared the gene expression profile of skeletal muscle obtain from both Crhr2 KO and WT littermates using gene expression microarray.

Aplidin synergizes with cytosine arabinoside: functional relevance of mitochondria in Aplidin-induced cytotoxicity

Aplidin (plitidepsin) is a novel marine-derived antitumor agent presently undergoing phase II clinical trials in hematological malignancies and solid tumors. Lack of bone marrow toxicity has encouraged further development of this drug for treatment of leukemia and lymphoma. Multiple signaling pathways have been shown to be involved in Aplidin-induced apoptosis and cell cycle arrest in G1 and G2 phase. However, the exact mechanism(s) of Aplidin action remains to be elucidated. Here we demonstrate that mitochondria-associated or -localized processes are the potential cellular targets of Aplidin. Whole genome gene-expression profiling (GEP) revealed that fatty acid metabolism, sterol biosynthesis and energy metabolism, including the tricarboxylic acid cycle and ATP synthesis are affected by Aplidin treatment. Moreover, mutant MOLT-4, human leukemia cells lacking functional mitochondria, were found to be resistant to Aplidin. Cytosine arabinoside (araC), which also generates oxidative stress but does not affect the ATP pool, showed synergism with Aplidin in our leukemia and lymphoma models in vitro and in vivo. These studies provide new insights into the mechanism of action of Aplidin. The efficacy of the combination of Aplidin and araC is currently being evaluated in clinical phase I/II program for the treatment of patients with relapsed leukemia and high-grade lymphoma.

Cyclophilin B supports the survuval of glioblastoma multiforme cells

We have found that cyclophilin B (CypB) expression is important for malignant glioblastoma multiforme (GBM) cell proliferation. To identify molecular mechanisms that could explain CypB-dependent survival in human GBM cells, a microarray analysis was performed using RNA prepared from U251MG GBM cells transduced with lentiviral CypB shRNA. These data revealed that about 130 genes were more than 2-fold affected by CypB depletion. Significant alterations in the expression of genes related to cell death, cell proliferation and cell migration were found in the shCypB cells.

Whole hearts from wild-type and Na,K-ATPase alpha 1 het. mice.  Adult male, 8-16 weeks old on a 129/BSwiss background.

Vanadium pentoxide induced gene expression in human lung fibroblasts

Exposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5 in vitro in order to identify candidate genes that could play a role in airway remodeling associated with V2O5-induced bronchitis. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Expression data were preprocessed using RMA with a log2 transformation. Statistical analysis was performed in R using the affylmGUI package using a linear model with contrasts between untreated control and V2O5-exposed fibroblasts. Genes identified as statistically significant were filtered by selecting only those genes that exhibited a > 2-fold change. Quantitative real-time RT-PCR was utilized to confirm expression of selected genes. More than 2000 genes were significantly changed in response to V2O5 over the time course of our experiment. Genes altered by V2O5 were involved in biologic processes related to cell growth and differentiation, oxidative stress responses, immune regulation, and interferon signaling and apoptosis. In particular, V2O5 induced genes that encode growth factors involved in epithelial repair (HB-EGF) or angiogenesis (VEGF), peroxide generating enzymes (SOD2), pro-inflammatory enzymes (PGHS2), while suppressing genes involved in growth arrest (GAS1, STAT-1) and cell cycle inhibition (CDKN1B). Our study also identified a variety of novel genes that could be used as biomarkers of V2O5-induced bronchitis or could serve as candidate genes for disease progression.
Keywords: time course, stress response

Synthetic lethal screening with small molecule inhibitors provides a pathway to rational combination therapies for melanoma

Recent data demonstrate that extracellular signals are transmitted through a network of proteins rather than hierarchical signaling pathways. This network model suggests why inhibition of a single component of a canonical pathway, even when targeting a mutationally activated driver of cancer, has insufficiently  dramatic effects on the treatment of cancer. The biological outcome of signals propagated through a network is inherently more robust and resistant to inhibition of a single network component due to compensatory and redundant signaling events. In this study, we performed a functional chemical genetic screen analogous to synthetic lethal screening in yeast genetics to identify novel interactions between signaling inhibitors that would not be predicted based on our current understanding of signaling networks. We screened over 300 drug combinations in nine melanoma cell lines and have identified pairs of compounds that show synergistic cytotoxicity. Among the most robust and surprising results was synergy between sorafenib, a multi-kinase inhibitor with activity against Raf, and diclofenac, a non-steroidal anti-inflammatory drug (NSAID). This synergy did not correlate with the known RAS and BRAF mutational status of the melanoma cell lines. The NSAIDs celecoxib and ibuprofen could qualitatively substitute for diclofenac. Similarly, the MEK inhibitor PD325901 and the Raf inhibitor RAF265 could qualitatively substitute for sorafenib. These drug substitution experiments suggest that inhibition of cyclo-oxygenase and MAP kinase signaling are components of the observed synergistic cytotoxicity. Genome-wide expression profiling demonstrates synergy-specific down-regulation of survival-related genes. This study provides proof of principle that synthetic lethal screening can uncover novel functional drug combinations and suggests that the underlying signaling networks that control responses to targeted agents can vary substantially depending on unexplored components of the cell genotype.

Nrg4 is a brown fat-enriched adipokine that binds to the liver and preserves metabolic homeostasis during chronic energy excess. Adipose tissue Nrg4 expression is reduced in mouse and human obesity. Nrg4 deficient mice develp more severe insulin resistance and hepatic fat accumulation upon high-fat diet feeding.
We used microarrays to elucidate the metabolic pathways that are regulated by Nrg4 in the liver.

G9a is an H3K9m2 methyltransferase, which is critical in controlling gene suppression and DNA methylation. We used microarray analysis to identify the target genes that are regulated by G9a in MDA-MB231 cells, in which E-cadherin is silenced.

mRNA expression profile modified by microRNA mir-517a (MIR517A) in human hepatocellular carcinoma cell line

mRNA expression profile modified by stable transfection of microRNA mir-517a (MIR517A) in a human hepatocellular carcinoma cell line Huh-7

Gene expression in alveolar macrophages from phenotypically normal smokers compared to non-smokers

Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, ~20 pack-yr smokers compared to healthy non-smokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to non-smokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related, signal transduction and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in alveolar macrophages in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of alveolar macrophages associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.
Keywords: Comparison of gene expression profile in smokers vs non-smokers

normal and diabetic rat muscle treated and untreated with vanadyl sulfate

RNA was obtained from rear leg muscle of normal and diabetic rats in the presence and absence of oral treatment with vandyl sulfate. Diabetes was induced with streptozotocin and expression profiles were determined using Affymetrix Rat Genechip U34A.  The effect of treatment and disease on gene expression was examined.
Keywords: vanadium treatment

Knockdown of TERC with siRNA in normal bone marrow stromal cells (BMSCs) recapitulates the defective BMSC phenotype in patients with telomere biology disorders

Dyskeratosis congenita (DC) is an inherited multi-system disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow failure, solid tumors, and other medical problems such as osteopenia.  DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells.  We found that skeletal stem cells (SSCs) within the bone marrow stromal cell population (BMSCs, also known as bone marrow-derived mesenchymal stem cells), may contribute to the hematological phenotype.
TBD-BMSCs exhibited reduced clonogenicity, reduced telomerase activity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro.  Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by siTERC-RNA recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony forming efficiency, and by accelerating senescence in vitro.  Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the mRNA level, and decreased secretion of factors at the protein level.  These findings are consistent with defects in SSCs/BMSCs contributing to bone marrow failure in TBD.

disease state analysis, gene therapy, comparative gene expression, muscular dystrophy, glycosylation, collagens, integrins, dysferlin, dystroglycan

Gene expression profiling of Galgt2 overexpression in mdx skeletal muscle

Transgenic overexpression of Galgt2 in the skeletal muscles of mdx mice inhibits the development of disease pathology associated with muscular dystrophy.  This is the case both in transgenic mice, where Galgt2 overexpression occurs from embryonic timepoints onward and in mdx mice where Galgt2 is overexpressed in the early postnatal period using Adeno-associated virus (AAV).  Here, we use gene expression profiling to compare transcriptional changes resulting from embryonic and postnatal Galgt2 overexpression in mdx skeletal muscle.  A surprising number of changes were in genes known to ameliorate muscular dystrophy when overexpressed (agrin, integrin alpha 7, ADAM12, Bcl2) or to cause muscular dystrophy when mutated (collagen VI (alpha1,alpha2), plectin 1, dystroglycan, selenoprotein N1, integrin alpha7, biglycan, dysferlin).    Several genes involved in calcium homeostasis were also changed.  In Galgt2 transgenic mice, where embryonic overexpression of Galgt2 in skeletal muscles alters neuromuscular development and muscle growth, the number of gene expression changes was vastly greater, however, 14% of genes altered in postnatal AAV-Galgt2 infected mdx muscles were also changed with embryonic overexpression.  These experiments suggest that postnatal overexpression of Galgt2 inhibits muscular dystrophy in mdx mice via induction of a group of genes that, in aggregate, can govern membrane stability, membrane repair, calcium homeostasis, and apoptosis.
Keywords: disease state analysis, gene therapy, comparative gene expression, muscular dystrophy, glycosylation, collagens, integrins, dysferlin, dystroglycan

The transcriptional activities of c-Myb and its oncogenic variant v-Myb were compared by expressing them in primary human monocytes using recombinant adenovirus vectors. All the samples were compared to cells infected with a control adenovirus expressing only GFP.  The results showed that v-Myb, which differs from c-Myb only by N- and C-terminal deletions and eleven amino acid substitutions, has a qualitatively different transcriptional activity.

Profile of Bmpr1a-/- embryonic stem cells in ESC media and N2B27

A fundamental question in developmental biology is whether there are mechanisms to detect stem cells with mutations that although do not adversely affect their viability, would compromise their ability to contribute to further development. Here we show that cell competition is a novel mechanism regulating the fitness of embryonic stem cells (ESCs). We find that ESCs displaying defective BMP signalling, defective autophagy or are tetraploid are eliminated at the onset of differentiation by wild-type cells. This elimination occurs in an apoptotic dependent manner and is mediated by secreted factors. Furthermore, during this process we find that establishment of differential cMyc levels is critical and that cMyc over-expression is sufficient to induce competitive behaviour in ESCs. Cell competition is therefore a process that allows recognition and elimination of defective cells during the early stages of development and is likely to play important roles in tissue homeostasis and stem cell maintenance.
We used microarrays to compare the gene expression profiles of Bmpr1a-/- and control embryonic stem cells (ESCs) in the ESC state and after differentiation in N2B27

Expression data from wild type and Ctip2-/- (Bcl11b) mutant mouse striatum at P0

Striatal medium spiny neurons (MSN) are critically involved in motor control, and their degeneration is a principal component of Huntington’s disease. We find that the transcription factor Ctip2 (also known as Bcl11b) is central to MSN differentiation and striatal development. Within the striatum, it is expressed by all MSN, while it is excluded from essentially all striatal interneurons. In the absence of Ctip2, MSN do not fully differentiate, as demonstrated by dramatically reduced expression of a large number of MSN markers, including DARPP-32, FOXP1, Chrm4, Reelin, MOR1, GluR1, and Plexin-D1. Furthermore, MSN fail to aggregate into patches, resulting in severely disrupted patch-matrix organization within the striatum. Finally, heterotopic cellular aggregates invade the Ctip2-/- striatum suggesting a failure by MSN to repel these cells in the absence of Ctip2.   In order to investigate the molecular mechanisms that underlie Ctip2-dependent differentiation of MSN and that underlie the patch-matrix disorganization in the mutant striatum, we directly compared gene expression between wild type and mutant striatum at P0. Because CTIP2-expressing MSN constitute 90-95% of the neurons within the striatum, we reasoned that we should be able to detect changes in medium spiny neuron gene expression in Ctip2 null mutants. We microdissected out small regions of striatum at matched locations in wild type and Ctip2-/- mutant littermates at P0 and investigated gene expression with Affymetrix microarrays. We selected the 153 most significant genes and further analyzed them to identify a smaller set of genes of potentially high biological relevance. In order to verify the microarray data and define the distribution of the identified genes in the striatum, we performed in situ hybridization or immunohistochemistry for 12 selected genes: Plexin-D1, Ngef, Nectin-3, Kcnip2, Pcp4L1, Neto1, Basonuclin 2, Fidgetin, Semaphorin 3e, Secretagogin, Unc5d, and Neurotensin. We find that all these genes are either specifically downregulated (Plexin-D1, Ngef, Nectin-3 Kcnip2, Pcp4L1, Neto1), or upregulated (Basonuclin 2, Fidgetin, Semaphorin 3e, Secretagogin, Unc5d, Neurotensin), in the Ctip2-/- striatum, confirming and extending the microarray results. Together, these data indicate that Ctip2 is a critical regulator of MSN differentiation, striatal patch development, and the establishment of the cellular architecture of the striatum.

Wilms' tumor 1 WT1(-KTS) isoform time course induction in U2OS cells

The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that plays important roles during development of several organs including metanephric kidneys. A number of WT1 target genes have been identified, but the detailed mechanisms by which WT1 orchestrates renal development remain elusive. To identify WT1 target genes relevant to development, genome-wide expression profiling was performed using oligonucleotide microarrays representing 39,000 human transcripts
Keywords: Wilms' tumor 1 (WT1) gene, -KTS isoform; time course

Pregnant C57Bl6N mice were treated with 0 (corn oil), 1.5, 3.0, or 6.0 ug/kg TCDD on gd14.5.  Fetal hearts were collected on gd17.5.  Hearts from each litter were pooled onto one chip.  4 replicates of each condition were run on affymetrix MG_U74Av2 chips, using standard affymetrix protocols and controls.
Keywords: dose-response, 3 doses plus corn oil control, 4 replicates

Androgen receptor (AR) is a ligand-dependent transcription factor that plays a key role in the onset and progression of prostate cancer. Surprisingly little is known of AR binding sites and collaborating transcription factors in the human genome. Here we have identified the DNA sequence motifs that are significantly enriched within the authentic 90 AR target regions found on chromosomes 21 and 22 in human prostate cancer cells by combining chromatin immunoprecipitation for AR with chromosome-scale tiled oligonucleotide microarrays. By integrating the DNA sequence motif data with the gene expression profiles from human prostate cancers we identified the transcription factors that recognize each of these motifs. These factors form complexes with AR, bind to specific AR target regions and govern androgen-dependent transcription. Together with AR these collaborating transcription factors form a regulatory network that directs prostate cancer growth and survival and identify potential new opportunities for therapeutic intervention.

RNA expression analysis of lineage-negative marrow progenitors after Cebpa knockdown.

Analysis of global RNA expression of Cebpa knockdown lineage-negative marrow cells reveals known and potentially novel C/EBPa targets.

Novel bio-marker discovery for stratification and prognosis of breast cancer patients

The study entails novel bio-marker discovery of Tumor Aggressive Grade signature (TAGs) genes and their role in recurrence free survival of breast cancer (BC) patients. Current BC dataset was used for co-expression analysis of TAGs genes and their role in BC progression. Additionally, recent findings have suggested an importance of structural organization of sense-antisense gene pairs (SAGPs) for transcription, post-transcriptional and post-translational events and their associations with cancer and disease. We studied SAGPs in which both gene partners are protein encoding genes (coding-coding SAGPs), their role in human BC development and demonstrated their potential for BC stratification and prognosis. Based on gene expression and correlation analyses we identified the robust set of breast cancer-relevant SAGPs (BCR-SAGPs). We isolated and characterized the sense-antisense gene signature (SAGS) and evaluated its prognostic potential in various gene expression datasets comprising 1161 BC patients. The methods used included the Cox proportional survival analysis, statistical analysis of clinicopathologic parameters and differential gene expression. The SAGS was effective in identification of BC patients with the most aggressive disease. Independently, we validated the SAGS using 58 RNA samples of breast cancer tumors purchased from OriGene Technologies (Rockville, MD).

Activation of oncogenic ras pathway accounts for up to 90% low-grade superficial urothelial carcinomas of bladder, and p53 deficiency is very common in high-grade muscle invasive carcinomas. These two pathways in bladder urothelial tumorigenesis used to be considered divergent and their potential collaboration has not been illustrated.
We did laser capture micro-dissection (LCM) to separate the tumor cells from the whole bladder tissues from H-ras transgenic and p53 knockout mice, as well as normal urothelial cells as control. Microarray was then performed to identify distinct classes of up-regulated genes during tumorigenesis and progression.

Analysis of expression profiles of pDCs from wild type and heterozygous E2-2 mice. Results show the control by E2-2 of the expression of pDC-enriched genes.

Estradiol Treated Breast Cancer Cells Expressing Mutant Estrogen Receptors

MDA-MB-231 Breast Cancer Cells expressing either wild-type estrogen receptor or the mutant estrogen recepor L540Q were treated with estradiol for 1 or 2 hours

PCOS, DNA microarray, global pathway analysis, mitochondrial oxidative metabolism, qantitative real time PCR, insulin resistance, skeletal muscle

Reduced expression of mitochondrial oxidative metabolism genes in skeletal muscle of women with PCOS

Recently, abnormalities in mitochondrial oxidative phosphorylation (OXPHOS) have been implicated in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes. In the present study, we hypothesized that decreased expression of OXPHOS genes could be of similar importance for insulin resistance in the polycystic ovary syndrome (PCOS).
Using the HG-U133 Plus 2.0 expression array from Affymetrix, we analyzed gene expression in skeletal muscle from obese women with PCOS (n=16) and age- and body mass index-matched control women (n=13) metabolically characterized by euglycemic-hyperinsulinemic clamp and indirect calorimetry. To identify pathways of importance for the pathogenesis of insulin resistance in PCOS, we performed biological pathway analysis using Gene Set Enrichment Analysis (GSEA 1.0) and Gene Microarray Pathway Profiler (GenMAPP 2.0). The expression of 9 genes, selected according to biological relevance, was evaluated by quantitative real time PCR (q-RT-PCR).
Women with PCOS were characterized by fasting hyperinsulinemia and impaired insulin-stimulated glucose disposal - caused by reduced glucose oxidation and storage - as well as impaired suppression of lipid oxidation (all P<0.01). GSEA and GenMAPP both revealed the same set of genes involved in OXPHOS, which was also the most downregulated biological pathway (P<0.01). These results were confirmed by q-RT-PCR of six genes from the OXPHOS gene set as well as three transcription factors known to regulate the transcription of these genes.
Our results, for the first time, provide evidence for an association between insulin resistance and impaired mitochondrial oxidative metabolism in skeletal muscle in women with PCOS. This may contribute to the increased risk of type 2 diabetes observed in these women

miR-142-3p is highly expressed in peripheral blood mononuclear cells (PBMCs) and has been described as a hematopoietic-restricted lineage, suggesting immune functions (Chen, Li et al. 2004; Landgraf, Rusu et al. 2007; Merkerova, Belickova et al. 2008). In order to determine the roles of miR-142-3p in B lymphocytes, we over-expressed this miRNA in the Raji B-cell line using a synthetic mimic of miR-142-3p and analyzed gene expression 24 hours after the transfection.

Genome-wide analysis of Phgdh inactivation in murine embryonic head

D-3-Phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95) is a necessary enzyme for de novo L-serine biosynthesis via the phosphorylated pathway. We demonstrated previously that Phgdh is expressed exclusively by neuroepithelium and radial glia in developing mouse brain and later mainly by astrocytes. Mutations in the human PHGDH gene cause serine deficiency disorders (SDD) associated with severe neurological symptoms such as congenital microcephaly, psychomotor retardation, and intractable seizures. We recently demonstrated that genetically engineered mice, in which the gene for Phgdh has been disrupted, have significantly decreased levels of serine and glycine, and exhibit malformation of brain such as microcephaly. The Phgdh null (KO) embryos exhibit lethal phenotype after gestational day 14, indicating that the phosphorylated pathway is essential for embryogenesis, especially for brain development. It is worth noting that the Phgdh knockout (KO) embryos primarily displayed microcephaly, which is the most conspicuous phenotype of patients with SDD.  Thus, Phgdh KO mice are a useful animal model for studying the effect of diminished L-serine levels on development of the central nervous system and other organs. To better understand the mechanism underlying the molecular pathogenesis of SDD, we sought to examine whether gene expression is altered in the Phgdh KO mouse model. We identify genes that have altered expression in the head of the Phgdh KO embryos using the GeneChip array. Some of the genes identified by this method belong in functional categories that are relevant to the biochemical and morphological aberrations of the Phgdh deletion.

Gene expression data from MGHU3 bladder cancer cells (FGFR3-Y375C) treated with TAK1 siRNA and/or PD173074

The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation.  A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1).  Here, we identify TAK1 as a novel interacting protein and direct target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity.
We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes which regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types.  Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

BP1 Expression Promotes Tumorigenesis and Aggression in MCF-7 Breast Cancer Cells

This study was designed to identify genes that are differentially expressed when BP1 homeobox gene is overexpressed in MCF-7 breast cancer cells. The goal is to understand the functional role of BP1 in breast tumorigenesis.

Expression data from wild type, Ring1B-/-, Eed-/-, and Ring1B/Eed double deficient mouse ES cells

Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb repressive complex 1 (PRC1) and PRC2 during the formation of differentiated cells. ES cells lacking the function of either PRC1 or PRC2 can differentiate into cells of the three germ layers, whereas simultaneous loss of PRC1 and PRC2 abrogates differentiation. On the molecular level the differentiation defect is caused by the derepression of a set of genes that is redundantly repressed by PRC1 and PRC2 in ES cells. Furthermore, we find that genomic repeats are Polycomb targets and show that in the absence of Polycomb complexes endogenous MLV elements can mobilize. This indicates a contribution of the PcG system to the defense against parasitic DNA and a potential role of genomic repeats in Polycomb mediated gene regulation.

Comparison of gene expression in Wild type and T cell-specific conditional Trim28 KO in TCR stimulated and un-stimulated naive CD4 positive and T regulatory cells

A microarray study was performed in unstimulated and TCR-stimulated CD4 + T cells and Treg in wild type and conditional Trim28 KO mice to identify genes that are regulated by Trim28.  These experiments constitute a portion of the study described below:     Paper Abstract: Peripheral T cell activation and differentiation into specialized effectors are regulated by TCR- and cytokine-mediated signals that induce clonal expansion and unique transcriptional factors. These processes may include active chromatin modification by nuclear factors. In search of such molecules, we found Trim28, a component of large nuclear chromatin-regulatory complex is tightly controlled upon TCR stimulation at the level of phosphorylation, and examined global impact of Trim28 loss in especially CD4+ T cells, by generating T cell-specific conditional Trim28 KO mice (CKO). CD4+ T cells from CKO mice showed defective IL-2 production and T cell proliferation associated with defective upregulation of cell-cycle associated proteins. Accordingly, young CKO showed T-lymphopenia. Surprisingly, Trim28 CKO mice eventually accumulated auto-reactive memory-phenotype T cells that produced inflammatory IL-17. CKO mice are also susceptible to induced auto-inflammatory disease with TH-17 dominant immune response.  Loss of Trim28 showed aberrant accumulation of TH-17 and FoxP3+ T cells, two key T cells in inflammation vs. tolerance. We found CKO T cells showed a cell-extrinsic promotion of TH-17 and FoxP3+ T cell development by a mechanism involving overproduction of TGF-beta. Our study revealed unexpected roles of Trim28, a global chromatin regulator in both T cell activation and tolerance.

Expression profiling in Williams-Beuren Syndrome patient fibroblast cell lines

Williams-Beuren Syndrome (WBS) is a neurodevelopmental disorder caused by aa 1.5 Mb microdeletion on human chromosome 7. Although the molecular cause of the disorder is well-established, little is known about the global impact of the deletion on gene expression. Here we profiled the transcriptomes of fibroblast cell lines from 8 young girls with WBS, and 9 sex- and age-matched control individuals

Expression data from adult rat tail MNs after spinal cord transection

Spinal cord injury leads to impaired motor and sensory functions. After spinal cord injury there is a an initial phase of hypo-reflexia followed by a developing hyper-reflexia, often termed spasticity. Previous studies have suggested a relationship between the reappearence of plateau potentials in motor neurons and the development of spasticity after spinalization. To understand the molecular mechanism behind this phenomenon we examined the transcriptional response of the motor neurons after spinal cord injury.
We used a rat tail injury model where a complete transection of the caudal (S2) rat spinal cord leads to an immidate flaccid paralysis of the tail and a subsequent appearence of spasticity 2-3 weeks post injury that develops into strong spasticity after 2 months.
Gene expression changes were studied in motor neurons 21 and 60 days after complete spinal transection where the tail exhibits clear signs of spasticity.

shRNA kinome screen identifies TBK1 as therapeutic target for HER2+ breast cancer

Microarray analysis was performed at the UHN Microarray Centre (UHNMAC, Ontario, Canada) using Illumina HumanHT-12 v4 BeadChip with 500 ng of total RNA prepared by RNeasy mini kit (QIAGEN, Cat. No. 74104). Samples from HCC1954 cells with 3-day treatment of TBK1-II at 4 uM were used to compare with vehicle-treated controls.  Microarray data was processed and normalized by lumi package from BioConductor in R with Quantile Method.  Difference between the samples were calculated by Bayesian statistic using limma package from BioConductor in R to obtain Moderated T value for subsequent Pathway analysis.

Expression data from WT mice and bitransgenic Pdx1-cre/Kras*A mice bearing Pancreatic Ductal Adenocarcinoma

We have carried out transcriptional profile analysis in WT MICE and bitransgenic Pdx1-cre/Kras*A MICE baring Pancreatic Ductal Adenocarcinoma
Mouse models faithfully simulating human cancer are valuable for genetic identification of potential  drug-targets but, among them, the most advantageous for practical use in subsequent preclinical  testing of candidate therapeutic regimes are those exhibiting rapid tumor development. Considering  that a KRAS mutation (predominantly in codon 12, such as KRASG12D; KRAS*) occurs with high  frequency (~90%) in cases of human pancreatic ductal adenocarcinoma (PDA)1, we sought to develop  a mouse PDA model that would exhibit high tumor incidence and short latency by ectopic  overexpression of Kras*.

Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines

We report the effect of motexafin gadolinium (MGd) and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, cell cycle, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines.  We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptotic response.  Treatment with exogenous zinc increased oxidative stress and, conversely, oxidative stress resulted in increased intracellular free zinc levels.  To better understand the molecular basis of these results, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate.  Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of MTF-1 and HIF-1 regulated genes.  Cultures co-treated with MGd and zinc acetate displayed further increases in the levels of MTF-1 and HIF-1 regulated transcripts as well as additional transcripts regulated by NRF-2.  Overall, these studies suggest that co-treatment of Ramos cells with MGd and zinc acetate increase intracellular zinc levels with a concomitant rise in oxidative stress levels that activate adaptive survival responses but eventually lead to cell death.
Keywords: Dose response

We aimed to identify genes that are regulated by FGFR1 in brown adipose tissues of adult male ob/ob mice by injecting 1 mg/kg anti-FGFR1 agonistic antibody.

Alveolar epithalial cells (A549) were used as a model of pulmonary parenchymal cell activation to quiery gene expression profile of activation with either cyclic stretch (20% elongation), TNF (20 ng/ml), LPS (1 mcg/ml) or TNF+STRETCH at both 1 and 4 hrs.

Expression data comparing azacitidine and decitabine in non-small cell lung cancer lines

Azacitidine (AZA) and decitabine (DAC) are cytidine azanucleoside analogs with clinical activity in myelodysplastic syndromes (MDS) and potential activity in solid tumors. To better understand the mechanism of action of these drugs, we examined the effects of AZA and DAC in a panel of non-small cell lung cancer (NSCLC) cell lines. Of 5 NSCLC lines tested in a cell viability assay, all were sensitive to AZA (EC50 of 1.8–10.5 µM), while only H1299 cells were equally sensitive to DAC (EC50 of 5.1 µM). In the relatively DAC-insensitive cell line A549, both AZA and DAC caused DNA methyltransferase I depletion and DNA hypomethylation; however, only AZA significantly induced markers of DNA damage and apoptosis, suggesting that mechanisms in addition to, or other than, DNA hypomethylation are important for AZA-induced cell death. Cell cycle analysis indicated that AZA induced an accumulation of cells in sub-G1 phase, whereas DAC mainly caused an increase of cells in G2/M. Gene expression analysis of AZA- and DAC-treated cells revealed strikingly different profiles, with many genes distinctly regulated by each drug. In summary, while both AZA and DAC caused DNA hypomethylation, distinct effects were demonstrated on regulation of gene expression, cell cycle, DNA damage, and apoptosis.

Using targeted gene deletion, we have firmly established that the Class IV POU domain transcription factor Brn-3.2 controls a developmental program regulating axon pathfinding in the mouse visual system. We have isolated and identified downstream gene targets of Brn-3.2, using Representational Difference Analysis (RDA), on cDNA populations derived from wildtype and Brn-3.2-/- retina at the appropriate embryonic stage. One of these candidate genes, the Homeodomain Interacting Protein Kinase 2 (HIPK2), is postulated to be a transcriptional coregulator, based on its in vitro interactions with repressor homeodomain proteins of the NK class, as well as other components of repressor complexes. HIPK2 has also been shown to be involved in post-translational modification of two major transcriptional regulators, p53 and CtBP. Expression of a dominant-negative form of HIPK2 in sensory neurons affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect.
The aim of this project is to identify targets for the Homeodomain Interacting Protein Kinase 2 (HIPK2), that we isolated as a downstream gene target of the class IV POU domain transcription factor Brn-3.2, and to investigate their function in the Brn-3.2 dependent pathway regulating axon pathfinding.
We hypothesize that the genes regulated by HIPK2 will play a critical role in axon pathfinding and that the results of this study will provide novel insights into the molecular mechanisms of axon pathfinding, and possibly neural plasticity and regeneration, and therefore, be of great interest to the field of neurobiology in general.
In order to uncover potential biological role(s) of HIPK2 in neural development, and particularly axon pathfinding, we generated transgenic mouse models for in vivo studies. Our preliminary observations in transgenic mice, indicate that a form of the molecule expected to act as a dominant-negative and designed to be expressed in sensory neurons, affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect. We plan to take advantage of this model to identify genes regulated by HIPK2, and which are likely to be involved in axon pathfinding. To this end, we will compare gene expression profiles in sensory neurons isolated wild-type and transgenic mice. In our experimental paradigm, the trigeminal ganglion represents the ideal sensory structure in which to perform such an analysis: 1) High levels of transgene can be expressed in the trigeminal ganglion, as assessed by the expression of LacZ from the bicistronic construct which contains IRES-LacZ downstream of a dominant negative form of HIPK2.  2) The trigeminal ganglion can be dissected at embryonic day 13.5, when the phenotype is apparent, with ease and free of contamination from surrounding tissues.  3) The amounts of RNA that can be isolated from a single ganglion are in the range of 200-300 ng, which should be sufficient for microarray analysis following linear amplification of RNA. One series will be carried out with transgenic embryos and a control series will be carried out with wildtype littermates. Animals will be prepared and sacrificed by a standard protocol. Tissue will be rapidly dissected from E13.5 trigeminal ganglion, frozen in liquid nitrogen, and stored at -80C until RNA is extracted. RNA will be prepared using RNeasy Micro Kit. We will be providing 4 tissue samples for each of the wildtype and transgenic animals to mitigate any expression differences resulting from mouse to mouse variation.

Gene expression profiles of patients with schizophrenia, bipolar disorder and healthy controls

Schizophrenia (SZ) and bipolar disorder (BD) are severe psychiatric conditions, with a lifetime prevalence of about 1%. Both disorders have a neurodevelopment component, with onset of symptoms occurring most frequently during late adolescence or early adulthood. Genetic findings indicate the existence of an overlap in genetic susceptibility across the disorders. These gene expression profiles were used to identify the molecular mechanisms that differentiate SZ and BP from healthy controls but also that distinguish both from healthy individuals. They were also used to expand an analysis from an experiment that searched molecular alterations in human induced pluripotent stem cells derived from fibroblasts from control subject and individual with schizophrenia and further differentiated to neuron to identify genes relevant for the development of schizophrenia (GSE62105).

In vivo gene expression data from wild type and CTLA-4 KO 5C.C7 T cells

CTLA-4 is thought to inhibit effector T cells both intrinsically, by competing with CD28 for B7 ligands, and extrinsically, through the action of regulatory T cells. We studied in vivo responses of normal and CTLA-4-deficient antigen-specific murine effector CD4+ T cells. In order to do these studies in a physiological model of immunity to foreign antigen, we transferred small numbers of congenically marked RAG2-deficient 5C.C7 T cells with either a normal or knockout allele of CTLA-4 into normal syngeneic B10.A recipient mice. The T cells were then activated by immunization with MCC peptide and LPS. To look for transcriptional signatures of negative regulation of T cell responses by CTLA-4, we used microarray analysis to compare transcripts in wild type and CTLA-4 KO 5C.C7 T cells four days after immunization. This is the first instance in which differences are observed in extent of accumulation of wild type and CTLA-4 KO 5C.C7 T cells.

Identification of an SRF- and androgen-dependent gene signature in prostate cancer

The androgen receptor (AR) is the principal target for treatment of non-organ confined prostate cancer (PCa).  Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes and point towards a role for secondary transcription factors (TFs) therein. We identified a novel indirect mechanism of androgen action in which effects of androgens on PCa cells are mediated by Serum Response Factor (SRF).    To identify and characterize genes and cellular processes that are androgen-regulated in an SRF-dependent manner in PCa, Affymetrix HG-U133 Plus 2.0 GeneChip Array analysis was performed starting from RNA obtained from LNCaP cells in which androgen stimulation was combined with siRNA-mediated SRF silencing.  To this end, LNCaP cells were seeded in 60 mm dishes at a density of 550,000 cells per dish in antibiotic-free medium.  The next day, cells were transfected with siGenome SmartPool siRNA targeting SRF (Dharmacon, Lafayette, CO) or a custom-made control SmartPool targeting luciferase (LUC condition) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions.  Forty-two hours after transfection, cells were treated with 5nM R1881 or ethanol vehicle.  3 biological triplicates were included per treatment group.  Forty-eight hours later, cells were harvested in Trizol reagent (Invitrogen). RNA was isolated, purified on RNeasy columns (Qiagen, Germantown, MD) and checked for integrity by Agilent testing (Affymetrix, Santa Clara, CA).  cDNA was generated and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the manufacturer’s instructions at the Mayo Clinic Advanced Genomics Technology Microarray Shared Resource core facility.

A High-Fat Diet and NAD+ Rescue Premature Aging in Cockayne Syndrome

Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in the genes encoding the DNA repair proteins CSA or CSB. Csbm/m mice were given a high-fat, caloric-restricted or resveratrol-supplemented diet. The high-fat diet rescued the phenotype of Csbm/m mice at the metabolic, transcriptomic and behavioral levels. Additional analysis suggests that the premature aging seen in CS mice, nematodes and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high-fat diet; and β-hydroxybutyrate, PARP inhibition, or NAD+ supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB is able to displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD+, through the deacetylase SIRT1 and suggests possible interventions for CS.

Negative regulation of the IFN/STAT signaling pathway by the Trim24 tumor suppressor protein through Rara inhibition

Recent genetic studies in mice have established a key role for the nuclear receptor coregulator Trim24 in liver tumor suppression and provided evidence that Trim24 suppresses hepatocarcinogenesis by inhibiting retinoic acid receptor alpha (Rara)-dependent transcription and cell proliferation. However, it is unknown which downstream targets of Rara regulated by Trim24 are critical for tumorigenesis. We report here that loss of Trim24 results in the overexpression of interferon (IFN)/STAT pathway genes in the liver, a process that occurs early in tumorigenesis and is more pronounced in tumors, despite the enhanced expression, late in the disease, of negative regulators such as Usp18, Socs1 and Socs2.
Remarkably, Rara haplodeficiency, which was previously shown to suppress tumor development in Trim24-/- mice, also suppresses overexpression of the IFN/STAT pathway,  thus providing evidence for a cross-pathway control that may be relevant to the transformation process. Biochemical studies revealed that Trim24 binds to the retinoic acid (RA)-responsive element in the Stat1 promoter in a RA-dependent manner and represses RA-induced transcription from this promoter. Together, these results identify Trim24 as a novel regulator of the IFN/STAT pathway and indicate that Trim24-mediated repression of the IFN/STAT signaling through Rara inhibition may play a critical role in preventing liver cancer.

F9 cells were exposed to two different differentiating agents (retinoic acid or trichostatin A both dissolved in 100% ethonal) for 24 hrs.  Control cells were exposed to ethonal for 24hrs

Empty vector, SRF-dM-VP16 (control construct lacking the SRF DNA binding domain), or SRF-VP16 were expressed in wild-type ES cells (E14wt), SRF heterozygotes (E99-/+) or two SRF ko cell lines (E81-/-,E100-/-). RNA was harvested 72h after transfection. Each condition was performed in duplicate, except E100-/- vector transfected.

PRDM14 Ensures a Naïve Pluripotency by a Dual Mechanism Involving Signaling and Epigenetic Pathways in Mouse Embryonic Stem Cells

Mouse embryonic stem cells (mESCs) fluctuate between a naïve inner cell mass (ICM)-like state and a primed epiblast-like state of pluripotency in serum, but are harnessed exclusively in a distinctive, apparently more naïve state of pluripotency (the ground state) with inhibitors for mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i).  Understanding the mechanism ensuring a naïve state of pluripotency would be critical in realizing a full potential of ESCs.  We show here that PRDM14, a PR domain-containing transcriptional regulator, ensures a naïve pluripotency by a dual mechanism: Antagonizing fibroblast growth factor receptor (FGFR) signaling that is activated paradoxically by the core transcriptional circuitry for pluripotency and directs a primed state and repressing de novo DNA methyltransferases that create a primed epiblast-like epigenome.  PRDM14 exerts these functions by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression.

Loss of BRMS1 promotes a mesenchymal phenotype through regulation of Twist1

Analysis of BRMS1 KD-induced EMT in non-samll cell lung cancer at gene expression level. The hypothesis tested in the present study was that BRMS1 KD induces EMT through differential regulation of EMT genes and Twist1 KD restores the epithelial phenotype in cells with BRMS1 KD. Results provide important information of biological functions in lung cancer which BRMS1 KD involves in, such as EMT, signaling, biological adhesion, immune system process, response to stimulus, and so on.

Susceptibility to DNA damage as a molecular mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip/palate (NSCL/P) is a complex, frequent congenital malformation, determined by the interplay between genetic and environmental factors during embryonic development. Previous findings have appointed an aetiological overlap between NSCL/P and cancer, and alterations in similar biological pathways may underpin both conditions. Here, using a combination of transcriptomic profiling and functional approaches, we report that NSCL/P dental pulp stem cells exhibit dysregulation of a co-expressed gene network mainly associated with DNA double-strand break repair and cell cycle control (p = 2.88x10-2 – 5.02x10-9). This network included important genes for these cellular processes, such as BRCA1, RAD51, and MSH2, which are predicted to be regulated by transcription factor E2F1. Functional assays support these findings, revealing that NSCL/P cells accumulate DNA double-strand breaks upon exposure to H2O2. Furthermore, we show that E2f1, Brca1 and Rad51 involved in DNA repair are co-expressed in the developing embryonic orofacial primordia, and may act as a molecular hub playing a role in lip and palate morphogenesis. In conclusion, we show that cellular defences against DNA damage may take part in the pathogenesis of NSCL/P, in accordance with the hypothesis of aetiological overlap between this malformation and cancer. These results provide more information regarding the aetiology of NSCL/P and have the potential tocan potentially assist incontribute to the development of future preventive strategies.

Comparison of hepatocellular carcinoma liver samples to non-cancerous cirrhotic and normal liver samples (average of 5 samples each)

A total of 10 male Fischer 344 rats, 5 weeks old, were purchased from Charles River Japan, Incorporated, Atsugi, Kanagawa, Japan.  Rats were divided into 2 groups each consisting of 5 animals housed in a plastic cage with white flake bedding in an air-conditioned room.  Rats were used for the experimentation after a 1-week acclimation on a basal diet (CRF-1, Oriental Yeast Corporation, Itabashi, Tokyo, Japan) and allowed free access to food and tap water throughout the acclimation and experimental periods.  Body weight, food consumption, and water intake were monitored weekly.  The CDAA diet was obtained from Dyets, Bethlehem, PA.
After acclimation, group 1 rats were administered the basal diet, while group 2 rats were placed on the CDAA diet for 70 weeks and then sacrificed.  The livers were taken and macroscopically examined.  Portions of group 1 livers, as well as the macroscopic tumors and their surrounding, non-cancerous tissues of group 2 livers were fixed in 10% neutrally-buffered-formalin for 24 hours, embedded in paraffin, processed for the routine hematoxylin-and-eosin staining procedure, and histologically examined.  Remaining portions of these 3 types of liver tissues were immediately frozen in liquid nitrogen and stored at -80C.  Group 1 liver tissues, group 2 liver tissues from non-cancerous areas, and group 2 liver tissues from tumors (after histologically diagnosed as HCCs; see the results section) were used as normal liver samples, surrounding non-cancerous liver samples, and HCC samples, hereafter referred to as CON, NC, and CA, respectively.  The microarray and RT-PCR experiments and the subsequent data analysis were performed using 5 CON samples from individual group 1 rats and 5 matched pairs of NC and CA samples from individual group 2 animals.
RNA Isolation and Probe Labeling.  Total RNA from liver tissues was isolated using RNeasy Midi kits (QIAGEN, Hilden, Germany).  The integrity of the RNA was checked by electrophoresis on 1% agarose-formaldehyde gels. Five micrograms of total RNA from individual samples were labeled with Cy3 (Amersham Biosciences, Uppsara, Sweden) using BD Atlas Powerscript Fluorescent Labeling kits (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer’s protocols.
Hybridization, Scanning, and Quantification.  Cy3-labeled probes were hybridized to Atlas Rat 3.8 I microarrays (BD Biosciences Clontech) containing 3757 genes for 16 hours at 50C.  After hybridization, microarrays were washed, dried, and scanned using a GMS 418 confocal laser scanner (Genetic MicroSystems, Woburn, MA).  Fluorescence intensities of the Cy3 channels were quantified using ImaGene 4.0 software (BioDiscovery, El Segundo, CA).
Data analysis was performed using the GeneSpring software, version 5.1 (Silicon Genetics, Redwood City, CA), including appropriate statistics.  Dividing by the median calculated from all of the signal intensities for the Cy3 in a given sample normalized the fluorescence signal for each gene.  The VALUEs for each gene was then calculated by dividing the normalized signal by the median of each gene to offset differences in expression levels between genes.

Disease mechanisms in peripheral neuropathies due to altered Pmp22 gene dosage or a Pmp22 point mutation

We compared gene expression at ages P4 and P60 in sciatic nerve of wild type mice and mice with peripheral neuropathies caused by altered Pmp22 gene dosage (homozygous knockout or transgene) or a point mutation (Trembler).

Altered de novo lipogenesis contributes to low adipose stores in cystic fibrosis mice.

Cystic fibrosis (CF) mouse models exhibit exocrine pancreatic function yet do not develop adipose stores to the levels of non-CF mice. CF mice homozygous for the Cftr mutation (F508del) at 3 weeks (post-weaning) and 6 weeks (young adult) of age had markedly less adipose tissue than non-CF mice. Both 3- and 6-week old mice had dietary lipid absorption and fecal lipid excretion comparable to non-CF controls. Fractional hepatic de novo synthesis of palmitate and stearate (de novo lipogenesis, DNL) as determined by deuterium incorporation was reduced in CF mice. At 3 weeks of age, F508del mice had significantly decreased DNL of palmitate and stearate, by 83% and 80%, respectively. By 6 weeks of age, DNL rates in non-CF mice remained unchanged as compared to 3-week old mice, while DNL rates of F508del mice were still reduced, by 33% and 40%, respectively. Adipose tissue fatty acid profiles were comparable in CF and non-CF mice, indicating that adipose differences are quantitative, not qualitative. A correspondingly lower content of deuterium-labeled fatty acids was found in CF adipose tissue, consistent with reduced deposition of newly made hepatic triglycerides and/or decreased adipose tissue lipogenesis. Hepatic transcriptome analysis revealed lower mRNA expression from several genes involved in fatty acid biosynthesis, suggesting down-regulation of several enzymes in fatty acids synthesis as a mechanism for the reduced lipogenesis. These novel data provide a model for altered fat and fatty acid metabolism in CF, independent of malabsorption, and may partly explain the inability of pancreatic enzyme replacement therapy to completely restore normal body mass to CF patients

The PARK2 gene was knocked down using 2 independent siRNAs in SNB19 and SF539 cell lines
A non-targeted scramble siRNA was used as the control.

In order to better understand signaling events following receptor dimerization involving HER2, we have generated an experimental system in which ErbB dimerization can be controlled.
We used gene expression microarrays to identify genes and pathways that are differentially activated by HER2 homodimers and HER2 containing heterodimers.

Cytochrome P450;NADPH-cytochrome P450 reductase;transgenic mice;liver;nuclear receptor;other

Altered hepatic gene expression in liver-Cpr-null and Cpr-low mice

NADPH-cytochrome P450 reductase (CPR) is important for the functions of many enzymes, such as microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. Two mouse models with deficient CPR expression in adults were recently generated in this laboratory: liver-Cpr-null (with liver-specific Cpr deletion) (Gu et al., J. Biol. Chem., 278, 25895–25901, 2003) and Cpr-low (with reduced CPR expression in all organs examined) (Wu et al. J. Pharmacol. Expt. Ther. 312, 35-43, 2005). The phenotypes included a reduced serum cholesterol level and an induction of hepatic P450 in both models, and hepatomegaly and fatty liver in the liver-Cpr-null mouse alone. Our aim was to identify hepatic gene-expression changes related to these phenotypes. Cpr-lox mice, which have normal CPR expression (Wu et al., Genesis, 36, 177-181, 2003.), were used as the control in microarray analysis. A detailed analysis of the gene-expression changes in lipid metabolism and transport pathways revealed potential mechanisms, such as an increased activation of constitutive androstane receptor (CAR) and a decreased activation of peroxisomal proliferators activated receptor alpha (PPAR-gamma) by precursors of cholesterol biosynthesis, that underlie common changes (e.g., induction of multiple P450s and inhibition of genes for fatty acids metabolism) in response to CPR-loss in the two mouse models. Moreover, we also uncovered model-specific gene-expression changes, such as the induction of a lipid translocase (CD36 antigen) and the suppression of carnitine O-palmitoyltransferase 1 (CPT1a) and acyl-CoA synthetase long-chain family member 1 (Acsl1), that are potentially responsible for the severe hepatic lipidosis observed in liver-Cpr-null, but not Cpr-low mice.
Keywords = Cytochrome P450

BRD4 Inhibition of spindle cell malignant peripheral nerve sheath tumor (sMPNST) tumor cells

Immunity and Defense Genes in Peripheral Blood Mononuclear Cells of Rheumatoid Arthritis patients

Large-scale gene expression profiling of peripheral blood mononuclear cells from Rheumatoid Arthritis (RA) patients could provide a molecular description that reflects the contribution of diverse cellular responses associated with this disease. The aim of our study is to identify peripheral blood gene expression profiles for RA patients, using Illumina technology, to gain insights into RA molecular mechanisms. The Illumina Human-6v2 Expression BeadChips were used for a complete genome-wide transcript profiling of peripheral blood mononuclear cells from 18 RA patients and 15 Controls. Differential analysis per gene was performed with one-way analysis of variance (ANOVA) and P values were adjusted to control the False Discovery Rate (FDR<5%). Genes differentially expressed at significant level between patients and controls were analyzed using Gene Ontology (GO) in the PANTHER database to identify biological processes. A differentially expression of 339 Reference Sequence genes (238 down-regulated and 101 up-regulated) between the two groups was observed. We identified a remarkably elevated expression of a spectrum of genes involved in Immunity and Defense in peripheral blood mononuclear cells of RA patients compared to Controls. This result is confirmed by GO analysis, suggesting that these genes could be activated systemically in RA. No significant down-regulated ontology groups were found. Microarrays data were validated by real time PCR in a set of nine genes showing a high degree of correlation. Our study highlighted several new genes that could contribute in the identification of innovative clinical biomarkers for diagnostic procedures and therapeutic intervention. Further studies on larger scale groups of patients should be performed with the same technology to replicate these results and to allow clinical stratification.

FGFR3-shRNA induced transcriptional changes in RT112 bladder cancer cells

Aberrant activation of FGFR3 via overexpression or mutation is a frequent feature of bladder cancer; however, its molecular and cellular consequences and functional relevance to carcinogenesis are not well understood.  In this study with a bladder carcinoma cell line expressing inducible FGFR3 shRNAs, we sought to identiy transcriptional targets of FGFR3 and investigate their contribution to bladder cancer development.

Investigate the role of Wnt/beta-catenin signaling in development of the exocrine pancreas

beta-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of beta-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that beta-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of beta-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of beta-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of beta-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed beta-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.

Identification of Nrf2-regulated genes in A549 lung cancer cells by oligonucleotide microarray

To elucidate the mechanisms by which Nrf2 regulates cell growth, we performed global gene expression profiling of A549 lung cancer cells with knockdown of Nrf2. Gene networks associated with carbohydrate metabolism and drug metabolism were significantly downregulated in Nrf2-depleted A549 cells. Gene Set Enrichment Analysis revealed significant enrichment of genes associated with carbohydrate catabolic processes, positive regulation of metabolic processes, PPP, and arachidonic acid metabolism. In summary, this analysis revealed that Nrf2 positively regulates transcription of genes that play key roles in central carbon metabolism.

Inflammation leads to loss of smooth muscle cells but fails to induce invasiveness in a prostate tumor model

Inflammation has a causal role in many cancers. In prostate cancers, epidemiological data suggest a link between prostatitis and subsequent cancer development, but a proof for this concept in a tumor model has been lacking. A constitutively active version of the IkappaB kinase 2 (IKK2), the molecule activated by a plethora of inflammatory stimuli, was expressed specifically in the prostate epithelium. Signaling of the IKK2/NF-kappaB axis was insufficient for transformation of prostate tissue. However, while PTEN+/- epithelia exhibited intraepithelial neoplasias only recognizable by nuclear alterations, additional IKK2 activation led to an increase in tumor size and formation of cribriform structures and to a fiber increase in the fibroblastic stroma. This phenotype was coupled with inflammation in the prostate gland characterized by infiltration of granulocytes and macrophages. Molecular characterization of the tissues showed a specific loss of smooth muscle markers as well as expression of chemokines attracting immune cells. Isolation of epithelial and stromal cells showed differential chemokine expression by these cells. Correlation studies showed the inflammatory phenotype coupled to loss of smooth muscle in infiltrated glands, but maintenance of the phenotype in glands where inflammation had decreased. Despite the loss of the smooth muscle barrier, tumors were not invasive in a stable genetic background. Data mining revealed that smooth muscle markers are downregulated in human prostate cancers and literature data show that loss of these markers in primary tumors is associated with subsequent metastasis. Our data show that loss of smooth muscle and invasiveness of the tumor are not coupled. Thus, inflammation during early steps of tumorigenesis can lead to increased tumor size and a potential change in the subsequent metastatic potential, but the tumor requires an additional transformation to become a carcinoma.

Effect of age, calorie restriction and resveratrol on gene expression in mouse heart, brain, and skeletal muscle

Resveratrol in high doses has been shown to extend lifespan in some studies in invertebrates and to prevent early mortality in mice fed a high-fat diet. We fed mice from middle age (14-months) to old age (30-months) either a control diet, a low dose of resveratrol (4.9 mg kg-1 day-1), or a calorie restricted (CR) diet and examined genome-wide transcriptional profiles.
We report a striking transcriptional overlap of CR and resveratrol in heart, skeletal muscle and brain. Both dietary interventions inhibit gene expression profiles associated with cardiac and skeletal muscle aging. Gene expression profiling suggests that both CR and resveratrol may retard some aspects of aging through alterations in chromatin structure and transcription. Resveratrol, at doses that can be readily achieved in humans, fulfills the definition of a dietary compound that mimics some aspects of CR.

Effect of Ursodeoxycholic acid on gene expression in the intestial epithelium

Background & Aims: Ursodeoxycholic acid (UDCA) attenuates chemical and colitis-induced colon carcinogenesis in animal models. We investigated its mechanism of action on normal intestinal cells, in which carcinogenesis- or inflammation-related alterations do not interfere with the result.   Methods: Alterations of gene expression were identified in Affymetrix arrays in isolated colon epithelium of mice fed with a diet containing 0.4% UDCA and were confirmed in the normal rat intestinal cell line IEC-6 by RT-PCR. The effect of the insulin receptor substrate 1 (Irs-1) expression and of ERK phosphorylation on proliferation was investigated in vitro by flow cytometry, western blotting, siRNA-mediated gene suppression or by pharmacological inhibition of the kinase activity. The ERK1-effect on Irs-1 transcription was tested in a  reporter system.  Results: UDCA-treatment in vivo suppressed potential pro-proliferatory genes including Irs-1 and reduced cell proliferation by more than 30%. In vitro it neutralised the proliferatory signals of IGF-1 and EGF and slowed down the cell cycle. Irs-1 transcription was suppressed due to  high ERK1 activation. Both Irs-1 suppression and the persistent high ERK activation inhibited proliferation. Conversely, the decrease of phosphorylation of ERK1 (but not ERK2) or of its expression partially abrogated the inhibitory effects of UDCA.   Conclusions: UDCA inhibits proliferation of intestinal epithelial cells by acting upon IGF-1 and EGF pathways and targeting ERK1 and, consequently, Irs-1. The inhibition of these pathways adds a new dimension to the physiological and therapeutic action of UDCA and,  since both pathways are activated in inflammation and cancer, suggests new applications of UDCA in chemoprevention and chemotherapy.

Syndromic features and mild cognitive impairment in mice with genetic reduction on p300 activity

Rubinstein-Taybi syndrome (RSTS) is a complex autosomal-dominant disease characterized by mental and growth retardation and skeletal abnormalities. A majority of the individuals diagnosed with RSTS carry heterozygous mutation in the gene CREBBP, but a small percentage of cases are caused by mutations in EP300. To investigate the contribution of p300 to RSTS pathoetiology, we carried out a comprehensive and multidisciplinary characterization of p300+/- mice. These mice exhibited facial abnormalities and impaired growth, two traits associated to RSTS in humans. We also observed abnormal gait, reduced swimming speed, enhanced anxiety in the elevated plus maze, and mild cognitive impairment during the transfer task in the water maze. These analyses demonstratethat p300+/- mice exhibit phenotypes that are reminiscent of neurological traits observed in RSTS patients, but their comparison with previous studies on CBP deficient strains also indicate that, in agreement with the most recent findings in human patients, the activity of p300 in cognition is likely less relevant or more susceptible to compensation than the activity of CBP.
To identify those genes whose expression was altered in the hippocampus of p300 deficient mutants, we performed a gene profiling analysis of hippocampal tissue using high-density oligonucleotide microarrays.

Retinal gene expression in Egr-1 knock-out mice during development (p30 and p42)

In chicks, the avian homologue of the early growth response protein-1 (ZENK) has been shown to be increased in a special cell type of the retina, the glucagonergic amacrine cells, under conditions that lead to a reduction in eye growth (myopic defocus, recovery of myopia) and decreased under conditions that enhance ocular growth (hyperopic defocus, form-deprivation). The investigation of Egr-1 knock-out mice showed that homozygous knock-out mice with no functional Egr-1 protein developed relative axial myopia at the age of 42 and 56 days, compared to heterozygous- and wildtype Egr-1 knock-out mice.
To clarify the role of Egr-1 in the retinal regulation of eye growth, and to get an idea about the biochemical pathways underlying this mechanism, we studied the role of Egr-1 in more detail using Affymetrix microarrays.

Marked Reduction of AKT1 Expression and Deregulation of AKT1-associated Pathways in Peripheral Blood Mononuclear Cells of Schizophrenia Patients

Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood Mononuclear Cells (PBMCs) of schizophrenia patients. We therefore examined PBMC expression levels of AKT1 in schizophrenia  patients  versus controls, and examined whether functional biological processes in which AKT1 plays an important role are deregulated in schizophrenia patients.   We performed a case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years) schizophrenia patients (N=43) as compared to controls (N=29). Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR)= 0.05).   Functional aspects of the deregulated set of genes were investigated with the Ingenuity Pathway Analysis (IPA) Software Tool.
From each participant 30 ml of blood was drawn into heparinized tubes. All blood samples were obtained between 10.00 and 11.00 am to minimize diurnal variation. PBMC’s were isolated by Ficoll gradient separation, started within 20 minutes after the drawing of blood and performed with minimum time variation. Cells were subsequently disrupted (Qiashredder kit; Qiagen), and RNA was isolated (RNeasy minikit; Qiagen) with an additional DNAse digestion step (RNase-free DNase set; Qiagen), all according to the manufacturer’s protocol, diluted in nuclease free water, and frozen at –80 C before use. Before freezing, RNA purity and quantity was assessed with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). After thawing the isolated RNA was biotinylated into cRNA using the One-Cycle Target Labeling and Control Reagents Kit (Affymetric Co) according to the manufacturer’s protocol. Before hybridisation RNA quality and integrity was assessed using the Agilent 2100 BioAnalyzer (Agilent). Biotinylated cRNA was hybridized to the Affymetrix Human Genome U133 plus 2.0 GeneChip© microarray containing 54,675 probe sets (Affymetrix Co). Each sample was individually biotinylated and hybridized to an individual microarray. Biotinylation was performed in batches with randomisation of patients and controls according to their ratios across the batches. The arrays were scanned and analyzed using Affymetrix Microarray Suite 4.2 software. Scanning was performed in three batches with randomization of patients and controls according to their ratios across the batches.

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme; however, recently metabolic properties had been attributed to it. Hereby, we examined the metabolic consequences of PARP-2 ablation in liver. Microarray analysis of PARP-2 knockdown HepG2 cells revealed the dysregulation of lipid and cholesterol metabolism genes. Induction of cholesterol biosynthesis genes stemmed from the enhanced expression of sterol-regulatory element binding protein (SREBP)-1. We revealed that PARP-2 is a suppressor of the SREBP-1 promoter, therefore ablation of PARP-2 induces SREBP-1 expression and consequently cholesterol synthesis. PARP-2-/- mice had higher SREBP-1 expression that was translated into enhanced hepatic and serum cholesterol levels.

Expression data from quadriceps muscle of young DMD patients and age matched controls

Albeit increased serum CK level and abnormal muscle histology are always present, boys with DMD are phenotipically indistinguishable from the normal ones at birth and, in their first years of life, acquire early motor milestones at normal times. A clear defect in muscle function becomes generally apparent by the end of the second year. As the disease is typically diagnosed between the ages of 3 and 7, the first two years are often considered and referred to as clinically presymptomatic.
As a defined gene expression signature was shown to characterize these symptomatic patients we sought to investigate whether and to which extent alterations may be also present in muscle from presymptomatic DMD infants. To this aim, we used the Affymetrix technology to compare the individual expression profiles of 19 DMD patients with age at biopsy scattered along the first two years of the disease with those of 14 age matched controls. This approach allowed us to describe with high resolution the altered transcriptional state that characterizes this early, presymptomatic phase of the disease and highlight some molecular pathways as potential critical targets in the pathophysiology of the disease.

The aim of this study was to find disease-associated genes in atopic eczema.
Keywords: Skin biopsy, DNA microarray, atopic eczema

polycomb, development, differentiation, cell fate, stem cells, siRNA

Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions

The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes.
Keywords: polycomb, development, differentiation, cell fate, stem cells, siRNA

Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little prospect for cure. Despite some advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, understanding the biological mechanisms underpinning EOC progression is critical for its treatment and to ameliorate patients survival. Recently, we reported that CD157 is expressed in EOC and controls tumor cell migration and invasion. Using stable overexpression and knockdown in OVCAR-3 and OV-90 ovarian cancer cell lines, we demonstrated that CD157 overexpression promotes morphological and functional changes, characterized by downregulation of epithelial marker and upregulation of mesenchymal ones. These are mediated at the transcriptional level by altering the expression of Snail and Zeb1 transcriptional repressors. The effects of CD157 overexpression on ovarian cancer phenotype translate into increased tumor cell motility and mesotelial invasion, while its knockdown significantly reduces the migratory potential, implying a direct correlation between CD157 expression levels and EOC aggressiveness.  The analysis of the transcriptomic profiling highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing EOC cells. The overall picture deduced from the analysis of these modulated transcripts indicated that high CD157 expression results in strengthening of a number of biological functions that favour tumor progression (including cell differentiation, cell motility and migration), and weakening of selected biological processes that hinder the tumor progression (such as apoptosis, cell death and response to stress). Collectively, these data support a causal role of CD157 in the control of ovarian cancer progression motivating the existence of a direct correlation between the expression levels of CD157 and the adverse clinical outcome in EOC patients, and suggest that CD157 may represent a valuable therapeutic target.

JAG1 is an Invasion and Metastasis promoting genes of Lung cancer

Background: JAG-1 is a ligand of Notch signaling and can regulate cell differentiation and proliferation in cancers. Recent studies indicated that JAG1 is a gene associated with cancer progression. Therefore, we investigated the role of JAG1 in lung cancer progression. Methods: The expression of JAG1 was manipulated by overexpression or RNA silencing in several human lung cell lines. The effect of JAG1 on tumorigenesis and invasion was assessed by the cell anchorage-independent growth, cell proliferation, cell migration and invasion assays in vitro as well as metastasis in vivo. The potential downstream genes of JAG1 were identified by oligonucleotide microarrays and quantitative reverse transcription¡Vpolymerase chain reaction (RT-PCR). We further measured JAG1 expression in lung cancer specimens by RT-PCR. Correlation between JAG1 expression and overall survival of lung cancer patients was determined by using the log-rank test and multivariable Cox proportional hazards regression analysis. All statistical tests were two-sided. Results: JAG1 enhanced anchorage-independent growth, cell migration, invasion in the lower invasive cells, CL1-0. JAG1 also increased the capability of migration and invasion in the other two lung cancer cell lines (A549 and NCI-H226). The silencing of JAG1 inhibited migration and invasion activities of the higher invasive cells, CL1-5, by siRNA technology. The invasion-promoting activity of JAG1 was also demonstrated in vivo by using a mouse metastasis model. By microarray analysis, we found that the expression of heat shock 70kDa protein 2 (HSPA2) was activated by JAG1 overexpression and eliminated by JAG1 silencing. Moreover, lung cancer patients with high JAG1 expressing tumors had shorter overall survival than those with low-expressing tumors. Conclusion: JAG1 might be an oncogene which promotes colonogenesis and metastasis, and high JAG1 expression is associated with shorten survival in lung cancer.

Parkin pathway activation mitigates glioma cell proliferation and predicts patient survival

Mutations in the parkin gene, which encodes a ubiquitin ligase, are a major genetic cause of parkinsonism. Interestingly, parkin also plays a role in cancer as a putative tumor suppressor, and the gene is frequently targeted by deletion and inactivation in human malignant tumors. Here, we investigated a potential tumor suppressor role for parkin in gliomas. We found that parkin expression was dramatically reduced in glioma cells. Restoration of parkin expression promoted G1 phase cell cycle arrest and mitigated the proliferation rate of glioma cells in vitro and in vivo. Notably, parkin-expressing glioma cells showed a reduction in levels of cyclin D1, but not cyclin E, and a selective downregulation of Akt serine-473 phosphorylation and VEGF receptor levels. In accordance, cells derived from a parkin null mouse model exhibited increased levels of cyclin D1, VEGF receptor and Akt phosphorylation and divided significantly faster when compared with wild type cells, with suppressionof these changes following parkin re-introduction. Clinically, analysis of parkin pathway activation was predictive for the survival outcome of glioma patients. Taken together, our study provides mechanistic insight into the tumor suppressor function of parkin in brain tumors, and suggests that measurement of parkin pathway activation may be used clinically as a prognostic tool in brain tumor patients.

Gene Expression Profiling of an Immortalized Human Neural Stem Cell Line HB1.F3

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. To efficiently induce neuronal lineage cells from NSC for neuron replacement therapy, we should clarify the intrinsic genetic programs involved in a time and place-specific regulation of human NSC differentiation. Recently, we established an immortalized human NSC clone HB1.F3 to provide an unlimited NSC source applicable to genetic manipulation for cell-based therapy. To investigate a role of neurogenin 1 (Ngn1), a proneural basic helix-loop-helix (bHLH) transcription factor, in human NSC differentiation, we established a clone derived from F3 stably overexpressing Ngn1. Genome-wide gene expression profiling identified 250 upregulated genes and 338 downregulated genes in Ngn1-overexpressing F3 cells (F3-Ngn1) versus wild-type F3 cells (F3-WT). Notably, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a novel stem cell marker, showed a robust increase in F3-Ngn1.

Nuclear receptor, RORa plays a critical role in obesity-associated inflammation, hepatic steatosis, and insulin resistance

We demonstrated that RORa-deficient staggerer mice (RORasg/sg) fed with a high fat diet (HFD) showed reduced adiposity and hepatic triglyceride levels compared to wild type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORasg/sg mice. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORasg/sg mice fed with a HFD. The infiltration of macrophages and the expression of many immune-response and pro-inflammatory genes, including those encoding various chemo/cytokines, toll-like receptors, and TNF signaling proteins, were significantly reduced in RORasg/sg WAT. Moreover, RORasg/sg mice fed with a HFD were protected from the development of insulin resistance. Together, these results indicate that RORa plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORa may provide a novel therapeutic target in the management of obesity and associated metabolic diseases.

In this study, we have explored microarray-based differential gene expression profile in mouse lung tissue 8 h after inducing polymicrobial sepsis and the effect of preprotachykinin-A (PPTA) gene deletion. A range of genes differentially expressed (> 2-fold) in microarray analysis was assessed, PPTA-knockout septic mice with their respective sham controls.

We performed the oligonucleotide microarray analysis in bipolar disorder, major depression, schizophrenia, and control subjects using postmortem prefrontal cortices provided by the Stanley Foundation Brain Collection. By comparing the gene expression profiles of similar but distinctive mental disorders, we explored the uniqueness of bipolar disorder and its similarity to other mental disorders at the molecular level. Notably, most of the altered gene expressions in each disease were not shared by one another, suggesting the molecular distinctiveness of these mental disorders. We found a tendency of downregulation of the genes encoding receptor, channels or transporters, and upregulation of the genes encoding stress response proteins or molecular chaperons in bipolar disorder. Altered expressions in bipolar disorder shared by other mental disorders mainly consisted of upregulation of the genes encoding proteins for transcription or translation. The genes identified in this study would be useful for the understanding of the pathophysiology of bipolar disorder, as well as the common pathophysiological background in major mental disorders at the molecular level.

Regulatory consequences of ZNF395 knock-down in MDA-LM2-shTARBP2 cell-line

shRNA-mediated knock-down followed by transcriptomic profiling revealed that silencing of ZNF395 resulted in a significant up-regulation in several key oncogenes.

Reversal of oncogene transformation and suppression of tumor growth by the novel IGF1R kinase inhibitor A-928605.

The insulin-like growth factor (IGF) axis is an important signaling pathway in the growth and survival of many cell types and has been implicated in multiple aspects of cancer progression from tumorigenesis to metastasis. The multiple roles of IGF signaling in cancer suggest that selective inhibition of the pathway might yield clinically effective therapeutics. Here we describe A-928605, a novel small molecule inhibitor of the receptor tyrosine kinase responsible for IGF signal transduction. This small molecule is able to abrogate activation of the pathway as shown by effects on the target and downstream effectors and is shown to be effective at inhibiting the proliferation of an oncogene addicted tumor model cell line (CD8-IGF1R 3T3) both in vitro and in vivo.

Molecular profiling of LGL leukemia reveals role of sphingolipid signaling of cytotoxic lymphocytes

We used microarrays to expression profile peripheral blood mononuclear cells (PBMCs) from LGL leukemia patients and control subjects to identify survival pathways that render leukemic LGL resistant to activation induced cell death.
Keywords: granular lymphocyte leukemia, PBMC

Genome-wide analysis of E17.5 pituitary gland gene expression of control and Insm1 mutant mice

The Insm1 gene encodes a zinc finger factor expressed in many endocrine organs. We show here that Insm1 is required for differentiation of all endocrine cell types in the pituitary. Thus, in Insm1 mutant mice, hormones characteristic of the different pituitary cell types (thyroid, follicle and melanocyte stimulating hormone, adrenocorticotrope hormone, growth hormone and prolactin) are absent or produced at markedly reduced levels. The differentiation deficit is accompanied by an up-regulated expression of components of the Notch signaling pathway. Further, skeletal muscle-specific genes are ectopically expressed, indicating that Insm1 blocks a muscle-specific expression program. Since Insm1 is also essential for differentiation of endocrine cells in the pancreas, intestine and adrenal gland, it is emerging as a transcription factor that acts in a pan-endocrine manner. The Insm1 factor contains a SNAG domain at its N-terminus, and we show here that the SNAG domain recruits histone modifying factors (Kdm1a, Hdac1/2 and Rcor1-3) and other proteins implicated in transcriptional regulation (Hmg20a/b and Gse1). Deletion of the SNAG domain in mice disrupted differentiation of pituitary endocrine cells, and resulted in an upregulated expression of components of the Notch signaling pathway and ectopic expression of skeletal muscle-specific genes. Our work demonstrates that Insm1 acts in the transcriptional network that controls differentiation of endocrine cells in the anterior pituitary gland, and requires the SNAG domain to exert this function in vivo.

Transcriptome-wide characterization of gene expression associated with unruptured intracranial aneurysms

The biological mechanisms by which cerebral aneurysms emerge, enlarge and rupture are not totally understood. In the present study, we analyzed the genome-wide gene expression profile in human intracranial aneurysms using cDNA microarrays.

Knockout of the Acyl CoA binding protein (ACBP) in mice - expression profile from the liver of 21 days old ACBP-/- and +/+ mice.

The ACBP knockout were created by targeted disruption of the gene in mice. The expression profiling was performed on liver tissue from ACBP-/- (KO) and +/+ (WT) mice at the age of 21 days, which in our study is the time immediately before weaning. The mice used for this experiment were taken directly away from their mother. Thus, having free access to chow and breast milk until sacrificed at 8-11am

Comparison of gene expression pattern between Wild-type and Setd2 knockout mESCs [Affymetrix]

SETD2/HYPB has been known as a histone H3K36 specific methyltransferase. However, its roles in physiology such as development and cellular function remain unclear. In this study, using mESCs as cellular model, we show that Setd2 mainly regulates differentiation of murine embryonic stem cells (mESCs) towards primitive endoderm. This study aimed at exploring how did Setd2 regulate primitive endoderm. differentiation.
We used microarrays to detail the global programme of gene expression controled by setd2, which is required for endoderm differentiation.

Microarray Analysis of Gene Expression in Human Normal and Breast Cancer Cells

Differential gene expression between normal breast epithelial cells and the malignant counterpart was studied using microarray technique. We utilized GeneAlbum GEM 1-6 from Incyte Pharmaceuticals, Inc., which contains which contains 65,873 cDNA clones (57,172 clones excluding controls), representing 33,515 individual genes. The normal human mammary epithelial cells (HMEC) from 3 donors were used to search different gene expression patterns among individuals, ages and races. In addition, 8 well-established breast cancer cell lines were used as probes against a common normal HMEC from 50 years old donor

A series of gene expression measurements of uterine fibroids with mutated or wild-type fumarate hydratase (FH) gene.

Human endometriosis vs normal endometrium study - transcriptional profiling

Normal and diseased human tissues were profiled for gene expression using the Affymetrix U133 plus 2.0 array
In total 20 samples were processed

Gene expression profiling of Notch1 knockout mouse liver samples and murine hepatic angiosarcoma cell lines.

This work is part of the paper: Generation of a murine hepatic angiosarcoma cell line and reproducible mouse tumor model, Rothweiler S et al, Laboratory Investigation, 2014
Hepatic Angiosarcoma (AS) is a rare and highly aggressive tumor of endothelial origin with dismal prognosis. Studies of the molecular biology of AS and treatment options are limited since animal models are rare. We have previously shown that inducible knockout of Notch1 in mice leads to spontaneous formation of hepatic AS. The aim of this study was to 1) establish and characterize a cell line derived from this murine AS, 2) to identify molecular pathways involved in the pathogenesis and potential therapeutic targets, and 3) to generate a tumor transplantation model. AS cells retained specific endothelial properties such as tube formation activity, as well as expression of CD31 and von Willebrand Factor. However, electron microscopy analysis revealed signs of dedifferentiation with loss of fenestrae and loss of contact inhibition. Microarray and pathway analysis showed substantial changes in gene expression and revealed activation of the Myc pathway. Exposing the AS cells to sorafenib reduced migration, filopodia dynamics, and cell proliferation but did not induce apoptosis. In addition, sorafenib suppressed ERK phosphorylation and expression of cyclin D2. Injection of AS cells into NOD/SCID mice resulted in formation of undifferentiated tumors confirming the tumorigenic potential of these cells.  Conclusion: We established and characterized a murine model of spontaneous AS formation and hepatic AS cell lines as a useful in vitro tool. Our data demonstrate antitumor activity of sorafenib in AS cells with potent inhibition of migration, filopodia formation, and cell proliferation, which support further evaluation of sorafenib as a novel treatment strategy. In addition, AS cell transplantation provides a subcutaneous tumor model useful for in vivo preclinical drug testing.

The Tight Junction Protein Claudin-2 Controls Mucosal Permeability, Immune Tolerance and Colitis in Mice

The mucosal epithelium plays a key role in regulating immune homeostasis. Dysregulation of epithelial barrier function is associated with mucosal inflammation. Expression of claudin-2, a pore-forming tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Furthermore, claudin-2 also regulates colonic epithelial cell proliferation and intestinal nutrient absorption. However, the precise role of claudin-2 in regulating colonic epithelial and immune homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic (Cl-2TG) mice, that increased colonic claudin-2 expression unexpectedly protects mice against experimentally induced colitis and colitis-associated cancer. Notably, Cl-2TG mice exhibited increased colon length and permeability as compared with wild type (WT) littermates. However, despite their leaky colon, Cl-2TG mice subjected to experimental colitis were immune compromised, with reduced induction of TLR-2, TLR-4, Myd-88 expression and NF-kB and STAT3 activation. Most importantly, colonic macrophages in Cl-2TG mice exhibited an anergic phenotype. Claudin-2 overexpression also increased colonocyte proliferation and provided protection against colitis-induced colonocyte death. Taken together, our findings have revealed a critical role of claudin-2 in regulating colonic homeostasis, suggesting novel therapeutic strategies for inflammatory conditions of the gastrointestinal tract.

wild type and aire -/- murine meduallary thymic epithelial cells

Mice used were B6/129 F2's, 3-5 weeks of age, either wild type or with both copies of the autoimmune regulator gene (aire, GenBank #AF079536) disrupted.  Thymi from five of these mice of both sexes were removed and pooled. After collagenase/dispase digestion, density gradient fractionation, and fluorescent antibody staining, cells with the phenotype CD45-, G8.8+, CDR1int and B7.1hi were FACS-sorted and total RNA was made from them. RNA was twice-amplified using a T7 polymerase-based method.

Normal human spermatogenesis concludes with the formation of large numbers of  morphologically well developed spermatozoa.  While transcriptionally quiescent these cells carry  an RNA payload that reflects the final spermiogenic phase of transcription. We report here the spermatozoal  transcript profiles characteristic of normally fertile individuals and infertile males suffering from a  consistent and severe teratozoospermia in which under 4% of spermatozoa are morphologically normal.  RNA was  extracted from the purified sperm cells of ejaculate and hybridized to Affymetrix U133 (v2) Microarrays.

We have determined the global gene expression upon loss of function of the Yy1 transcription factor in mouse embryonic stem cells

Profiling gene expression in HeLa cells  by hsa-mir-26b overexpression

Total RNA samples from three biological replicates in which the hsa-mir-26b was overexpressed in HeLa cells  were profiled by gene expression. As negative control, we used total RNA samples from HeLa cells transfected with cel-mir-67

Elevated pulmonary arterial pressure and altered expression of Ddah1 and Arg1 in mice lacking Cavin-1/PTRF

Lungs from newborn cavin-1/PTRF-deficient and wild-type mice were analyzed

Iron plays a central role in the regulation of many cellular functions. Dysregulation of its metabolism leads an iron overload situation and iron depletion leads to an inhibition of cell proliferation. Recent reports demonstrated that ICL670 (Novartis) acts as a potent NF-kappa-B inhibitor and improves hematological data in a subset of MDS patients (Cilloni et al, Haematologica, s1: 238, 2007). However, the precise mechanism of anti-cancer effect of ICL670 is still uncertain.
To evaluate the effect of ICL670, and to identify the moleclular pathways responsible for the observed reduced transfusion requirement during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anti-cancer effect of ICL670.

Ubiquitylation plays an important role in the control of Na+ homeostasis by the kidney. It is well established that the epithelial Na+ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney, and is also a circadian output gene. In heterologous expression systems USP2-45 binds to ENaC, deubiquitylates it and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na+ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that the USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO  mice did not show any differences to wild-type littermates with respect to the diurnal control of Na+ or K+ urinary excretion and plasma levels neither on standard diet, nor after acute and chronic changes to low and high Na+ diets, respectively. Moreover, they had similar aldosterone levels either at low or high Na+ diet. Blood pressure measurements using telemetry did not reveal variations as compared to control mice. Usp2-KO did neither display alternations in ENaC or Na+,Cl--cotransporter (NCC) expression, nor were there any changes in regulatory protein levels, as evidenced by immunoblotting and transcriptome analysis. We conclude that USP2-45 is not crucial for the regulation of Na+ balance or maintenance of blood pressure in vivo.

Identification of novel transcriptional networks in response to the treatment D3T (3H-1, 2-dithiole-3-thione)

3H-1, 2-dithiole-3-thione (D3T), an inducer of antioxidant and phase 2 genes, is known to enhance the detoxification of environmental carcinogens, prevent neoplasia, and to elicit other protective effects. However, a comprehensive view of the regulatory pathways induced by this compound has not yet been elaborated. Fischer F344 rats were gavaged daily for 5 days with vehicle or D3T (0.3 mmol/kg). The global changes of gene expression in liver were measured with Affymetrix RG-U34A chips. Using functional class scoring, a semi-supervised method exploring both the expression pattern and the functional annotation of the genes, the Gene Ontology classes were ranked according to the significance of the impact of D3T treatment. Two unexpected functional classes were identified for the D3T treatment, cytosolic ribosome constituents with 90% of those genes increased, and cholesterol biosynthesis with 91% of the genes repressed. In another novel approach, the differentially expressed genes were evaluated by the Ingenuity computational pathway analysis tool to identify specific regulatory networks and canonical pathways responsive to D3T treatment. In addition to the known glutathione metabolism pathway (ρ = 0.0011), several other significant pathways were also revealed, including antigen presentation (ρ = 0.000476), androgen/estrogen biosynthesis (ρ = 0.000551), fatty acid (ρ = 0.000216) and tryptophan metabolism (ρ = 0.000331) pathways. These findings showed a profound impact of D3T on lipid metabolism and anti-inflammatory/immune-suppressive response, indicating broader cytoprotective effect of this compound than previously expected.
Keywords: Response to antioxidant compound

gene expression profile of the prefrontal cortex in response to induction of arthritis.

profiling of prefrontal cortex from arthritic C57Bl/6 mice s.c. vs. control C57Bl/6 mice

The main function of the nervous system is to maintain homeostasis by sensing and reacting to signals that reach a certain threshold. For example, the brain can sense immune peripheral events through soluble compounds or the vagus nerve and can react through activation of the hypothalamus-pituitary-adrenal axis, resulting in the modulation of an ongoing immune response. Using microarrays we studied the gene expression profile of the prefrontal cortex in C57BL/6 mice strain that developed collagen induced-arthritis vs control mice.
Keywords: gene expression profile of the prefrontal cortex in response to induction of arthritis.

Molecular Genetic Classification of clear-cell Renal Cell Carcinoma (ccRCC) based on the Gene Expression Profiling of Tumors and Tumorgrafts deficient for BAP1 or PBRM1

Renal cell carcinoma (RCC) exhibits some unusual features and genes commonly mutated in cancer are rarely mutated in clear-cell RCC (ccRCC), the most common type. The most prevalent genetic alteration in ccRCC is the inactivation of the tumor suppressor gene VHL. Using whole-genome and exome sequencing we discovered BAP1 as a novel tumor suppressor in ccRCC that shows little overlap with mutations in PBRM1, another recent tumor suppressor. Whereas VHL was mutated in 81% of the patients (142/176), PBRM1 was lost in 58% and BAP1 in 15% of the patients analyzed. All these tumor suppressor genes are located in chromosome 3p, which is partially or completely lost in most ccRCC patients. However, BAP1 but not PBRM1 loss was associated with higher Fuhrman grade and, therefore, poorer outcome. Xenograft tumors (tumorgrafts) implanted orthotopically in mice exhibited similar gene expression profiling to corresponding primary tumors. Gene expression profiling of tumors and tumorgrafts displayed different signatures for BAP1- and PBRM1-deficient samples. Thus, after inactivation of VHL, the acquisition of a mutation in BAP1 or PBRM1 defines a different program that might alter the fate of the patient. Our results establish the foundation for an integrated pathological and molecular genetic classification of about 70% of ccRCC patients, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

ERBB3 expression levels were down-regulated by shRNA. Two such stable lines were made and gene expression was measured by Affymetrix arrays.

Expression data from E protein deficient double-positive (DP) thymocytes

We wanted to test the role of mammalian E proteins E2A and HEB in the development of T cells.
Using a conditional deletion system in which these proteins are deleted at the DP stage of T cell development, we compared DP thymocytes deficient for E2A, HEB or both to wild-type thymocytes

Gene expression profile of the mammary stroma in Rarb -/- mice compared to their wild type littermates

Inactivation of RAR-b, which has been reported as a tumor suppressing gene by numerous studies, results in protective effect against the tumorigenesis induced by activated ErbB2. Moreover, tissue recombination indicates that the RAR-b deficient-microenvironment, rather than the RAR-b status of mammary epithelial cells, plays a key-determining role in the initiation and progression of the mammary carcinoma. Ablation of RAR-b extensively modulates the remodeling of stroma during tumor progression through suppressing the activation and transdifferetiation of myofibroblasts.
RNA microarray has been employed to identify the gene expression signature in the mammary stroma of our RAR-b null animals. We want to find out the underlining mechanism of the protective effects resulted from Rarb abliton.

Expression in wild type and TgDREAM mouse B cells unstimulated or 2 days after  LPS+IL4 stimulation

DREAM/KChIP-3 is a calcium-dependent transcriptional repressor highly expressed in immune cells. Transgenic mice expressing a dominant active DREAM mutant show reduced serum immunoglobulin levels. In vitro assays show that reduced immunoglobulin secretion is an intrinsic defect of transgenic B cells that occurs without impairment in plasma cell differentiation but with an accelerated entry in cell division and an increase in class switch recombination. B cells from DREAM knockout mice did not show any phenotype, due to compensation by endogenous KChIP-2. Expression arrays revealed modified expression of Edem1 and Derlin3, two proteins related to the ER-associated degradation pathway and of Klf9, a cell-cycle regulator. Our results disclose a function of DREAM and KChIP-2 in Ig subclass production in B lymphocytes.

Gene expression profile of hepatocellular carcinoma in low-dose, repeated diethylnitrosamine (DEN)-treated rat treated with erlotinib

Gene-expression profiles of rat hepatocellular carcinoma induced by diethylnitrosamine (DEN) and the effect of erlotinib
Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and the third leading cause of cancer-related death. Given the lack of successful treatment options, chemoprevention in high-risk patients has been proposed as an alternative strategy. Mounting evidence supports a role for epidermal growth factor (EGF) during chronic liver disease and hepatocellular transformation. We address the hypothesis that blocking the EGF-EGF receptor (EGFR) pathway may be an effective strategy for inhibiting fibrogenesis and hepatocarcinogenesis. A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the effects of erlotinib on underlying chronic liver disease and HCC formation. The DEN-induced rat model closely resembles disease progression in humans both pathologically and molecularly. Erlotinib significantly prevented the development of HCC tumor nodules in a dose-dependent fashion. Further, erlotinib inhibited the activation of hepatic stellate cells and prevented fibrogenesis. Erlotinib also reduced hepatotoxicity and improved liver function. Finally, a gene expression signature predictive of poor survival in human cirrhosis patients was reversed in response to erlotinib. Our data demonstrate for the first time that EGFR inhibition prevents liver fibrogenesis. Further, our results suggest that erlotinib is a potentially effective HCC chemoprevention strategy through inhibition of cirrhosis progression which can be monitored at the molecular level.

The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 tyrosine kinase, which is present in almost every patient with chronic myeloid leukemia. In this study, the tyrosine kinase inhibitor Imatinib was used for pharmacological inhibition of BCR-ABL1. Gene expression profiles of CML cell lines were analyzed in response to Imatinib treatment.

Gene expression changes induced by heavy metals and other chemicals

From the results of gene expression analyses of HepG2 under the exposure of 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ), N-nitrosodimethylamine (DMN), phenol and six heavy metals We showed that biological action of six heavy metals were clearly related to that of DMNQ and distinguishable from the other chemicals. These results suggest that oxidative stress is major apparent biological action of high dose heavy metals, supporting the previous reports.

A time course of the effect of overexpressing GPI-PLD on global gene expression in HepG2 cells was compared to control virus.
Keywords: Expression profiling

Two different mouse models of cardiac-specific ILK expression (ILKS343D and ILKR211A) were used to investigate the role of ILK in cardiac regeneration

Gene expression changes in the heart of MCH3-KO mouse (HDAC3 f/f, Muscle Creatine Kinase-Cre) versus control WT mouse (HDAC3 f/f).
Histone deacetylases (HDACs) play important roles in cardiac development and function. We show here that mice deficient of HDAC3 in heart and skeletabl muscle are relatively normal on normal chow, but develop hypertrophic cardiomyopathy and heart failure that leads to death on high-fat diet. This microarray experiment is to explore the underlying molecular mechanism.

Gene expression data from AML cell lines, MOLM-14, U937, THP-1 and HL-60, that were infected with a scrambled control hairpin (shControl), two shRNAs directed against GSK-3B (shGSK3B_1 and shGSK3B_2), or two shRNAs directed against GSK-3A (shGSK3A_5 and shGSK3A_6).
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetic-based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3A (GSK-3A) in AML by performing two independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3A induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3A–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3B has been well studied in cancer development, these studies support a role for GSK-3A in AML.

Gene expression profiling with microarrays was used to identify genes differentially expressed in the lungs of B6 and BALB CF mice compared to non-CF littermates

mRNA expression profiling in mouse bronchoalveolar stem cells (BASC)

We performed miRNA and mRNA profiling in BASC cells and c-Myc depleted BASC cells. We built potential miRNA-mRNA interaction networks specific to c-Myc regulation in BASCs

Differential gene expression in placentae (E10.5) from adra2bKO and adra2bWT mice

alpha2-adrenoceptors are essential presynaptic regulators of norepinephrine release from sympathetic nerves. Previous studies in mice with targeted deletions in the three alpha2-adrenoceptor genes have indicated that these receptors are essential for embryonic development. In the present study, we searched for the alpha2-adrenoceptor subtype(s) involved in placental development and its molecular mechanism using mice carrying targeted deletions in alpha2-adrenoceptor genes.
Congenic alpha2B-adrenoceptor-deficient mice (Adra2b-/-) developed a defect in fetal and maternal vessel formation in the placenta labyrinth at embryonic day E10.5. This defect was accompanied by reduced endothelial cell proliferation and decreased ERK1/2 phosphorylation levels in Adra2b-/- as compared with Adra2b+/+ placenta. Microarray analysis of wild-type and mutant placentae (maternal genotype Adra2b+/-) revealed 179 genes which were significantly up- or downregulated >1.5-fold in alpha2B-deficient placenta. The type 1 receptor for vascular endothelial growth factor (Flt1), which is coexpressed with alpha2B-adrenoceptors in spongiotrophoblast and giant cells of the placenta, was found to be 2.3-fold upregulated in alpha2B-deficient placenta. Neutralization of Flt1 and its soluble splice variant sFlt1 by a specific antibody in vivo prevented the vascular defect in alpha2B-deficient placenta at E10.5. Thus, alpha2B-adrenoceptors are essential to suppress antiangiogenic (s)Flt1 in spongiotrophoblasts to control the coordinated formation of a vascular labyrinth of fetal and maternal blood vessels in the murine placenta during development.

Gene expression alterations in the cerebellum and granule neurons of Cstb-/- mouse are associated with early synaptic changes and inflammation

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an inherited neurodegenerative disease with myoclonus, seizures and ataxia, caused by the mutations in cystatin B (CSTB) gene. In an approach towards understanding the molecular basis of pathogenic events in EPM1 we have utilized the cystatin B  deficient mice (Cstb-/-), a model for the disease. We have characterized the gene expression changes from the cerebellum of Cstb-/- mouse at postnatal day 7 (P7) and P30 as well as in cultured cerebellar granule cells using a pathway-based approach. A marked upregulation of immune response genes was seen at P30, reflecting the ongoing neuropathology, however, the observed alterations in complement cascade genes could also imply defects in synaptic plasticity. Differentially expressed genes in pre-symptomatic Cstb-/- animals at P7 were connected to synaptic function and plasticity and in cultured cerebellar granule cells to cellular biogenesis, cytoskeleton and intracellular transport. Especially GABAergic pathways were affected.

gene expression signatures of Akt1 +/- mice at 8weeks and 35weeks

To know the difference of transcriptome between Wild type and Akt1+/- mice, 8weeks and 35weeks female mice were subjected to microarray analysis.

Braf inhibitors reverse the unique molecular signature and phenotype of hairy cell leukemia, and exert anti-leukemic activity

Hairy cell leukemia (HCL) shows unique clinico-pathological and biological features. HCL responds well to purine analogues but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-MEK-ERK pathway in HCL by exposing in vitro primary leukemic cells purified from  26 patients to clinically available BRAF (Vemurafenib; Dabrafenib) or MEK (Trametinib) inhibitors. Results were  validated in vivo in samples from Vemurafenib-treated HCL patients within a phase-2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, TRAP and cyclin-D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by co-culture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.

Frequent driver mutations in histone H3.3 and chromatin remodeling genes in paediatric glioblastoma

Whole exome sequencing identified frequent driver mutations in a series of paediatric glioblastomas
We used microarray-based profiling to investigate differences in gene expression according to mutational status of driver genes

Dicer1 deletion in myeloid-committed progenitors causes neutrophil dysplasia and blocks macrophage/dendritic cell development in mice

MiRNAs have the potential to regulate cellular differentiation programs. However, miRNA-deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicer1, which encodes an essential RNaseIII enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein alpha (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multi-potent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating a regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion towards myeloid differentiation in GMPs.

Parietal cell gene expression in the presence and absence of gastrin

Parietal cells were isolated from WT and Gastrin Knockout mice.  Targets from three biological replicates of each were generated and the expression profiles were determined using Affymetrix murine U74Av2 arrays.  Comparisons between the sample groups allow the identification of genes with gastrin responsiveness.

We performed global scale microarray analysis to identify detailed mechanisms by which bisphenol A (BPA) induce cell death by using an Affymetrix GeneChip system. Testicular Sertoli TTE3 cells used in the present study were derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen. Cell death accompanying endoplasmic reticulum stress was observed in the cells treated with 0.2 mM BPA. Of the 22,690 probe sets analyzed, approximately 1,300 genes were down- and up-regulated by a factor of 2.0 or greater in the cells treated with BPA.
Keywords: bisphenol A, gene expression, testicular Sertoli cell

Comparison of two sets of microarray experiments to define allergic asthma expression pattern

Allergic asthma is a complex trait. Several approaches have been used to identify biomarkers involved in this disease. This study aimed at demonstrating the relevance and validity of microarrays in the definition of allergic asthma expression pattern. The authors compared the transcript expressions of bronchial biopsy of 2 different microarray experiments done 2 years apart, both including nonallergic healthy and allergic asthmatic subjects (n = 4 in each experiment). U95Av2 and U133A GeneChips detected respectively 89 and 40 differentially expressed genes. Fifty-five percent of the U133A genes were previously identified with the U95Av2 arrays. The immune signaling molecules and the proteolytic enzymes were the most preserved categories between the 2 experiments, because 3/4 of the genes identified by the U133A were also significant in the U95Av2 study for both categories. These results demonstrate the relevance of microarray experiments using bronchial tissues in allergic asthma. The comparison of these GeneChip studies suggests that earlier microarray results are as relevant as actual ones to target new genes of interest, particularly in function categories linked to the studied disease. Moreover, it demonstrates that microarrays are a valuable technology to target novel allergic asthma pathways as well as biomarkers.

Glycerol kinase (GK) is at the interface of fat and carbohydrate metabolism and has been implicated in insulin resistance and type 2 diabetes mellitus (T2DM). To define GK’s role in insulin resistance, we examined gene expression in brown adipose tissue in a glycerol kinase (Gyk) knockout (KO) mouse model using microarray analysis. Global gene expression profiles of KO mice were distinct from wild type (WT) with 668 genes that were differentially expressed. These included genes involved in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Real-Time (RT) PCR analysis confirmed the differential expression of selected genes involved in lipid and carbohydrate metabolism. PathwayAssist analysis confirmed direct and indirect connections between GK and genes in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Network Component Analysis (NCA) showed that the transcription factors, peroxisome proliferator-activated receptor gamma (PPAR-γ), sterol regulatory element binding factor 1 (SREBP-1), SREBP-2, signal transducer and activator of transcription 3 (STAT3), STAT5, trans-acting transcription factor 1 (SP1),  CCAAT/enhancer binding protein alpha (CEBP-α), cAMP responsive element binding protein 1 (CREB), glucocorticoid receptor (GR), and PPAR-α have altered activity in the KO mice.  NCA also revealed the individual contribution of these transcription factors on the expression of genes altered in the microarray data. This study elucidates the transcription network of Gyk and further confirms a role for Gyk, a simple Mendelian disorder, in insulin resistance and T2DM, a common complex genetic disorder.
Keywords: genotype state analysis

Expression and neurobehavioral analyses in prosaposin deficient mice: Molecular alterations precede neuronal deficits

Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipids hydrolases in lysosomes. To explore the molecular mechanism(s) of disease progression, temporal transcriptome microarray analyses of cerebrum and cerebellum tissues were conducted using mRNA from three prosaposin deficiency mouse models: PS-NA (hypomorphic prosaposin deficiency), PS-/- (prosaposin null) and 4L/PS-NA (a V394L/V394L glucocerebrosidase mutation and PS-NA) mice. Our results indicate that regionally specific gene expression abnormalities preceded the histological and behavioral changes and CEBPD is a candidate regulator of brain disease in prosaposin deficiency. The alterations of gene expression are detected at birth and are more profound in cerebellum than cerebrum.
Keywords: disease-state analysis

Ogt chromatin recruitment is mediated by TET proteins in mouse ES cells [expression array]

O-linked N-acetylglucosamine (O-GlcNAc ) transferase (OGT) activity is essential for embryonic stem (ES) cell viability and mouse development. OGT is present in both cytoplasm and nucleus of different cell types and mediates serine or threonine glycosylation. The Ogt gene locus resides on the X-chromosome and its activity is required for the viability of male ES cells. Using Ogt conditional knock out (KO) ES cells it was shown the failure of establishing stable KO ES clones further suggesting that Ogt activity is required for ES cell self-renewal and pluripotency. For understanding these changes, we performed global gene expression upon silencing of Ogt mediated by esiRNA in mouse Embryonic Stem Cells.

Mast cells, activated by antigen via the high affinity receptor for IgE (FcεRI), release an array of pro-inflammatory mediators that contribute to allergic disorders such as asthma and anaphylaxis. The KIT ligand, stem cell factor (SCF), is critical for mast cell expansion, differentiation and survival, and, under acute conditions, enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal antigen-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of FcεRI-mediated degranulation and cytokine production. The hypo-responsive phenotype was not a consequence of altered signals regulating calcium flux or protein kinase C, but of ineffective cytoskeletal reorganization, with evidence implicating a down-regulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders.

Whole genome expression data comparison between WT and Cebpg-/- MEFs.

C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence. Cebpg-/- MEFs proliferated poorly, entered senescence prematurely, and expressed a pro-inflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β~β) inhibited the proliferation and tumorigenicity of RasV12-transformed NIH3T3 cells, activated SASP gene expression, and recruited the CBP co-activator in a Ras-dependent manner, whereas γ~β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg-/- MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg-/- MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg-/- bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.

Microarray profiling of WT or PDE10A KO mice treated with vehicle or a PDE10 inhibitor

Inhibition of phosphodiesterase 10A (PDE10A) promotes cyclic nucleotide signaling, increases striatal activation, and decreases behavioral activity. Enhanced cyclic nucleotide signaling is a well established route to producing changes in gene expression. We hypothesized that chronic suppression of PDE10A activity would have significant effects on gene expression in the striatum. A comparison of the expression profile of PDE10A knockout (KO) mice and wild-type mice after chronic PDE10A inhibition revealed altered expression of 19 overlapping genes with few significant changes outside the striatum or after administration of a PDE10A inhibitor to KO animals. Chronic inhibition of PDE10A produced up-regulation of mRNAs encoding genes that included prodynorphin, synaptotagmin10, phosphodiesterase 1C, glutamate decarboxylase 1, and diacylglycerol O-acyltransferase and a down-regulation of mRNAs encoding choline acetyltransferase and Kv1.6, suggesting long-term suppression of the PDE10A enzyme is consistent with altered striatal excitability and potential utility as a antipsychotic therapy. In addition, up-regulation of mRNAs encoding histone 3 (H3) and down-regulation of histone deacetylase 4, follistatin, and claspin mRNAs suggests activation of molecular cascades capable of neuroprotection. We used lentiviral delivery of cAMP response element (CRE)-luciferase reporter constructs into the striatum and live animal imaging of 2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid (TP-10)-induced luciferase activity to further demonstrate PDE10 inhibition results in CRE-mediated transcription. Consistent with potential neuroprotective cascades, we also demonstrate phosphorylation of mitogen- and stress-activated kinase 1 and H3 in vivo after TP-10 treatment. The observed changes in signaling and gene expression are predicted to provide neuroprotective effects in models of Huntington's disease.

Lack of local vitamin D synthesis accelerates breast cancer development after Cyp27B1 gene ablation in mammary epithelium

The ablated Cyp27b1 mice showed no difference in mammary development but tumorigenesis was significantly accelerated. Cell proliferation, angiogenesis, cell cycle progression and survival markers were upregulated by Cyp27b1 ablation, a pattern confirmed by microarray analysis.

atherosclerosis, smooth muscle cell, ß-receptor antagonist, inflammation, microarray, nebivolol

Effects of metoprolol and nebivolol on gene expresion in human coronary smooth muscle cells

Human coronary smooth muscle cells were treated with two different ß-blocker (metoprolol and nebivolol). RNA from three replicates of each, treated and the untreated control group, were isolated and the expression profiles were determined using Affymetrix Human Genechip U133A arrays.  Comparisons between the sample groups allow the identification of genes with different expression patterns between the treated and untreated control cells.
Keywords: atherosclerosis, smooth muscle cell, ß-receptor antagonist, inflammation, microarray, nebivolol

Expression data of sandwich cultured primary rat hepatocytes incubated to piperazine designer drugs

The piperazine derivatives most frequently consumed for recreational purposes are 1-benzylpiperazine (BZP), 1-(3,4-methylenedioxybenzyl)piperazine (MDBP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP), and 1-(4-methoxyphenyl)piperazine (MeOPP). Currently it is unclear whether piperazine designer drugs induce mechanisms of hepatotoxicity and, if so, whether they act by a common mechanism.
We performed a gene array study with rat hepatocytes incubated with the four designer drugs for 24 h in order to detail the global programme of gene expression

Gene expression under normoxia/hypoxia in mouse embryonic fibroblasts with mutations in the CH1 domains of p300 and CBP

The CH1 (TAZ) domain of the transcriptional coactivators p300 and CBP has been reported to interact with the transcription factor HIF-1alpha and this interaction is thought to be critical for HIF-1alpha target gene expression in response to hypoxia.  To determine the requirement for the CH1 domain in hypoxia-responsive gene expression, primary mouse embryonic fibroblasts (MEFs) were generated from e14.5 C57B/6x129/Sv F2 embryos that were either wildtype or bore deletion mutations in the CH1 protein binding domains of both alleles of p300 and one allele of CBP (tri_CH1). Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction.
Keywords: genetic modification, dose response

Strain (Wt versus Ko) and disease state (DNFB treated versus control).

Cannabinoid receptor double knockout mice (Cnr1 -/- /Cnr2 -/-) in CHS model

We evaluated cutaneous contact hypersensitivity (CHS) in Cnr1-/-/Cnr2-/- animals using the obligate contact allergen 2,4-dinitrofluorobenzene (DNFB), which generates a specific cutaneous T-cell mediated allergic response upon repeated allergen contact. Allergic contact dermatitis affects about 5% of men and 11% of women in industrialized countries and is one of the leading causes for occupational diseases. In an animal model for cutaneous contact hypersensitivity we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. In contrast, fatty acid amide hydrolase deficient mice, which have increased levels of the endocannabinoid anandamide, displayed reduced allergic responses in the skin. Cannabinoid receptor antagonists exacerbated whereas receptor agonists attenuated allergic inflammation. These results demonstrate a protective role of the endocannabinoid system in contact allergy in the skin, and suggest a novel target for therapeutic intervention.
Keywords: Strain (Wt versus Ko) and disease state (DNFB treated versus control).

Identification of amyotrophic lateral sclerosis (ALS) associated genes. Post mortem spinal cord grey matter from sporadic and familial ALS patients compared with controls.

Microarray analysis of genes regulated by SHP in wild-type and ob/ob mice.

Liver gene expression was compared in wild type, SHP knockout, ob/ob, and ob/ob SHP double mutant mice.

Folic acid supplementation normalizes the endothelial progenitor cell transcriptome of patients with type 1 diabetes

Background: Endothelial progenitor cells play an important role in vascular wall repair. Patients with type 1 diabetes have reduced levels of endothelial progenitor cells of which their functional capacity is impaired. Reduced nitric oxide bioavailability and increased oxidative stress play a role in endothelial progenitor cell dysfunction in these patients. Folic acid, a B-vitamin with anti-oxidant properties, may be able to improve endothelial progenitor cell function. In this study, we investigated the gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes compared to endothelial progenitor cells from healthy subjects. Furthermore, we studied the effect of folic acid on gene expression profiles of endothelial progenitor cells from patients with type 1 diabetes. Methods: We used microarray analysis to investigate the gene expression profiles of endothelial progenitor cells from type 1 diabetes patients before (n=11) and after a four week period of folic acid supplementation (n=10) compared to the gene expression profiles of endothelial progenitor cells from healthy subjects (n=11). The probability of genes being differentially expressed among the classes was computed using a random-variance t-test. A multivariate permutation test was used to identify genes that were differentially expressed among the two classes. Functional classification of differentially expressed genes was performed using the biological process ontology in the Gene Ontology database.   Results: Type 1 diabetes significantly modulated the expression of 1591 genes compared to healthy controls. These genes were found to be involved in processes regulating development, cell communication, cell adhesion and localization. After folic acid treatment, endothelial progenitor cell gene expression profiles from diabetic patients were similar to those from healthy controls. Genes that were normalized by folic acid played a prominent role in development, such as the transcription factors ID1 and MAFF. Few oxidative-stress related genes were affected by folic acid. Conclusion: Folic acid normalizes endothelial progenitor cell gene expression profiles of patients with type 1 diabetes. Signaling pathways modulated by folic acid may be potential therapeutic targets to improve endothelial progenitor cell function.

A role for Prenylated rab acceptor 1 in vertebrate photoreceptor development

The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day P10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. Using a microarray approach, we performed gene profiling comparing rd1 and wild type retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

Activation of the Mitogen activated protein kinase (MAPK) cascade following Toll-like receptor (TLR) stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase Dual specificity phosphatase-1 (DUSP1) as an essential endogenous regulator of the inflammatory response to LPS. DUSP1-deficient (DUSP1-/-) bone marrow derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1-/- mice with LPS caused increased lethality and overshooting production of IL-6 and TNF?. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, that includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4 and CXCL2. In contrast, the expression of  the important mediators of endotoxin lethality, IFN? and IL-12,  was not  significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock.
Keywords: in vivo analysis of splenic mRNA expression

Transcript analysis in response to ozone in mice deficient in TLR4

We previously identified toll-like receptor 4 (Tlr4) as a candidate gene responsible for ozone (O3)-induced pulmonary hyperpermeability and inflammation. The objective of this study was to determine the mechanism through which TLR4 modulates O3-induced pulmonary responses and to utilize transcriptomics to determine TLR4 effector molecules. C3H/HeJ (HeJ; Tlr4 mutant) and C3H/HeOuJ (OuJ; Tlr4 normal), mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48 or 72 hr. Affymetrix Mouse430A_MOE gene arrays were used to analyze lung homogenates from HeJ and OuJ mice followed using a bioinformatic analysis. Inflammation was assessed by bronchoalveolar lavage and molecular analysis by ELISA, immunoblotting, and transcription factor activity. TLR4 signals through both the MYD88-dependent and independent pathways in OuJ mice, which involves MAP kinase activation, NF-kappaB, AP-1, and KC. Microarray analyses identifiedTLR4 responsive genes for strain and time in OuJ versus HeJ mice (p<0.05). One significantly upregulated cluster of genes in OuJ were the heat shock proteins (Hspa1b; Hsp70), Hsp90ab1). Furthermore, O3-induced expression of HSP70 protein was increased in OuJ compared to HeJ mice following 24-48 h O3. Moreover, BAL polymorphonuclear leukocytes (PMN) and total protein were significantly reduced in response to O3 in Hspa1a/Hspa1btm1Dix (Hsp70-/-) compared to Hsp70+/+ mice (p<0.05). TLR4 signaling (MYD88-dependent), ERK1/2, AP-1 activity, and KC protein content were also significantly reduced after O3 exposure in Hsp70-/- compared to Hsp70+/+ mice (p<0.05). These studies suggest that HSP70 is involved in the regulation of O3-induced lung inflammation through the TLR4 pathway and provide evidence that HSP70 is an endogenous in vivo TLR4 ligand.

Expression data from HeLa cells treated with Casiopeina Cas-II-gly

Copper-based chemotherapeutic compounds Casiopeinas, have been presented as able to promote selective programmed cell death in cancer cells, thus being proper candidates for targeted cancer therapy. DNA fragmentation and apoptosis -in a process mediated by reactive oxygen species- for a number of tumor cells, have been argued to be the main mechanisms. However, a detailed functional mechanism (a model) is still to be defined and interrogated for a wide variety of cellular conditions; before establishing settings and parameters needed for their wide clinical application.
Microarrays were used to determine the expression profile from HeLa cells in order to propose a model for the role played by intrinsic apoptosis triggered by the oxidative stress caused by Cas-II-gly.

NOTCH signaling in T-cell acute lymphoblastic leukemia cell lines

In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 ICD to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels were low. In contrast, pharmacologically blocking Notch signaling reversed this picture and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, which leads to more rapid cell cycle progression.
Keywords: comparative genomic hybridization

NPTX1 is a Critical Regulator of Neural Induction in Human Pluripotent Stem Cells

NPTX1 is a key inducer of neural lineages from the human ESC.
We used microarrays to detail the global programme of gene expression underlying neural induction by NPTX1.

Transcriptomic modulation by adenosine 2A receptors in normal and endotoxemic myocardium

Analysis of venticular myocardium from adenosine 2A receptors (A2AR) knockouts following LPS stimulation. Results provide insight into the molecular components of A2AR mediated protection, but also reveal pathogenetic components of endotoxemic myocarditis as a result of LPS exposure. These findings demonstrate that intrinsic A2AR activity exerts limited transcriptional effects in unstressed heart, modifying G-coupled cAMP/PKA signal paths. LPS-dependent injury and dysfunction is associated with profound up-regulation of inflammatory/immune processes, fibrotic and cell death paths, and NF-kB, Erk/MAPK and JAK/Stat signaling, with shifts in multiple determinants of cardiac contraction and survival. Intrinsic A2AR activity modulates key aspects of these inflammatory responses, involving MAPK, JAK/Stat and NF-kB signaling

T-cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we used a mouse model of T-ALL through the overexpression of the intarcellular transcriptionally active part of Notch1 (N1-IC). This model faithfully recapitulates the major characteristics of the human disease. Comparison of the leukemic cells from peripheral tumors(thymoma) of this mouse model to normal thymic cells Double Positive (DP) for the markers CD4 and CD8 that express very low levels of Notch1 showed major expression changes in pathways controlling the transition from physiology to disease. Further correlation of the data to ChIP-Seq data from the same cell populations led us to identify a hitherto unknown antagonism of the Notch1 oncogenic pathway and the polycomb complex (PRC2) in leukemia. Importantly exome sequencing in primary samples from human patients with T-ALL revealed that the PRC2 complex is frequently mutated and inactivated, further supporting the tumor suppressor role of the complex in this disease.

Gene expression data of skin from psoriatic patients and normal controls

Gene expression has been proposed as an intermediate phenotype that can increase power in complex trait gene-mapping studies. Psoriasis, an immune-mediated, inflammatory and hyperproliferative disease of the skin and joints, provides an ideal model system to evaluate this paradigm, as conclusive evidence demonstrates that psoriasis has a genetic basis and the disease tissue is readily accessible.
To better understand the complex nature of processes in psoriasis, we characterize gene expression profiles in uninvolved and involved skin from affected individuals as well as normal skin from control individuals.

Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis

Adult-onset diseases can be associated with in utero events, but mechanisms for such temporally distant dysregulation of organ function remain unknown.  The polycomb histone methyltransferase, Ezh2, stabilizes transcription by depositing repressive histone marks during development that persist into adulthood, but the function of Ezh2-mediated transcriptional stability in postnatal organ homeostasis is not understood. Here, we show that Ezh2 stabilizes the postnatal cardiac gene expression program and prevents cardiac pathology, primarily by repressing the homeodomain transcription factor Six1 in differentiating cardiac progenitors. Loss of Ezh2 in embryonic cardiac progenitors, but not in differentiated cardiomyocytes, resulted in postnatal cardiac pathology, including cardiomyocyte hypertrophy and fibrosis. Loss of Ezh2 caused broad derepression of skeletal muscle genes, including the homeodomain transcription factor Six1, which is expressed in cardiac progenitors but is normally silenced upon cardiac differentiation. Many of the deregulated genes are direct Six1 targets, implying a critical requirement for stable repression of Six1 in cardiac myocytes. Indeed, upon de-repression, Six1 promotes cardiac pathology, as it was sufficient to induce cardiac hypertrophy. Furthermore, genetic reduction of Six1 levels almost completely rescued the pathology of Ezh2-deficient hearts. Thus, repression of a single transcription factor in cardiac progenitors by Ezh2 is essential for stability of the adult heart gene expression program and homeostasis. Our results suggest that epigenetic dysregulation during discrete developmental windows can predispose to adult disease and dysregulated stress responses.

TTP mRNA targets identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice, and cells derived from them, showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target
mRNAs for TTP include tumor necrosis factor alpha (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin 2 beta (IL2 beta). Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained conserved TTP binding sites; 10 of these were shown by secondary analyses to be stabilized. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly, and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.

whole brains from nicotinic acetylcholine receptor subunit mutant mice and littermate controls

Studies using mice with beta4 nicotinic acetylcholine receptor (nAChR) subunit deficiency (beta4-/- mice) helped reveal the roles of this subunit in bradycardiac response to vagal stimulation, nicotine-induced seizure activity and anxiety. In order to identify genes that might be related to beta4-containing nAChRs activity, we compared the mRNA expression profiles of brains from beta4-/- and wild-type mice using Affymetrix U74Avr2 microarray.
Keywords: knockout mice, nicotinic acetylcholine receptor

Gene expression profiling of lung tissue from undiseased (NML), 'usual' emphysema (EML), and Alpha-1 Antitrypsin Deficiency-related emphysema (ADL).
Keywords = Emphysema

The Notch/Hes1 pathway sustains NF-κB activation through CYLD repression in T cell leukemia

The NF-κB pathway is a critical regulator of the immune system and has been implicated in cellular transformation and tumorigenesis. NF-κB response is regulated by the activation state of the IκB kinase (IKK) complex and triggered by a wide spectrum of stimuli. We previously reported that NF-κB is downstream of Notch1 in T cell acute lymphoblastic leukaemia (T-ALL), however both the mechanisms involving Notch1-induced NF-κB activation and the potential importance of NF-κB in the maintenance of the disease are unknown. Here we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models of this type of leukemia. We show that it is not Notch1 itself but Hes1, a canonical Notch target, the responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by a direct transcriptional repression of the deubiquitinating enzyme CYLD, a well-characterized IKK inhibitor. Consistently, CYLD expression is significantly reduced in primary T-ALL leukemias. Finally, we demonstrate that IKK complex inhibition is a promising option for the targeted therapy of T-ALL as suppression of IKK function affected both the survival of human T-ALL cells in vitro and the maintenance of the disease in vivo.
Transcriptional consequences of NF-kB inactivation in human T-ALL1 cell line

Expression data from U-87 human glioma cells with proficient and deficient NF-кB after treatment with temozolomide

The transcription factor, NF-кB, plays a central role in the response to DNA damage. This ubiquitous family of proteins is made up of five subunits: p50 (NF-κB1, p105), p52 (NF-κB2, p100), p65 (relA), relB, and crel that appear in their mature form as dimers. Following stimulation, NF-κB dimers translocate to the nucleus where they bind specific consensus elements (κB-sites) in the promoter region of genes involved in cell survival, inflammation and the immune system. While there is a general propensity of NF-кB to mediate survival, this is not always the case and several reports note the pro-apoptotic nature of the NF-кB pathway. In examining the NF-кB response to DNA damage, we have found that the p50 subunit plays a central role in modulating cytotoxicity following TMZ treatment in malignant glioma. In the current study, given the importance of p50 to the cytotoxic response to TMZ, we set out to identify NF-кB-dependent factors that modulate the response to TMZ.

Obesity results from a chronic imbalance between energy intake and energy expenditure, with excess calories stored as fat. As such, weight loss has long been considered as a primary goal of treatment for obesity. A surgical treatment of severe obesity such as gastric bypass provides the most dramatic reductions in body weight, and a well-known effect of weight loss is an improvement in insulin sensitivity. However, the molecular mechanism underlying this association remains unclear. Thus, we profiled skeletal muscle of morbidly obese patients before and after gastric bypass surgery. Results from this project will provide global patterns of gene expression with weight loss, which help to understand the pathogenesis of obesity at the molecular level.

Gene Profiling of Testosterone-Regulated Genes in the Skeletal Muscle of Hiv-Infected Men Experiencing Weight Loss

Context: Although androgen treatment increases muscle mass in HIV-infected men, the underlying mechanisms are poorly understood. Analysis of genome-wide microarray data obtained from skeletal muscle biopsies can be used to profile androgen-regulated pathways.
Objective: To identify genes and pathways associated with testosterone treatment and myogenesis in the context of HIV-infected men using genome-wide microarray analysis of skeletal muscle biopsies.
Results: A significant weight gain was observed in subjects treated with testosterone compared to placebo (+2.05 kgs and –1.07 kgs, respectively; P = 0.003) as well as gains in DEXA lean
mass (2.93 kgs vs. 0.35 kgs, respectively; P = 0.003). Microarray expression profiles and RTPCR validation of RNA after 14 days of treatment indicated that several gene sets including
transcriptional control, myogenesis, adipogenesis, insulin signaling, apoptosis, cell cycle, chromatin remodeling, and stress response genes were differentially expressed with testosterone treatment. Protein expression analysis of myogenic differentiation and protein synthesis markers (MyoD, Myogenin, phosphorylated p38 MAPK and phosphorylated AKT) in muscle biopsies and skeletal muscle cells treated with DHT confirmed that testosterone engages
a network of pro-myogenic genes.
Conclusions: Testosterone-associated gain in muscle mass in HIV-infected men engaged a network of regulatory pathways involved in broad transcriptional control, myogenesis, insulin signaling, chromatin remodeling, and stress response. Further evaluation of precise signaling intermediates within androgen regulated pathways may help to better define improvements in muscle mass, both in healthy and HIV infected patients.

PTEN imparts tumor suppression in mice by cell autonomous and non-autonomous mechanisms. Whether these two tumor suppressor roles are mediated through similar or distinct signaling pathways is not known. Here we generated and analyzed knockin mice that express a series of human cancer-derived mutant alleles of PTEN in either stromal or tumor cell compartments of mammary glands. We find that cell non-autonomous tumor suppression by Pten in stromal fibroblasts strictly requires activation of P-Akt signaling, whereas cell autonomous tumor suppression in epithelial tumor cells is independent of overt canonical pathway activation
These findings expose distinct Akt-dependent and independent tumor suppressor functions of PTEN in stromal fibroblasts and tumor cells, respectively, that can be used to guide clinical care of breast cancer patients

RNA was prepared from papilomas of 2-3 mice (2 for WT and 3 for Knockout) and this was pooled to perform the array. Each pooled sample was run in triplicates.   The mice were treated once with DMBA, followed by TPA application for 30 wks. These mice are mixed background of FVB and C57bl6 and the age when they got the papillomas was about 9 months.

Mouse ESCs depleted of the epigenetic modifying enzyme Usp22 fail to differentiate properly.  Ectopic expresison of Usp22 results in spontaneous differnetiation.
In order to understand the transcriptional program underlying this biological defect, whole genome expression analysis was performed.

Comparison of Differences in Mouse Mammary gland gene expression data at POSTNATAL DAY 70 in Akt1wildtype versus Akt1-deficient mice

Akt1, a serine-threonine protein kinase member of the PKB/Akt gene family, plays a critical role in the regulation of several cellular processes including cell proliferation and apoptosis. In this study, we utilized Akt1+/+ and Akt1¬-/- C57/Bl6 female mice to demonstrate that Akt1 is required for normal mammary gland postnatal development and homeostasis. Akt1 deficiency resulted in severely delayed postnatal mammary gland growth as well as a significant decrease in the number of terminal end buds during puberty. Adult Akt1-/- mammary glands exhibited significantly fewer alveolar buds coupled with a significant increase in epithelial cell apoptosis compared to their wild-type counterparts. Microarray analysis revealed that Akt1 deficiency resulted in several altered gene expression changes and biological processes in adult mammary glands, including organismal development, cell death, and tissue morphology. Of particular importance, a significant decrease in expression of Btn1a1, a gene involved in milk lipid secretion, was observed in Akt1-/- mammary glands by both microarray and RT-PCR validation.
Transcriptome analysis of Akt1 wild type and akt1-homozygous mouse mammary glands

Glucocorticoids, which activate glucocorticoid receptor signaling and thus modulate gene expression, are widely used to treat asthma. Glucocorticoids exert their therapeutic effects in part through modulating airway smooth muscle structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. Here, we used transcription profiling to characterize the effects of a potent glucocorticoid, dexamethasone, on cultured human airway smooth muscle gene expression at 4 and 24 hours.

Gene expression changes in neonatal rat ventricular myocytes plated in fibronectin (2 samples) or uncoated plates (2 samples).
Keywords = Hypertrophy Cardiac Myocyte Fibronectin

Difference in skeletal muscle function in Males vs Females; Role of Estrogen Receptor Beta

Male skeletal muscles are generally faster and have a higher maximum power output than female muscles. Conversely, during repeated contractions female muscles sre generally more fatigue resistant and recover faster. we hypothesized that estrogen receptor beta is involved in this gender difference. The Affymetrix micorarray were used to deteced the differently expressed genes between male BERKO and WT mice in soleus muscles. Soleus muscles from 3 individual mouse of each genotype were used to perform the experiment.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGFβ is known to be a potent inducer of EMT, a process involved in tumor invasion. TIF1γ has been reported to participate to TGFβ signaling. In order to understand the role of TIF1γ in TGFβ signaling and its requirement for EMT, we analyzed the TGFβ1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGFβ1 treatment, whereas Smad4 inactivation completely blocked this process. In support of these observations, microarray data show that the functions of several TIF1γ target genes can be linked to EMT. As a negative regulator of Smad4, TIF1γ could be critical for the regulation of TGFβ signaling. This work highlights the molecular relationship between TIF1γ and Smad4 in TGFβ1 signaling and EMT.

Transcriptional profiling of colon epithelial biopsies from ulcerative colitis patients and healthy control donors.
Study aims to survey and analyze variation from disease in different GI regions.

Expression profile of lung adenocarcinoma, A549 cells following induction of non metastatic 2 (NME2/NM23 H2)

It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression.

CXCL4 induces a unique transcriptome in monocyte-derived macrophages

Human blood monocytes were differentiated over six days with either 100 ng/ml M-CSF or 1 umol/l CXCL4
In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.

Whole genome array expression analysis was performed to identify changes in gene expression associated with the downregulation of the Core Binding Factor subunit CBFbeta.

To determine the expression of genes in cells following silencing of CDK9 by siRNA.
U2OS human osteosarcoma cell lines were utilized.

The pluripotency factor NANOG regulates the mitotic program of adult stratified epithelia.

NANOG is a key transcription factor for pluripotency in embryonic stem cells. However, its role in adult tissues remains largely unexplored. Here, we show that mouse NANOG is expressed in the progenitor layer of stratified epithelia, including esophagus, forestomach and skin. Accordingly, the Nanog promoter is hypomethylated in the epithelial layers of the esophagus and forestomach. Interestingly, ubiquitous transgenic overexpression of NANOG in mice induces hyperplasia in stratified epithelia, but not in other tissues. Mechanistically, we show that NANOG transcriptionally activates the mitotic program selectively in stratified epithelia, and endogenous NANOG directly binds the Aurora kinase A (Aurka) promoter in keratinocytes. Finally, human and mouse squamous cell carcinomas (SCCs) express NANOG and its levels positively correlate with those of AURKA in human head and neck SCCs. Together, these results implicate a lineage-restricted mitogenic role of NANOG in normal stratified epithelia and its derived carcinomas.

Stress-response pathways are altered in the hippocampus of chronic alcoholics.

Comparison of gene expression in post-mortem hippocampus from 20 alcoholics and 19 controls.
The chronic high-level alcohol consumption seen in alcoholism leads to dramatic effects on the hippocampus, including decreased white matter, loss of oligodendrocytes and other glial cells, and inhibition of neurogenesis. Examining gene expression in post mortem hippocampal tissue from 20 alcoholics and 19 controls allowed us to detect differentially expressed genes that may play a role in the risk for alcoholism or whose expression is modified by chronic consumption of alcohol. We identified 639 named genes whose expression significantly differed between alcoholics and controls at a False Discovery Rate (FDR) ≤ 0.20; 52% of these genes differed by at least 1.2-fold. Differentially expressed genes included the glucocorticoid receptor and the related gene FK506 binding protein 5 (FKBP5), UDP glycosyltransferase 8 (UGT8), urea transporter (SLC14A1), zinc transporter (SLC39A10), Interleukin 1 receptor type 1 (IL1R1), thioredoxin interacting protein (TXNIP), and many metallothioneins. Pathways related to inflammation, hypoxia, and stress showed activation, and pathways that play roles in neurogenesis and myelination showed decreases. The cortisol pathway dysregulation and increased inflammation identified here are seen in other stress-related conditions such as depression and post-traumatic stress disorder and most likely play a role in addiction. Many of the detrimental effects on the hippocampus appear to be mediated through NF-κB signaling. Twenty-four of the differentially regulated genes were previously identified by genome-wide association studies of alcohol use disorders; this raises the potential interest of genes not normally associated with alcoholism, such as suppression of tumorigenicity 18 (ST18), BCL2-associated athanogene 3 (BAG3), and von Willebrand factor (VWF).

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emergning as key regulatory proteins with great promise as therapeutic agents for bone disorders. Until recently Sost and its paralog Sostdc1 have been described as growth factors with highly restricted expression in the adult where Sost was assumed 'osteocyte-' and Sostdc1 'kidney-' specific. Here we show that these two proteins emerged throgh ancestral genome duplication and their expression patterns have diverged to span complimentary domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme.
While Sostdc1-/- mice lack any obvious limb and skeletal defects, Sost-/- mice recapitulate the hand defects described for sclerosteosis patients. However, elevated WNT signaling in Sost-/-; Sostdc1-/- mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and ultimately preaxial polydactyly. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost-/- and Sost-/-; Sostdc1-/- mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanisms and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Effect of chronic fluoxetine treatment on hippocampal gene expression

Both the mechanism of action and the factors determining the behavioral response to antidepressants are unknown. It has been shown that antidepressant treatment promotes the proliferation and survival of hippocampal neurons via enhanced serotonergic signaling, but it is still unclear whether hippocampal neurogenesis is responsible for the behavioral response to antidepressants. Furthermore, a large subpopulation of patients fails to respond to antidepressant treatment due to presumed underlying genetic factors. In the present study, we have used the phenotypic and genotypic variability of inbred mouse strains to show that there is a genetic component to both the behavioral and neurogenic effects of chronic fluoxetine treatment, and that this antidepressant induces an increase in hippocampal cell proliferation only in the strains that also show a positive behavioral response to treatment. The behavioral and neurogenic responses are associated with an upregulation of genes known to promote neuronal proliferation and survival.  These results suggest that inherent genetic predisposition to increased serotonin-induced neurogenesis is a determinant of antidepressant efficacy.

Acne lesion vs. normal skin. Also includes non-acne patients' normal skin samples.

The pathogenesis of acne has been linked to multiple factors such as increased sebum production, inflammation, follicular hyperkeratinization, and the action of Propionibacterium acnes within the follicle. In an attempt to understand the specific genes involved in inflammatory acne, we performed gene expression profiling in acne patients. Skin biopsies were obtained from an inflammatory papule and from normal skin in six patients with acne. Biopsies were also taken from normal skin of six subjects without acne. Gene array expression profiling was conducted using Affymetrix HG-U133A 2.0 arrays comparing lesional to nonlesional skin in acne patients and comparing nonlesional skin from acne patients to skin from normal subjects. Within the acne patients, 211 genes are upregulated in lesional skin compared to nonlesional skin. A significant proportion of these genes are involved in pathways that regulate inflammation and extracellular matrix remodeling, and they include matrix metalloproteinases 1 and 3, IL-8, human beta-defensin 4, and granzyme B. These data indicate a prominent role of matrix metalloproteinases, inflammatory cytokines, and antimicrobial peptides in acne lesions. These studies are the first describing the comprehensive changes in gene expression in inflammatory acne lesions and are valuable in identifying potential therapeutic targets in inflammatory acne.
Keywords: acne lesion, normal skin

LOX-1-dependent transcriptional regulation after oxidized LDL (OxLDL) treatment of human aortic endothelial cells.

LOX-1 is the primary endothelial receptor for oxidized LDL in endothelial cells and plays a role in the development of atherosclerosis.  Expression profiling was carried out on samples from HAECT cells over-expressing either LOX-1 or GFP control and treated with or without OxLDL over a time course of  2, 6, 12 and 24 Hours.  The goal of the study was to identify genes expression changes activated by OxLDL binding to LOX-1 at several different time points.

In vitro knockdown of LAMP3 leads to down-regulation of  interferon-inducible genes

We identified LAMP3 as a key driver gene of anti-viral subnetwork genes in cervical cancer patients. Therefore we tested this prediction using an in vitro system. This is the first direct demonstration of LAMP3 regulatory role in interferon-dependent immune response.

Acrylamide is a type-2 alkene monomer with established human neurotoxic effects.  While the primary source of human exposure to acrylamide is occupational, other exposure sources include food, drinking water, and smoking.  In this study, neurobehavioral assays coupled with transcriptional profiling analysis were conducted to assess both behavioral and gene expression effects induced by acrylamide neurotoxicity in rats when administered during early postnatal life.  Acrylamide administration in rat pups induced significant characteristic neurotoxic symptoms including increased heel splay, decrease in grip strength, and decrease in locomotor activity.   Transcriptome analysis with the Affymetrix Rat Genome 230 2.0 array indicated that acrylamide treatment caused a significant alteration in the expression of genes involved in muscle contraction, pain regulation, and dopaminergic neuronal pathways.  First, in agreement with the observed behavioral effects, expression of the Mylpf gene involved in muscle contraction was downregulated in the spinal cord in response to acrylamide. Second, in sciatic nerves, acrylamide repressed the expression of the opioid receptor gene Oprk1 that is known to play a role in neuropathic pain regulation. Finally, in the cerebellum, acrylamide treatment caused a decrease in the expression of the nuclear receptor gene Nr4a2 that is required for development of dopaminergic neurons.   Thus, our work examining the effect of acrylamide at the whole-genome level on a developmental mammalian model has identified novel genes previously not implicated in acrylamide neurotoxicity that can be further developed into biomarkers for assessing the risk of acrylamide exposure.

Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are still poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype that reminiscent of psoriasis and seborrheic dermatitis. We defined ZNF750 as a nuclear effector that is strongly activated in and essential for keratinocyte terminal differentiation. ZNF750 knockdown in HaCaT keratinocytes markedly reduced the expression of  epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, ZNF750 over-expression in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation, and with its downstream targets can serve in future elucidation of therapeutics for common disease of skin barrier

ZFP36L2, zinc finger protein 36, C3H type-like 2 (also known as Brf2, Erf2, Tis11D) is a member of the tristetraprolin (TTP; Zfp36) family of tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in the 3'-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation.  We have generated Zfp36l2 knockout mice.  Knockout mice were born at the expected Mendelian frequency, but within several weeks of birth they died rather suddenly with pallor and frequent intestinal hemorrhage.  These mice exhibited pancytopenia, decreased hematopoietic progenitor cells from fetal liver and yolk sac, and ineffective hematopoietic stem cells.  Since ZFP26L2 is likely to function as an ARE-containing mRNA destabilizing protein, we were interested in identifying any abnormally stabilized transcripts in fetal livers from the Zfp36l2 knockout mice whose protein product may directly or indirectly affect hematopoietic stem cell function.

Gene expression in Bach2-deficient and wildtype CD4 single-positive thymocytes from mixed chimeric animals

The role of FoxP3+ regulatory T (Treg) cells in the maintenance of immunological tolerance is well established. Recently, genome-wide association studies (GWAS) in humans have associated polymorphisms within the BACH2 locus encoding the transcription factor BTB and CNC homology 1, basic leucine zipper transcription factor 2 (Bach2) with diverse allergic and autoimmune diseases including asthma, multiple sclerosis, Crohn's disease, celiac disease, generalized vitiligo and type 1 diabetes. Common to these diseases is a failure to adequately maintain immunological tolerance. However, a role for Bach2 in this process has not been established. Here, by assessing the phenotype of mice in which the Bach2 gene is disrupted, we demonstrate a non-redundant role for Bach2 in the prevention of a spontaneous lethal inflammatory disorder predominantly affecting the lung and gut with excessive T helper 2 (Th2) responses and formation of circulating autoantibodies. Bach2 was necessary for efficient induction of FoxP3 expression both during thymopoesis and upon stimulation of naïve peripheral CD4+ T cells under Treg polarizing conditions in vitro. Consequently, in bone marrow reconstitution experiments, Bach2 expression within the haematopoetic system was necessary for suppression of lethal autoimmunity in a manner that was FoxP3 dependent. These findings demonstrate a requirement for Bach2 in early lineage commitment of both thymic and induced Treg cells and point to shared mechanisms that underlie diverse allergic and autoimmune disorders that may serve as targets in the development of novel therapeutic strategies.

Analysis of gene expression in differentiated human primary keratinocytes depleted for MLL2

Human primary keratinocytes were depleted of MLL2 by siRNA and induced to differentiated for 2 days by addition of Calcium

Purpose:
Accurate prediction of clinical response is the prerequisite for individualized therapy in chronic lymphocytic leukemia (CLL). We hypothesized that sequential assessment of gene expression changes early during therapy may well reflect behaviour of the leukemic clone in response to specific drugs.
Patients and Methods:  Gene expression profiles (GEP) were determined in CD19+ selected B-cells from 20 patients treated with fludarabine and cyclophosphamide (FC) (N=10) or FC plus rituximab (FCR) (N=10). Samples were collected in the first cycle before and within 48hours after initiation of treatment. GEP analysis was stratified by clinical response 3 months after start of therapy.
Results: GEP before treatment detected high expression of 34 genes correlated with response and 32 genes correlated with resistance to therapy. These genes were related to regulation of apoptosis, cell cycle, cell adhesion, and signal transduction. Different results were obtained with sequential GEP: Sixteen genes were up-regulated after rituximab infusion in non-responders. Rituximab therapy resulted in down-regulation of AKT1 indicating involvement of the PI3-kinase pathway in CD20-signaling. Up-regulation of 24 genes after FC (including ITPKB (inositol 1,4,5-trisphosphate 3-kinase) and CD44) and of 36 genes after FCR (including CD49d) was associated with resistance. Down-regulation of CTLA4 correlated with poor response to FC. CD44, CD49d and the PI3-kinase signaling pathway were confirmed as potential therapeutic targets to overcome resistance by (protein analysis) or functional experiments.   Conclusion  Sequential GEP provides rapid and relevant information for prediction of response and resistance. This approach could be used to guide and adapt individualized therapy in CLL.

Polycomb-like 2 associates with PRC2 and is required for mouse embryonic stem cell

Polycomb group (PcG) proteins are highly conserved epigenetic transcriptional repressors important for the control of numerous developmental gene expression programs and have recently been implicated in the modulation of embryonic stem cell (ESC) identity. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for novel regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a novel Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation and altered patterns of histone methylation. Through integration of global gene expression and promoter occupancy analyses of both PCL2 and PRC2 components EZH2 and SUZ12, we have predicted PCL2 target genes and formulated regulatory networks describing the role of PCL2 both in modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.

Expression data from H157 non-small cell lung carcinoma cells with and without DJ-1 expression knocked-down

DJ-1 expression protects H157 cells from cell death, using siRNA oligomers we knocked down DJ-1 and evaulated gene expression using Affymetrix U133A GeneChip arrays.
Keywords: siRNA comparison

Cooperative dependencies between mutant oncoproteins and wild-type proteins are critical in cancer pathogenesis and therapy resistance. Although spleen tyrosine kinase (SYK) has been implicated in hematologic malignancies, it is rarely mutated. We used kinase activity profiling to identify collaborators of SYK in acute myeloid leukemia (AML) and determined that FMS-like tyrosine kinase 3 (FLT3) is transactivated by SYK via direct binding. Highly activated SYK is predominantly found in FLT3-ITD positive AML and cooperates with FLT3-ITD to activate MYC transcriptional programs. FLT3-ITD AML cells are more vulnerable to SYK suppression than FLT3 wild-type counterparts. In a FLT3-ITD in vivo model, SYK is indispensable for myeloproliferative disease (MPD) development, and SYK overexpression promotes overt transformation to AML and resistance to FLT3-ITD-targeted therapy.

Rat diaphyseal femoral fracture healing:  Plate fixation vs. intramedullary nail

This is a study of femoral fracture healing in female rats 16 weeks old at fracture to compare intramedullary nailing, screw and plate fixation, and sham surgery.  The sham surgery group received a surgical exposure of the femur, but no fracture, no plate, and no nail.  Samples were collected at 1 day, 3 days, 1 week, 2 weeks, 4 weeks, and 6 weeks after surgery.  Each sample is a pool of RNA from three rats from the same surgery group at the same time point after fracture.  The middle third of the femur was collected with the cortical bone, fracture callus, and marrow elements.  Mid-diaphyseal, simple, transverse fractures were induced by a Gigli saw.  The no fracture sample was a time 0 control collected on the day of surgery from intact rats.

Expression data from mouse ES cells after control RNAi (scramble siRNAs) or RNAi specific for Kdm6a treatment.

To address the functional role of KDM6A in the regulation of Rhox genes, male and female mouse ES cells were transfected with a mixture of three small interfering RNA duplexes, each of which targets a different region of Kdm6a mRNA. We found that Kdm6a knockdown in mouse ES cells caused a decrease in expression of a subset of Rhox genes, Rhox6 and 9. Furthermore, Rhox6 and 9 expression was decreased in female ES cells but not male ES cells indicating that KDM6A regulates Rhox gene expression in a sexually dimorphic manner.

Effect of CCL3 on target ablation-induced macrophage recruitment into the olfactory epithelium

The aim was to determine the identity of genes regulated at 3 d post-olfactory bulbectomy in the olfactory mucosa of carrier-injected Ccl3-/- mice compared with carrier-injected C57BL/6 mice.  Total RNA from the OE was hybridized to Affymetrix microarrays.  A number of chemokine-, cytokine-, and growth factor-related genes were significantly regulated in the Ccl3-/- mice, and were restored in CCL3 protein-injected Ccl3-/- mice.  The results illustrated that CCL3 played a key role in recruitment of macrophages to the OE and provided insight into the genomic regulation involved in OE remodeling.

Expression data from infected with Neisseria gonorrhoeae (GC) and uninfected bone marrow derived macrophages from wild type (C67BL/6) and Nod2 knock out mice

In this dataset we include the data obtained from 3 hour stimulation with Neisseria gonorrhoeae (GC) of bone marrow macrophages(BMDM) from wild type (C57BL/6) and Nod2 knock out mice (in C57BL/6 background).

The samples are a part of a study aiming at diagnosing ulcerative colitis from genome-wide gene expression analysis of the colonic mucosa. Colonic mucosal samples were collected as endoscopic pinch biopsies from ulcerative colitis patients and from control subjects. Samples with and without macroscopic signs of inflammation were collected from the patients.
Keywords: Disease state analysis

HSF1 mediated Gene regulation in T cells at normal (37C) and febrile (40C) temperatures

HSF1 is a major transcriptional regulator of heat shock responses. Many cells activate HSF1 in response to heat shock temperatures (>42oC) and other cellular stress causing agents. Unlike other cell types, T cells activate HSF1 in response to T cell activation or when exposed to febrile (40oC) temperatures, suggesting a role for HSF1 beyond the heat-shock response.
We used microarray analysis and HSF1 knock-out mice to study the HSF1 mediated gene regulation in activated T cells under normal and fever temperatures.

A study of the effects of Hoxc13 overexpression in skin using Hoxc13 transgenic mice (GC13) known to develop severe hair growth defects and alopecia. Total skin from GC13 mice 5 dpn is compared to total skin from normal FVB mice 5 dpn using Affymetrix HG-U74Av2 GeneChips.

Expression data from HeLa cells transiently transfected with control siRNA or SON siRNA

SON is a large Ser/Arg (SR)-related protein localized in nuclear speckles. SON siRNA causes defects in mitotic progression and genome instability. We used microarrays to detail the pattern of gene expression after SON knockdown.

Keap1-dependent gene expression determined in the liver using conditional Keap1 knockout mice vs. genetic control mice

To compare hepatic gene expression in conditional Keap1 knockout (Alb-Cre:Keap1(flox/-)) and genetic control mice. Disruption of Keap1-mediated repression of Nrf2 signaling was expected to result in increased expression of Nrf2-regulated genes.

Stroke is a “brain attack” cutting off vital blood, and consequently the nutrients and oxygen vital to the brain cells that control everything we do. Stroke is a complex disease with unclear pathogenesis resulting from environmental and genetic factors.
To better understand IS´s etiology, we performed genomic expression profiling of patients and controls.

Effect of mir-210 overexpression or down-modulation on human umbilical vein cells

MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism.  In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.

Bronchoalveolar lavage samples collected from lung transplant recipients.  Numeric portion of sample name is an arbitrary patient ID and AxBx number indicates the perivascular (A) and bronchiolar (B) scores from biopsies collected on the same day as the BAL fluid was collected.  Several patients have more than one sample in this series and can be determined by patient number followed by a lower case letter.  Acute rejection state is determined by the combined A and B score - specifically, a combined AB score of 2 or greater is considered an acute rejection.
Keywords = Bronchoalveolar lavage

Rsk2 controls synovial fibroblast hyperplasia and the course of arthritis.

To understand differences in the pathogenesis of synovial hyperplasia during TNF-induced arthritis, we compared the global gene expression of hTNFtg and hTNFtg;Rsk2-/y primary synovial fibroblasts.

The DNMT1 associated lncRNA Dali is an epigenetic regulator of neural differentiation [3]

Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.

Targeted ErbB3 loss in mammary organoids harvested from ErbB3 DOX-KO mice +/- DOX treatment

Mammary organoids harvested from ErbB3 DOX-KO mice, which utilize MMTV-Cre transgene expression in the LE to cause genomic recombination at floxed ErbB3 alleles in ErbB3FL/FL were cultured in the presence or absence of doxycycline to induce ErbB3 loss.  The gene expression shift following DOX-induced ErbB3 loss in the 3D organoids was examined by microarray.

Human prolactinomas (n=4, 3 males and 1 female) were obtained during trans-sphenoidal surgery as part of an ongoing accession of human pituitary tumors.  The study was approved Institutional Review Board (IRB) of Emory University, and informed consent obtained for all subjects. Tumors were microdissected and removed using the surgical microscope, rinsed in sterile saline, snap-frozen in liquid nitrogen, and stored (-80 ℃) until analysis. Each tumor fragment was confirmed independently by a neuropathologist by histology and immunohistochemistry prior to molecular analysis. Three normal pituitary glands from cadavers were obtained from the National Resource Center (NDRI, www.ndriresource.org).   Each human tissue sample was analyzed using Affymetrix Human Genome U95Av2 arrays.

High fat diet (HF) rodent models have contributed significantly to the dissection of the pathophysiology of the insulin resistance syndrome, but their phenotype varies distinctly between different studies. Here, we have analyzed gene expression patterns in livers of animals fed with different HF with varying fatty acid compositions.
Keywords: diet-gene interaction

Loss of Caveolin-3 Induces the Development of a Lactogenic Microenvironment

Here, we show that functional loss of a single gene is sufficient to confer constitutive milk protein production and protection against mammary tumor formation. Caveolin-3 (Cav-3), a muscle-specific caveolin-related gene, is highly expressed in striated and smooth muscle cells. We demonstrate that Cav-3 is also expressed in myoepithelial cells within the mammary gland. To determine if genetic ablation of Cav-3 expression affects adult mammary gland development, we next studied the phenotype(s) of Cav-3 (-/-) null mice. Interestingly, detailed analysis of Cav-3 (-/-) virgin mammary glands shows dramatic increases in ductal thickness, side-branching, and the development of extensive lobulo-alveolar hyperplasia, akin to the changes normally observed during pregnancy and lactation. Analysis by genome-wide expression profiling reveals the upregulation of gene transcripts associated with pregnancy/lactation, mammary stem cells, and human breast cancers, consistent with a constitutive lactogenic phenotype. The expression levels of three key transcriptional regulators of lactation, namely Elf5, Stat5a, and c-Myc are also significantly elevated. Experiments with pregnant mice directly show that Cav-3 (-/-) mice undergo precocious lactation. Finally, using orthotopic implantation of a transformed mammary cell line (known as Met-1), we demonstrate that virgin Cav-3 (-/-) mice are dramatically protected against mammary tumor formation. Interestingly, Cav-3 (+/-) mice also show similar protection, indicating that even reductions in Cav-3 levels are sufficient to render these mice resistant to tumorigenesis. Thus, Cav-3 (-/-) mice are a novel preclinical model to study the protective effects of a constitutive lactogenic microenviroment on mammary tumor onset and progression. Our current studies have broad implications for using the lactogenic micro-environment as a paradigm to discover new therapies for the prevention and/or treatment of human breast cancers. Most importantly, a lactation-based therapeutic strategy would provide a more natural and nontoxic approach to the development of novel anti-cancer therapies.

Gene expression analyses of mir-30a knockdown in ovarian clear cell cancer cell line

A variety of human cancers demonstrate alterations in microRNA expression.  We hypothesized that regulatory defects in microRNAs play a central early role in organizing the molecular changes involved in ovarian cancer (OvCa).  Using both gene arrays and deep sequencing, we comprehensively profiled mRNA and microRNA expression, respectively, in human clear cell epithelial OvCa cell lines and short-term primary cultures of normal ovarian surface epithelium.  We expected that over-expression of a specific microRNA would lead to lower expression of its mRNA targets, and under-expression of a specific microRNA would lead to higher expression of its target genes.  Using our expression data in conjunction with established in silico algorithms, we found putative microRNA:mRNA functional pairs.  Furthermore, gene expression profiles were taken of clear cell cultures having functional knockdown or over-expression of specific microRNAs of interest.  Knockdown of mir-30a (found over-expressed in clear cell OvCa) resulted in up-regulation in vitro of a significant number of the in silico predicted mir-30a target genes that were normally under-expressed in OvCa.

Identification of downstream transcriptional targets of Dlx5 during early mouse inner ear (otocyst/otic vesicle) development

Several transcription factors are known to be expressed in discrete regions of the otic vesicle and Dlx5 is one of those that is expressed highly in the presumptive dorsal vestibular region. Mice lacking Dlx5 have vestibular defects. Specifically, they fail to form the endolymphatic duct (a defect visible as early as E10) as well as the anterior and posterior semi-circular canals. The lateral canal does form but is smaller, whereas the saccule, the utricle and the cochlea appear relatively normal. The goal of this study was to use microarrays to identify differentially expressed genes between wild-type and Dlx5-null otic vesicles microdissected from E10 and 10.5 and identify downstream targets of Dlx5 by searching the immediate 3kb promoter regions of the differentially expressed genes for homeodomain binding sites followed by chromatin immunoprecipitation in an otic vesicle-derived cell line over-expressing Dlx5.
Normal vestibular morphogenesis is compromised in mice lacking Dlx5, a member of the Distal-less family of homeobox transcription factors.  We identified its direct downstream targets in the developing mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5.  Four hundred genes were differentially expressed in mutants when compared to wild-type in at least one of the two stages. To further constrain the list of likely direct targets of Dlx5, we examined the genomic DNA sequences in the 3kb promoter regions immediately proximal to the transcriptional start sites of these genes.   We searched for (i) one or more previously described binding site for Dlx5, (ii) one or more novel 12bp-long motifs with a canonical homeodomain response element (HDRE) shared by promoters of two or more genes, and (iii) 100% conservation of the 12bp-long HDRE-containing motifs in promoter regions of human orthologs.  Forty genes passed one or more of these filters, 12 of which are known to be expressed in the developing otic vesicle in domains that at least partially overlap with that of Dlx5 in one or both stages that we examined. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoter regions of seven of these genes (Atbf1, Bmper, Large, Lrrtm1, and Msx1, all of which were down-regulated in mutants, and Ebf1 and Lhx1, both of which were up-regulated in mutants) in an otic vesicle-derived cell line over-expressing Dlx5. Gene expression profiling of this cell line showed that Bmper and Lrrtm1 transcripts were up-regulated, further supporting their identification as direct targets of Dlx5 activity.

Expression data from AtT20 mouse pituitary gland cells following overexpression or down regulation of NSBP1

Chromatin architectural protein NSBP1/HMGN5 belongs to the family of HMGN proteins which specifically interact with nucleosomes via Nucleosome Binding Domain, unfold chromatin and affect transcription. Mouse NSBP1 is a new and uncharacterized member of HMGN protein family. NSBP1 is a nuclear protein which is localized to euchromatin, binds to linker histone H1 and unfolds chromatin.
We analyzed the effect of altered expression levels of mouse NSBP1 protein on global gene expression profile in AtT20 pituitary cells. We found that NSBP1 modulates the fidelity of cellular transcription in the nucleosome binding-dependent manner.

Expression data from mouse neonatal ovarian xenobiotic culture experiments

Mammalian females are born with a finite number of non-renewing primordial follicles, the majority of which remain in a quiescent state for many years. Due to their non-renewing nature, these “resting” oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes.  In this study we characterised the mechanisms of ovotoxicity for three ovotoxic agents, 4-Vinylcyclohexene Diepoxide (VCD), Methoxychlor (MXC), and Menadione (MEN), all of which target immature follicles. Neonatal mouse ovaries (PND3-4) were cultured in the presence of 4-Vinylcyclohexene Diepoxide (50uM), Methoxychlor (50uM), and Menadione (5uM) for 96 hours to observe their effects on the ovarian transcriptome. This was done in the hopes of gaining a better understanding of the mechanisms underpinning xenobiotic induced pre-antral ovotoxicity.

Genes differentially regulated by the glucocorticoid receptor in developing skin of the GR knock out and wt embryos.

To understand the transcriptional program by which GR regulates skin development, we performed a microarray analysis using the skin of E18.5 GR-/- and GR+/+ mouse embryos.

The study was performed in liver-endothelial cells (LECs) isolated from 6 adult male Wistar rats, undergoing a cirrhosis induction protocol as described previously by our group, and LECs isolated from 6 control Wistar rats, according to the criteria of the Investigation and Ethics Committee of the Hospital Clínic Universitari (Spain).The aim of this study was to evaluate differences in the gene expression pattern between LECs from control and cirrhotic rats. Methods: LECs were isolated from livers by collagenase perfusion, isopycnic centrifugation and incubation of cells with magnetic beads coated with specific antibodies (Ab).Total RNA was extracted from LECs with Trizol reagent (Life Technologies, Rockville, MD) after the cell-isolation step described above. RNA quality and concentration were confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). cRNA samples were hybridized to the Rat Expression 230A oligonucleotide microarray (Affymetrix). Microarray samples preparation and processing procedures where performed at the IDIBAPS Genomic Unit (Hospital Clinic Universitari, Barcelona) following the protocol as described in the Affymetrix GeneChip Expression Analysis Manual (Affymetrix, Inc, Santa Clara, CA).

KLF1, KLF2 and c-myc control a regulatory network essential for embryonic erythropoiesis

The Krüppel-like factors, KLF1 and KLF2, positively regulate embryonic β-globin expression, and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1-/-KLF2-/- double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1-/- and KLF1-/-KLF2-/-. Among these, c-myc emerged as a central node in the most significant gene network. c-myc expression is synergistically regulated by KLF1 and KLF2, and both factors bind the c-myc promoters. To characterize the role of c-myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia analogous to KLF1-/-KLF2-/-. In the absence of c-myc, circulating erythroid cells do not show the normal increase in α- and β-like globin expression, but interestingly, have accelerated erythroid maturation, between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate c-myc, to control the primitive erythropoietic program.

Non muscle myosin light chain kinase isoform is an attractive molecular target in acute inflammatory lung injury

Acute lung injury (ALI), a major cause of acute respiratory failure with high morbidity and mortality, isare characterized by significant pulmonary inflammation and both alveolar and vascular barriers dysfunction.  In Pprior studies have highlighted the role of nonmuscle myosin light chain kinase (nmMLCK) as an essential element of inflammatory response with MYLK polymorphisms associated withwhich alters ALI susceptibility. In the present study we sought to further define nmMLCK in acute inflammatory syndromes and examined We examined nmMLCK as a molecular target involved in increase of lung epithelial and endothelial barrier permeability. We utilized in two muirine models of inflammatory lung injury: intratracheal administration of endotoxin/lipopolysaccharide (LPS, 2.5 mg/kg) and VILI (ventilator-induced lung injury, tidal volume 40ml/kg).  Two complementary strategies were used to reduce nmMLCK activity or expression. We found that membrane permeant oligopeptide, PIK, inhibited MLC kinase activity in vitro in aand displayed dose-dependent mannerinhibition of MLC kinase activity.. Intravenous delivery of PIK significantly attenuated LPS-induced lung inflammation reflected by decreasing accumulation of bronchoalveolar lavage (BAL) albumin (~ 50% reduction) as well as reduction in BAL cells, tissue MPO activity and tissue albumin in lung homogenates. A second regulatory approach explored targeting murine nmMLCK by administration of siRNA (5mg/kg) 3 days prior to LPS challenge. siRNA decreased of nmMLCK expression in lungs (~ 70% reduction) and resulted in significant attenuation LPS-induced lung inflammation (~ 40% reduction) as reflected by decreased BAL protein level and BAL cells. For targeting pulmonary vessels nmMLCK we used ACE antibody-conjugated liposomes with nmMLCK siRNA in murine ventilator-induced lung injury (VILI) model. Protein silencing of nmMLCK was evident by immunohistochemical analysis with a decrease in relative intensity of fluorescence in lung vessels compared with control animals. Furthermore, the inhibition of nmMLCK expression by siRNA in vessels significantly attenuated VILI lung injury as reflected by decreased BAL protein level (40% reduction). Finally, MLCK knockout mice were significantly protected (reduced BAL protein and albumin) when exposed to a model of severe VILI (4h, 40ml/kg tidal volume). Conclusion: the MLCK gene KO and chemical biology results indicate that the targeting of nmMLCK in vivo attenuate the severity of LPS-induced or VILI acute lung injury.
We used microarrays to detail the global programme of gene expression induced by VILI in Wild type and nmMLCK-/- mouse.

Regeneration of differentiated tissue in mammals is rare.  In an effort to identify genes that affect the healing process, we screened G3 mice containing germline point mutations for closure of an ear punch wound.  One particular line was identified with a heritable hole closure phenotype containing differentiated tissue. Mapping and sequencing efforts revealed that the mutant mice harbor a R244Q point mutation coded by the TGFBR1 gene which leads to enhanced signaling activity in a reporter gene assay.  Although there was no obvious effect on the immune system, bone marrow stromal cells from the mutant mice revealed accelerated chondrogenesis, mimicking the in vivo development of cartilage islands in the regenerated ears. This genetically well-defined mouse model should help to further dissect the role of TGF-beta signaling in vertebrate healing and regeneration.

Gene expression changes induced by overexpression of EVI1 in Lin- hematopoietic cells [Lin]

The transcription factor Evi1 is essential for the formation and maintenance of hematopoietic stem cells, and induces clonal dominance with malignant progression upon constitutive activation by chromosomal rearrangements or transgene integration events. To understand the immediate and adaptive response of primary murine hematopoietic cells to the transcriptional upregulation of Evi1, we developed an inducible lentiviral vector system with a robust expression switch. We found that Evi1 delays differentiation and promotes survival in myeloid culture conditions, orchestrating a battery of genes involved in stemness (Aldh1a1, Ly6a [Sca1], Abca1, Epcam, among others). Importantly, Evi1 suppresses Cyclins and Cyclin-dependent kinases (Cdk), while it upregulates Cdk inhibitors, inducing quiescence in various proliferation-inducing cytokine conditions and operating in a strictly dose-dependent manner. Hematopoietic cells with persisting Evi1-induction tend to adopt a relatively low expression level. We thus classify Evi1 as a dormancy-inducing oncogene, likely requiring epigenetic and genetic compensation for cell expansion and malignant progression.

Given the very substantial heterogeneity of most human cancers, it is likely that most cancer therapeutics will be active in only a small fraction of any population of patients. As such, the development of new therapeutics, coupled with methods to match a therapy with the individual patient, will be critical to achieving significant gains in disease outcome. One such opportunity is the use of expression signatures to identify key oncogenic phenotypes that can serve not only as biomarkers but also as a means of identifying therapeutic compounds that might specifically target these phenotypes. Given the potential importance of targeting tumors exhibiting a stem-like phenotype, we have developed an expression signature that reflects common biological aspects of various stem-like characteristics.  The Consensus Stemness Ranking (CSR) signature is upregulated in cancer stem cell enriched samples, at advanced tumor stages and is associated with poor prognosis in multiple cancer types. Using two independent computational approaches we utilized the CSR signature to identify clinically useful compounds that could target the CSR phenotype. In vitro assays confirmed selectivity of several predicted compounds including topoisomerase inhibitors and resveratrol towards breast cancer cell lines that exhibit a high-CSR phenotype. Importantly, the CSR signature could predict clinical response of breast cancer patients to a neoadjuvant regimen that included a CSR-specific agent.  Collectively, these results suggest therapeutic opportunities to target the CSR phenotype in a relevant cohort of cancer patients.

ZMYM2/FGFR1, BCR/FGFR1 or BCR/ABL1 in human cord blood CD34+ cells reveals similar but distinct gene expression profiles

The 8p11 myeloproliferative syndrome (EMS), also referred to as the stem cell leukemia/lymphoma syndrome, is a chronic myeloproliferative disorder that rapidly progresses into an acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinase activity within FGFR1. The two most common fusion genes in human EMS are ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) and BCR/FGFR1. To study the transcriptional programs becoming deregulated by the FGFR1 fusion genes, global gene expression analysis on human CD34+ cord blood cells expressing either of the fusion oncogenes ZMYM2/FGFR1 and BCR/FGFR1 was performed. As a reference gene we also included the more studied BCR/ABL1 fusion oncogene associated with chronic myeloid leukemia. We found that the 3 different fusion oncogenes had in common the upregulation of several genes involved in the JAK/STAT signalling pathway and also other sets of genes. However, the gene expression profiles were not identical, suggesting that both the tyrosine kinase containing gene and the partner gene would affect the transcription of downstream target genes.

HIPK2 and MED19 are new regulators of androgen receptor in prostate cancer cells

The androgen receptor (AR) is a mediator of both androgen-dependent and castration- resistant prostate cancers.  Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined.  Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells.  We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex).  Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells.  We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation.  Importantly, targeting of HIPK2 by an FDA approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR negative, treatment-resistant prostate cancer cells.  Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.
HIPK2 and MED19 were identified via a genome-wide RNAi screen as new androgen receptor (AR) reulators.  Our goal in performing this microarray was to identify the gene regulated by HIPK2 and MED19 in a late stage prostate cancer cell line (LNCaP-abl), and to see what genes are in common with known genes to be regulated by AR, and what genes are unique to HIPK2 or MED19.

Table of all 3329 changed genes in Ad-E2F-1 gene transferred vs LAC-Z control. (SK-MEL-2 melanoma cells)

E2F-1 gene expression profiling by adenovirus-mediated gene transfer in SKMEL-2 melanoma cell line.

Overexpression of disease-associated CYFIP1 alters cellular and synaptic morphology through mTOR dysregulation of mTOR signaling

Rare genomic gains at 15q11-q13 are observed in 1-2% of individuals with an Autism Spectrum Disorder (ASD). Because many genes are included here and breakpoints vary between cases, the potential contribution of specific genes is unclear. Cytoplasmic FMR1 interacting protein 1 (CYFIP1) is interesting in this regard given the association of smaller overlapping deletions with each of schizophrenia and intellectual disability. Towards an understanding of how increased CYFIP1 dosage might predispose to neurodevelopmental disease we investigated the consequence of overexpression in multiple systems. We show that CYFIP1 mRNA is increased in lymphoblastoid cells and human brain as a function of 15q dosage. Towards mechanisms, we determined that overexpression of CYFIP1 results in cellular abnormalities in SY5Y cells and mouse neuronal progenitors. Identical abnormalities, as well as anomalies in synaptic morphology, were seen after comparing two BAC transgenic strains to controls. Gene expression profiling at embryonic day 15 identified genes differentially expressed between transgenic and control mice and highlighted dysregulation of mTOR signaling. Finally, treatment of mouse neuronal progenitors with an mTOR inhibitor (Rapamycin) rescued morphologic abnormalities resulting from CYFIP1 overexpression. Together, these data are consistent with the notion that normalization of mTOR signaling, emerging as an important point of convergence in the ASDs, may be of clinical utility in genetically selected populations with a variety of neurodevelopmental disorders.

ACTL6a Enforces the Epidermal Progenitor State by Suppressing SWI/SNF-Dependent Induction of KLF4

Somatic progenitors suppress differentiation to maintain tissue self-renewal. While epigenetic regulators of DNA and histone modifications can support such repression, a role for nuclear actin-like proteins is unclear. In epidermis, ACTL6a/BAF53A was found enriched in progenitors and down-regulated during differentiation. Conditional ACTL6a deletion abolished epidermal self-renewal and induced terminal differentiation, whereas ectopically expressed ACTL6a suppressed differentiation. Among known activators of epidermal differentiation, KLF4 was found to control 227 genes also regulated by ACTL6a. ACTL6a loss upregulated KLF4 and its target genes, effects that were blocked by KLF4 depletion. Among multiple ACTL6a-interacting epigenetic regulators, the SWI/SNF complex was required for KLF4 activation and differentiation. In progenitors, ACTL6a loss led to enhanced SWI/SNF binding to the promoters of KLF4 and other differentiation genes. ACTL6a thus maintains the undifferentiated progenitor state, in part by suppressing SWI/SNF complex-enabled induction of KLF4.

Mycophenolic acid (MPA), an immunosuppressive drug widely used in kidney transplantation, has been suggested to have anti-fibrotic effects.
To analyze at a genomic level these effects, we prospectively studied a group of stable kidney transplant recipients (n=35) on cyclosporine (CyA) and azathioprine treatment. Twenty patients were converted from azathioprine to MPA (MPA group) and 15 patients continued on azathioprine (AZA group). RNA was extracted by peripheral blood mononuclear cells at baseline and 3 months thereafter. Genomic analysis, performed on 5 randomly-selected MPA patients, revealed that 17 genes discriminated the transcriptomic profile after conversion. Neutral endopeptidase (NEP), an enzyme degrading angiotensin-II, was the most significant up-regulated gene. NEP expression level was inversely correlated to proteinuria at baseline and after conversion. Immunohistochemistry on graft biopsy of 33 independent patients demonstrated higher glomerular and tubular NEP protein expression in CyA+MPA (n=13) compared to CyA+AZA (n=12) and CyA alone (n=8). Glomerular NEP levels were inversely correlated to proteinuria and glomerulosclerosis. Tubular NEP expression was inversely correlated to interstitial fibrosis. Incubation of proximal tubular cells with MPA led to a dose- and time-dependent increase of NEP gene expression. The direct  influence of MPA on NEP expression may suggest a novel therapeutic effect of this drug.

Gene profile in H1299 cells treated with PTD-DRBD GAPDH siRNA or treated with Lipofection GAPDH siRNA

Whole genome microarrays were probed with total mRNA from PTD-DRBD GAPDH siRNA treated H1299 cells at 12 h and 24 h. Using a 1.6x fold increase/decrease filter of cellular mRNAs, we detected a dramatic reduction in the target GAPDH mRNA along with a limited number of both up and down regulated genes. The up regulated genes were reduced in numbers and to nearly background 1.6x levels at 24 h, while the down regulated genes increased slightly in numbers and maintained a similar magnitude at 24 h. In contrast, lipofection treated cells showed both a dramatic increase in both the total number of genes altered and the magnitude of the increase. In addition, the numbers of genes affected increased between 12 h and 24 h, suggesting that lipofection of siRNAs into cells results in a substantial alteration to the transcriptome and may thereby confound interpretation of experimental outcomes. Moreover, the GAPDH specific knockdown was significantly smaller than PTD-DRBD mediated knockdown.

Effect of Twist-box mutation on gene expression induced by Twist1 overexpression

Twist1 is a transcription factor that induces EMT and drives metastasis in prostate cancer. We examined global gene expression in Myc-CaP mouse prostate cancer cells following overexpression
of Twist1 and the Twist1 mutants F191G, AQA, and DQD.

Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCa on resistance to methotrexate in human HT29 colon cancer cells

A summary of the work associated to these microarrays is the following:

Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy.

Activation of SREBP in Alveolar Type II Cells Enhances Lipogenesis Causing Pulmonary Lipotoxicity

Background:  Lung function is dependent upon the precise regulation of the synthesis, storage, and catabolism of tissue and alveolar lipids.
Results:  Activation of SREBP (Sterol Response Element Binding Protein) induced lipogenesis in alveolar epithelial cells, causing neutral lipid accumulation, lung inflammation, and tissue remodeling.
Conclusions:  The accumulation of neutral lipids in type II epithelial cells and alveolar macrophages caused lung inflammation, consistent with findings in lipid storage disorders.
Significance:  Pulmonary lipotoxicity may contribute to the pathogenesis of lung dysfunction associated with diabetes, obesity, and other metabolic disorders.

Gene expression profiling of anterior and posterior palatal tissue from Tgfbr2 mutant mouse models

The overall goal of this project is to investigate the role of TGF-beta signaling in epithelial cells as it pertains to the orientation of muscle fibers in the soft palate during embryogenesis. Here, we first conducted gene expression profiling of the anterior and posterior portions of the palate from wild-type mice. In addition, we also conducted gene expression profiling of the posterior palate in mutant mice with an epithelium-specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of submucosal cleft palate, which is a congenital birth defect commonly observed in many syndromic conditions.

Gene expression regulation of transporters and phase I/II metabolic enzymes in murine small intestine during fasting

Keywords: metabolic state analysis

Hepatic gene expression of HCV related Hepatocellular carcinoma and non-cancerous tissue with Il28B rs8099917 TT genotype and TG/GG genotype

IL28B genotype was shown to be associated with treatment outcome of antiviral thearpy for HCV infection. We tried to clarify the molecular feature that was asocciated with IL2B genotype by comparing Hepatic gene expression of HCV related Hepatocellular carcinoma and non-cancerous tissue with Il28B rs8099917 TT genotype and TG/GG genotype.

Notch1 deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced. Microarray was performed to study downstream mechanism of Notch1-deficiency

Thiazolidinediones, PPAR-gamma agonists, renal, type II diabetes, obesity

Thiazolidinediones increase tissue insulin sensitivity and are protective against worsening of nephropathy and hypertension in diabetes. Mechanisms underlying protection at the renal level likely involve a variety of unknown changes in gene expression.   We examined kidney gene expression in obese and lean Zucker rats in response to rosiglitazone (Avandia®), a peroxisome proliferator activated receptor (gamma-subtype) agonist.  Lean and obese Zucker rats were treated with either control chow or chow with added rosiglitazone (3 mg/kg•bw) for 12 weeks (n = 3/group).  Total kidney mRNA expression was evaluated using the Affymetrix Rat Genome 230 2.0 GeneChip. 903 probe sets were significantly (P < 0.05) altered with at least 1.5-fold changes between groups.  In untreated obese rats, 300 probe sets were increased and 244 decreased, relative to lean.  Increased genes included the β-subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter, and aquaporin 3.  Decreased genes included angiotensin converting enzyme, type 1 (ACE1).  FatiGO analysis showed that the highest number of altered genes between lean and obese belonged to the categories: ion binding, hydrolase activity, and protein binding.  RGZ increased expression of uncoupling protein 1 (UCP1), CD36, and fatty acid binding protein 4 (FAbp4) in both lean and obese rats.  In obese rats, 33 genes were normalized by RGZ (no longer different from lean) including ACE1, fatty acid synthase (Fasn), and stearoyl-coenzyme A desaturase 2 (Scd2).  Ingenuity Pathways System analysis of genes upregulated by RGZ in obese rats revealed two major nodes affected:  PPAR-gamma and tumor necrosis factor alpha (TNF-alpha).
Keywords: Thiazolidinediones, PPAR-gamma agonists, renal, type II diabetes, obesity

Altered gene expression in the neocortices and hippocampi of the adult S1P2-deficient and S1P3-deficient mice

Altered gene expression in the sphingosine 1-phosphate receptor 2 (S1P2)-deficient or sphingosine 1-phosphate receptor 3 (S1P3)-deficient brain.

CTCF is a multifunctional nuclear factor involved in epigenetic regulation.  We have used transgenic RNA interference to deplete maternal stores of CTCF from growing mouse oocytes, and identified the potential target genes

Total RNA was isolated from 3 WT and 3 ERRa null hearts and independent hybridizations were performed using MOE430 2.0 microarrays. Expression profiling was conducted to determine changes in gene expression in hearts lacking ERRa. The expression of genes involved in heart and muscle development, muscle contraction, lipid metabolism, OxPhos, protein metabolism and transcription were affected by the loss of ERRa.
Keywords: microarray and genetic modification

Classification of carcinoma in situ lesions in human bladder cancer

The presence of carcinoma in situ (CIS) lesions in the urinary bladder is associated with a high risk of disease progression to a muscle invasive stage. In this study, we used microarray expression profiling to examine the gene expression patterns in superficial transitional cell carcinoma (sTCC) with surrounding CIS (13 patients), without surrounding CIS lesions (15 patients), and in muscle invasive carcinomas (mTCC; 13 patients). Hierarchical cluster analysis separated the sTCC samples according to the presence or absence of CIS in the surrounding urothelium. We identified a few gene clusters that contained genes with similar expression levels in transitional cell carcinoma (TCC) with surrounding CIS and invasive TCC. However, no close relationship between TCC with adjacent CIS and invasive TCC was observed using hierarchical cluster analysis. Expression profiling of a series of biopsies from normal urothelium and urothelium with CIS lesions from the same urinary bladder revealed that the gene expression found in sTCC with surrounding CIS is found also in CIS biopsies as well as in histologically normal samples adjacent to the CIS lesions. Furthermore, we also identified similar gene expression changes in mTCC samples. We used a supervised learning approach to build a 16-gene molecular CIS classifier. The classifier was able to classify sTCC samples according to the presence or absence of surrounding CIS with a high accuracy. This study demonstrates that a CIS gene expression signature is present not only in CIS biopsies but also in sTCC, mTCC, and, remarkably, in histologically normal urothelium from bladders with CIS. Identification of this expression signature could provide guidance for the selection of therapy and follow-up regimen in patients with early stage bladder cancer.
Keywords: Tissue group comparisons (+-CIS)

Host directed activity of Pyrazinamide in Mycobacterium tuberculosis infection

Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB).  we have used human monocyte and a mouse model of pulmonary TB to investigate whether treatment with PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during Mycobacterium tuberculosis (Mtb) infection.

Expression data from 2-3 month old brain-specific IκBα conditional knockout mice

The activation of NFkB pathway is commonly observed in many neurodegenerative disease and contributes to the disease pathogenesis. However, with hundreds of target genes expressed in the brain, the mechanism of NFkB signaling transduction pathway is bearly understood.
We were interested in revealing the downstream mediators of NFkB in the brain and chose the IkBa-dificient model as our tool. IkBa is the essential negative regulator of NFkB activity and deletioon of IkBa induced NFkB hyperactivation. We used Nestin-Cre to conditionally knockout IkBa in the brain cells of neural lineage and performed the microarray to give us candidate genes.

Gene regulation profile of Medroxyprogesterone acetate (MPA)-treated late pregnant cervix

Noval and traditional signaling pathways involved in cervical ripening that were regulated by MPA were identified.
Keywords: Mouse, translational research

Integrative Transcriptome Analysis Reveals Common Molecular Subtypes of Human Hepatocellular Carcinoma (HT-HG_U133A)

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and prior attempts to develop genomic-based classification for HCC have yielded highly divergent results, indicating difficulty in identifying unified molecular anatomy. We performed a meta-analysis of gene expression profiles in data sets from eight independent patient cohorts across the world. In addition, aiming to establish the real world applicability of a classification system, we profiled 118 formalin-fixed, paraffin-embedded tissues from an additional patient cohort. A total of 603 patients were analyzed, representing the major etiologies of HCC (hepatitis B and C) collected from Western and Eastern countries. We observed three robust HCC subclasses (termed S1, S2, and S3), each correlated with clinical parameters such as tumor size, extent of cellular differentiation, and serum alpha-fetoprotein levels. An analysis of the components of the signatures indicated that S1 reflected aberrant activation of the WNT signaling pathway, S2 was characterized by proliferation as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation. Functional studies indicated that the WNT pathway activation signature characteristic of S1 tumors was not simply the result of beta-catenin mutation but rather was the result of transforming growth factor-beta activation, thus representing a new mechanism of WNT pathway activation in HCC. These experiments establish the first consensus classification framework for HCC based on gene expression profiles and highlight the power of integrating multiple data sets to define a robust molecular taxonomy of the disease.

Expression data from heart failure vs control peripheral blood mononuclear cells.

Inflammatory mediators play a role in the pathogenesis/progression of chronic heart failure (CHF). The aim of the present study was to identify diagnostic/prognostic markers and gene expression profiles of CHF vs control.
Keywords: CHF patients profiling vs controls.

Effect of H11 Kinase/Hsp22 deletion in response to cardiac stress

The expression of the small molecular weight heat shock protein (Hsp) H11 kinase/Hsp22 (Hsp22) is restricted to a limited number of tissues, including the heart and skeletal muscle, both in rodents and in humans. We generated a mouse knockout (KO) model, and investigated the role of Hsp22 in regulating cardiac hypertrophy in response to pressure overload. We compared gene expression profiles between WT and KO mice in basal condition and three days pressure overload after transverse aortic constriction (TAC). These data illustrated a novel mechanism of Hsp22-related gene expression in response to cardiac stress.

Recapitulation of human premature aging by using iPSCs from Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature aging disease1-5, characterized by premature atherosclerosis and degeneration of vascular smooth muscle cells (SMCs)6-8. HGPS is caused by a single-point mutation in the LMNA gene, resulting in the generation of progerin, a truncated mutant of lamin A. Accumulation of progerin leads to various aging-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin9-12. Here, we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature aging. Upon differentiation of HGPS-iPSCs, progerin and its associated aging consequences are restored. In particular, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescent SMC phenotypes associated with vascular aging. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs) as a component of the progerin-containing protein complex. The absence of nuclear DNAPKcs correlates with premature as well as physiological aging. Since progerin also accumulates during physiological aging6,12,13, our results provide an in vitro iPSC-based model with an acceleration progerin accumulation to study the pathogenesis of human premature and physiological vascular aging.

Gene expression associated with gemcitabine resistance and its reversal by bexarotene

Resistance of Calu3 NSCLC cells to the cytotoxic nucleoside analog gemcitabine (2',2'-difluorodeoxycytidine) can be prevented as well as reversed by the rexinoid X receptor selective agonist bexarotene.  This study was designed to investigate the changes in gene expression associated with gemcitabine resistance and its reversal by bexarotene.  In addition to the parental Calu3 cells and the 10 cycles of treatment of the gemcitabine resistant Calu3 cells with vehicle or bexarotene, analogous treatment paradigms with gemcitabine alone as well as the combination of both compounds have been included as controls.  (However, it has to be noted that in the combination treatment, cells that were re-sensitized by bexarotene have largely been removed from the culture before harvest due to the cytotoxic activity of gemcitabine.)
Keywords: cell type comparison, compound effects

Transcriptional Alterations of Hamstring Muscle Contractures in Children with Cerebral Palsy

Cerebral palsy is primarily an upper motor neuron disease that results in a spectrum of progressive movement disorders.  Secondary to the neurological lesion, muscles from patients with cerebral palsy are often spastic and form debilitating contractures that limit range of motion and joint function.  With no genetic component, the pathology of skeletal muscle in cerebral palsy is a response to aberrant neurological input in ways that are not fully understood.  This study was designed to gain further understanding of the skeletal muscle response to cerebral palsy using microarrays and correlating the transcriptional data with functional measures.  Hamstring biopsies from gracilis and semitendinosus muscles were obtained from a cohort of patients with cerebral palsy (n=10) and typically developing patients (n=10) undergoing surgery.  Affymetrix HG-U133A 2.0 chips (n=40) were used and expression data was verified for 6 transcripts using quantitative real-time PCR, as well as for two genes not on the microarray.  Chips were clustered based on their expression and those from patients with cerebral palsy clustered separately.  Significant genes were determined conservatively based on the overlap of three summarization algorithms (n=1,398).  Significantly altered genes were analyzed for over-representation among gene ontologies, transcription factors, pathways, microRNA and muscle specific networks.  These results centered on an increase in extracellular matrix expression in cerebral palsy as well as a decrease in metabolism and ubiquitin ligase activity.  The increase in extracellular matrix products was correlated with mechanical measures demonstrating the importance in disability.  These data lay a framework for further studies and novel therapies.

Expression data from mouse heart with and without GATA4 S105A mutation; with and without adrenergic stress

Serine 105 phosphorylation of GATA4 is necessary for stress-induced cardiac hypertrophy in vivo.
We used microarrays to detail global gene expression program alterations that arise in response to the ablation of the Gata4 S105 phosphorylation site in basal and adrenergic-stressed heart ventricles.

We expressed a constitutively active mutant of MEK5 (MEK5D) in human primary endothelial cells (EC) to study the transcriptional and functional responses to Erk5 activation under static conditions.

Pten loss and RAS/MAPK activation cooperate to promote EMT and prostate cancer metastasis initiated from stem/progenitor cells

PTEN loss or PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, in preclinical murine models, deletion of Pten alone fails to mimic the significant metastatic burden that frequently accompanies the end stage of human disease.  To identify additional pathway alterations that cooperate with PTEN loss in prostate cancer progression, we surveyed human prostate cancer tissue microarrays and found that the RAS/MAPK pathway is significantly elevated both in primary and metastatic lesions.  In an attempt to model this event, we crossed conditional activatable K-rasG12D/WT mice with the prostate conditional Pten deletion model we previously generated. Although RAS activation alone cannot initiate prostate cancer development, it significantly accelerated progression caused by PTEN loss, accompanied by epithelial-to-mesenchymal transition (EMT) and macrometastasis with 100% penitence. A novel stem/progenitor subpopulation with mesenchymal characteristics was isolated from the compound mutant prostates, which was highly metastatic upon orthotopic transplantation.  Importantly, inhibition of RAS/MAPK signaling by PD325901, a MEK inhibitor, significantly reduced the metastatic progression initiated from transplanted stem/progenitor cells.  Collectively, these data indicate that activation of RAS/MAPK signaling serves as a potentiating second hit to alteration of the PTEN/PI3K/AKT axis and co-targeting both pathways is highly effective in preventing the development of metastatic prostate cancers.

Expression data from GNMT knockout mice in Hepatocytes and OKER cell lines

Hepatocellular carcinoma (HCC) is the first cause of death in cirrhotic patients. Liver cirrhosis at a high risk of HCC has abnormal S-Adenosylmethionine (SAMe) levels. Catabolism of SAMe is mainly mediated by Glycine N-methyltransferase (GNMT), Lack in GNMT expression leads to epigenetic regulation of critical carcinogenesis pathways and mounting evidence assigns an essential role of GNMT in HCC. Methods: Here we have studied the role of LKB1-AMPK and RAS in the proliferation and transformation of GNMT-deficient HCC. Results. GNMT-deficient HCC was characterized by LKB1-AMPK misconnection, leading to resistance of apoptosis response mediated by a positive regulation of cAMP-PKA-CaMKKβ cascade. Additionally, Ras-mediated hyperactivation of LKB1 contributes to the proliferation of GNMT-deficient HCC in an ERK/p90RSK-dependent manner. The observed LKB1-induced Ras activation, was due to the regulation of RASGRP3 expression. Notably, the human HCC tumors with poorer prognosis showed the lowest levels of GNMT, p-AMPKα(Thr172) and the highest activation of Ras/LKB1/RASGRP3 axis. The present data suggest a correlation between LKB1 and RAS activity in a context of HCC with low GNMT expression, indicating that activation of the RAS/LKB1/RASGRP3 cascade might possess an important prognostic role in human liver cancer. Furthermore, these findings open the possibility to design new therapeutic strategies for the treatment of liver cancer.

Gene expression in different thymic stromal cells and subsets thereof was analyzed in 6-12 week old wild type (C57BL/6) and Aire knock-out (mixed background) mice. Thymic stromal cells were purified by sequential enzymatic digestion (collagenase, collagenase/dispase and trypsin) followed by gradient centrifugation and FACS sorting. Sort criteria were as follows: dendritic cells (CD11c+, F4/80 -), macrophages (F4/80+, CD11c-), cTECs (CD45–/lo, CDR1/Ly51+, Ep-CAM+) and mTECs (CD45–/lo, CDR1/Ly51–, Ep-CAM+). mTECs of wild-type and Aire knock-out mice were further subdivided according to CD80 expression levels.
For microarray analysis total RNA from thymic stromal cell samples of two independent experiments was pre-amplified and biotinylated by two rounds of cDNA synthesis and in vitro transcription. Fluorescence readings were evaluated by using Microarray Suite 5.0 software.

Alterations in DNA-methylation are a hallmark of human tumor cells. DNMT1 is expressed in three different splice variants and exhibits considerable preference for hemimethylated DNA and is therefore referred to as the maintenance methylase. DNMT3b is generally considered to be responsible for de novo methylation during embryonic development and probably also during tumor development. Recent studies using somatic knock-out of DNA-methyltransferases have shown a reduction in overall methylation and a re-expression of tumor suppressor genes. Cells deficient of both, DNMT1 and DNMT3b may exhibit almost complete demethylation and growth suppression. In the present study, we performed MEK-inhibition via U0126 treatment for the wildtype and every DNMT-knockout to focus on MEK/RAS-target genes regulated by methylation.

Genomic Strategies Identify the Antitumor Agent Apratoxin A as a Potent Antagonist of FGF Signaling and STAT3 Activation

Total RNA was extracted from apratoxin A or vehicle treated HT29 cells using the RNeasy Mini Kit (Qiagen).  Probe values from CEL files were condensed to probe sets using Rosetta Resolver software.  Resolver ANOVA analysis was then performed between groups.
Keywords: time course and dose

Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos obtained from heterozygous PKBa mice intercrosses (Yang et al., 2003). Briefly, after dissection of head and visceral organs for genotyping, embryos were minced and trypsinized for 30 min at 37°C. Embryonic fibroblasts were then plated and maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) (Life Technologies), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with wild-type and PKBa-/- MEFs between 15-20 passages. To induce adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with DMEM supplemented with 10% FCS, 8 mg/ml biotin, 4 mg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone and 10 mg/ml insulin (all from Sigma). Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer’s instructions.

IL-10 is an anti-inflammatory cytokine that has been shown to be produced by antigen-specific CD8 T cells at the peak of viral encephalitis. We found that IL-10+CD8 T cells are more activated and cytolytic than IL-10-CD8 T cells.
We used microarrays to detect gene expression changes in directly ex vivo sorted CNS IL-10+ and IL-10- CD8 T cells from a neurotropic J2.2-V-1-infected mouse.

Although HSF1 is known to play an important role in regulating the cellular response to proteotoxic stressors, little is known about the structure and function of the HSF1 signaling network under both stressed and unstressed conditions.  In this study, we used a combination of chromatin immunoprecipitation (ChIP) microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 comprehensively identify genes directly and indirectly regulated by HSF1 and examine the structure of the extended HSF1 signaling network. Correlation between promoter binding and gene expression was not significant for all genes bound by HSF1 suggesting that HSF1 binding per se is not sufficient for expression.  However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown allowing the identification of direct transcriptional targets of HSF1.  Among promoters bound by HSF1 following heat shock, a gene ontology (GO) analysis showed significant enrichment only in categories related to protein folding.  In contrast, analysis of the extended HSF1 signaling network showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitination and others, highlighting a complex transcriptional program directly and indirectly regulated by HSF1.
Keywords: Time Course, Heat Shock, siRNA

integrin alpha7 overexpression effects on skeletal muscle transcriptions

Analysis of integrin alpha7 transgenic mice skeletal muscle transcription profiles comparing to wild type controls. Integrin alpha7 is the major laminin binding integrin in muscle cells. Enhancing its expression has been demonstrated to alleviate pathology in a murine model of Duchenne muscular dystrophy. Results of this study provide insights into the effects of increasing integrin alpha7 expression on skeletal muscle transcription and physiology in vivo.  This analysis also evaluates any potential possible side effects associate with enhancing integrin alpha7 in skeletal muscle.
Keywords: transgenic mice comparision to wild type controls

Total Gene expression analysis of H3f3b constitutive knockout testis RNA

Total gene expression analysis was performed on RNA from testes extracted from two litters of constitutive homozygous and heterozygous H3f3b knockout mice compared to WT littermates.

STAT3 expression, activity and functional consequences of STAT3 inhibition in esophageal squamous cell carcinomas and Barrett’s adenocarcinomas

Signal transducer and activator of transcription 3 (STAT3) is altered in several epithelial cancers and represents a potential therapeutic target. Here, STAT3 expression, activity and cellular functions were examined in two main histotypes of esophageal carcinomas. In situ, immunohistochemistry for STAT3 and STAT3-Tyr705 phosphorylation (P-STAT3) in esophageal squamous cell carcinomas (ESCC) and Barrett’s adenocarcinomas (BAC) revealed similar STAT3 expression in ESCCs and BACs, but preferentially activated P-STAT3 in ESCCs. In vitro, strong STAT3 activation was seen by EGF-stimulation in OE21 (ESCC) cells, whilst OE33 (BAC) cells showed constitutive weak STAT3 activation. STAT3 knockdown significantly reduced cell proliferation of OE21 and OE33 cells and reduced cell migration in OE33, but not in OE21 cells. Transcriptome analysis identified STAT3-knockdown associated down-regulation of cell cycle processes and the selective down-regulation of cyclins and cyclin dependent kinaes  associated genes in both OE21 and OE33 cells. Moreover, the transcriptome response showed changes in cell migration/invasion related genes that correlated with the associated phenotype measurements.  This study demonstrates the importance of STAT3 expression and activation in esophageal carcinomas, whereby the extent differs between ESCCs and BACs. STAT3 knockdown significantly reduces cell proliferation in both types of esophageal cancer cells and inhibits migration in BAC cells. Thus, STAT3 may be further exploited as potential novel therapeutic target for esophageal cancers.

REST has been initially described as a repressor of neuronal genes in non-neuronal cells by binding to its recognition sequence RE1. Over-activation of this factor has been shown in several diseases such as Huntington disease or central nervous system cancers. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to the identification of one benzoimidazole-5-carboxamide derivative (X5050) that inhibited REST silencing in a RE1-dependent manner.
Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST.

Expression Profiling of Mixed Lineage Leukemia Cells Treated with a Potent Small-Molecule DOT1L Inhibitor

Cell lines bearing MLL translocations (MV4-11 and MOLM-13) were treated with a potent, selective inhibitor of the DOT1L histone methyl transferase.  Treatment of MLL-rearranged cell lines with the DOT1L inhibitor selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes.  Here we provide expression profiling data of cells treated with DOT1L inhibitor or vehicle control.

breast cancer;class prediction;doxorubicin;etoposide;5-fluorouricil;gene expression;microarrays;dose response

Prediction of toxicant-specific gene expression signatures following chemotherapeutic treatment of breast cell lines

Global gene expression profiling has demonstrated that the predominant cellular response to a range of toxicants is a general stress response.  This stereotyped environmental stress response commonly includes repression of protein synthesis and cell cycle regulated genes and induction of DNA damage and oxidative stress responsive genes.  Our laboratory has recently characterized the general stress response of breast cell lines derived from basal-like and luminal epithelium following treatment with doxorubicin (DOX) or 5-fluorouracil (5FU) and showed that each cell type has a distinct response.  However, we expected that some of the expression changes induced by DOX and 5FU were unique to each compound and might reflect the underlying mechanisms of action of these agents. Thus, we employed supervised analyses (Significance Analysis of Microarrays) to identify genes that showed differential expression between DOX- and 5FU-treated cell lines. We then used cross-validation analyses and identified genes that afforded high predictive accuracy in classifying samples into the two treatment classes.  To test whether these gene lists had good predictive accuracy in an independent data set, we treated our panel of cell lines with etoposide, a compound that is mechanistically similar to DOX.  We demonstrated that using expression patterns of 100 genes, we were able to obtain 100% predictive accuracy in classifying the etoposide samples as being more similar in expression to DOX- than 5FU-treated samples. These analyses also showed that toxicant specific gene expression patterns, similar to general stress responses, vary according to cell type.
Keywords = breast cancer

Molecular characterization of PERK's role in the mouse exocrine pancreas.  Postnatal days 16, 19 and 32 were examined.  Postnatal day 16 represents a stage immediately prior to severe cytological changes in the exPKO mice.  Postnatal day 19 represents the initial stage of severe phenotypes in the exPKO mice.  Postnatal day 32 represents the middle stage of severe phenotypes (about 2 wks from the initiation) in the exPKO mice.
Keywords: transgenic mouse

Altered transcriptome in a motor system spared by ALS: protective vs. adaptive strategies

Amyotrophic lateral sclerosis (ALS) spares the ocular motor system. In this study, we tested the hypothesis that the oculomotor neurons are intrinsically protected in ALS. Using high-density cDNA microarrays, we examined the transcriptome of oculomotor nuclei and spinal cords in mice expressing a human mutant SOD1, the SOD1(G93A) ALS model, at 6 and 10 weeks of age. Comparison of gene expression profiles of these pre-symptomatic SOD1(G93A) mice showed a shift to a proapoptotic state in spinal cords, while the opposite was true in oculomotor nuclei. Seventeen members of the A, B, C and D Hox clusters increased in oculomotor nuclei from 6 to 10 weeks of age; 15 were downregulated in spinal cord. Although only the first 4 classes of a given Hox cluster (e.g., Hoxa1-4) are normally expressed in the developing hindbrain, we found differential expression of mostly the latter classes in both oculomotor nuclei and spinal cords. Also, semaphorin 3B was expressed at 28-fold greater levels in oculomotor nuclei and 61-fold less in spinal cords in 10-week old SOD1(G93A) mice compared to 6-week old mice. Semaphorins 3A and 3E were also differentially regulated. Comparison of gene expression profiles of control SOD1 mice of 6 and 10 weeks of age did not show these changes. Based on these results, we rejected our hypothesis and conclude that the oculomotor nuclei actively adapt to the ALS-inducing mutation.
Supported by NEI and ALSA.

To study effect of niacin bound chromium in changing genetic profile of subcutaneous fat from diabetic mice
Keywords: nutritional supplemetation, obesity

Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors.
This study is described more fully in Perou CM, et al. 1999. Proc Natl Acad Sci USA 96:9212-7

Comparison of mammary glands of 16 wk old wild type and LH-overexpressing mice

LH-overexpressing mice display elevated levels of mammogenic hormones and consequently develop spontaneous mammary tumors with a median latency of 41 weeks. Tumors occur in the absence of p53 mutations and in the presence of a seemingly functional p53 signaling pathway. At 16 weeks of age, no histological evidence of tumors is observed in these transgenic mammary glands; however, mammary cells do demonstrate centrosome amplification at this age suggesting that early tumorigenic processes may have occured. Thus comparison of the transcriptome of the mammary glands from 16 week old LH-overexpressing mice to that of wild type mice will provide insight into early events occuring in p53-independent hormone-induced tumorigenesis.

This project investigates the mechanisms that underlie asthma-related phenotypes associated with allergen exposure. See http://www.hopkins-genomics.org/asthma/asthma006/index.html

The goal of the experiment was to identify genes that change their levels of expression as a consequence of challenge with allergen. The experiments utilized a murine model of human asthma that employed BALB/CJ mice. In this particular sensitization-challenge protocol, the response produced by BALB/CJ mice is representative of a majority of the mouse strains tested. The antigen complex used to sensitize and challenge the mice was ragweed pollen an allergen that is highly relevant to human disease.

Serum Response Factor (SRF) is a transcriptional regulator required for mesodermal development. Numerous studies have implicated SRF as a central regulator of muscle gene expression and myogenesis. In this present study we used a loss of function approach to delineate the role of SRF in cardiac myocyte gene expression and function. In SRF null neonatal cardiomyocytes, we observe severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric proteins. Consistent with this, gene array and RT-PCR analyses show downregulation of genes encoding key cardiac transcriptional regulatory factors and proteins required for the maintenance of sarcomeric structure, function, and regulation. Chromatin IP analysis reveals that at least a subset of these proteins are likely regulated directly by SRF. Together the results presented here reveal new cellular and genetic mechanisms through which SRF exacts control over the contractile apparatus in cardiac myocytes.

Expression data from human Ishikawa cells treated with Bisphenol A

This study provides a comprehensive evaluation of changes in gene expression during treatment with Bisphenol A in vitro.
Ishikawa cells were maintained and grown (to confluency or at a desired cell density between 500,000 - 1 million cells/mL) in DMEM/F-12 Medium (supplemented with 10% Fetal Bovine Serum + 1X penicillin/streptomycin) in xenoestrogen-free Corning plasticware.  Cells were gently washed in warm PBS and transferred to phenol red-free DMEM/F-12 Medium (supplemented with 10% Charcoal-Stripped Fetal Bovine Serum + 1X penicillin/streptomycin) in Corning Cell Culture Cluster wells overnight and then challenged with 1pM (very low, vL), 100pM (low, L), 10nM (high, H), and 1uM (very high, vH) levels of BPA.  Prior to collection, cells were washed in warm PBS, resuspended & briefly incubated in TRI-Reagent, & finally collected (in quintuplicate replicates) at each specific time point: 8 hours, 24 hours, and 48 hours.  Following RNA isolation, the best RNA yields for each quadruplicate set was selected for target preparation and GeneChip processing.

Direct Recruitment of Polycomb Repressive Complex 1 (PRC1) to Chromatin by Core Binding Transcription Factors (microarray)

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFbeta transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate considerable chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFbeta and functional regulation of a significant fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFbeta deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

Novel genes associated with malignant melanoma but not benign melanocytic lesions

A number of melanoma specific genes were identified to differentiate clinical relevant tissue samples containing benign from malignant melanocytes.

Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/ß-catenin activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/ß-catenin transcriptional program and tumourigenesis in the gut epithelium. The mechanisms by which TCF4/ß-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the Germinal centre kinase family, as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a ß-catenin dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential role of the kinase activity in Wnt target gene activation. siRNA depletion of TNIK followed by expression array analysis demonstrated that TNIK is an essential and exclusive activator of Wnt induced transcriptional program. As an essential component in the TCF4/ß-catenin activator complex, the kinase TNIK may present an attractive candidate for drug targeting in colorectal cancer.

Remission in Juvenile Idiopathic Arthritis Achieved on Different Drug Regimens

Remission has become both the gold standard for clinical care and the end point for clinical trials for children with juvenile idiopathic arthritis (JIA). Using gene expression microarrays, we found that when remission induced by methotrexate (MTX) or MTX plus a TNF inhibitor (etanercept, Et) (MTX+Et) was compared with healthy controls, there were notable differences in gene expression patterns, demonstrating that remission is not a restoration of immunologic normalcy. Differences were detected in PBMC as well as in granulocytes.

Expression data comparing the effect of IKK2 KO on a KRAS mouse model

Given that the NF-κB pathway plays an important role in tumor development and that IKK2 is the seminal kinase responsible for NF-κB pathway activation, we were particularly interested in exploring the therapeutic potential of IKK2 inhibition in non-small cell lung cancers.
We crossed IKK2fl/fl and KrasG12D mice so that we could simultaneously activate KrasG12D to initiate tumor formation and inactivate IKK2, upon transduction by Cre lentiviral vectors.

Expression data from control and LRF (leukemia/lymphoma related factor)-deficient mouse LT-HSCs

LRF, which is encoded by the ZBTB7A gene and formerly known as POKEMON (POK erythroid myeloid ontogenic factor), was originally identified as a PLZF (promyelocytic leukemia zinc finger) homologue interacting with BCL6 (B-cell lymphoma 6). LRF is a transcription factor that is broadly expressed in hematopoietic lineage cells, but its expression is particularly high in erythroblasts and germinal center (GC) B-cells. The goal of this study is to assess the effect of LRF loss on the LT-HSC transcriptome. Nine days after injection of adult mice with polyinosinic polycytidylic acid (pIpc) to activate Cre, total RNAs were isolated from double-sorted LT-HSCs from LRF Flox/+ Mx1-Cre+ and LRF Flox/Flox Mx1-Cre+ mice and processed for microarray analysis.

The transcription factor MyoD can coax na?e fibroblasts or otherwise committed cells to adopt the skeletal muscle phenotype by activating the muscle gene expression program. Activation of muscle gene expression occurs in quantal steps with not all the target genes of MyoD being activated at the same time. Some genes are induced in the initial phases, others at later stages despite the fact that MyoD is present throughout the differentiation process. MyoD is post-translationally modified by phosphorylation, ubiquitination, and acetylation. Here, we have employed a model system in which MyoD and its non-acetylatable version were inducibly expressed in mouse embryonic fibroblasts derived from mice to investigate how MyoD acetylation may contribute to differential gene activation.

Deregulated expression of the Myc transcription factor is a frequent causal mutation in human cancer. Thousands of putative Myc target genes have been identified in in vitro studies, indicating that Myc exerts highly pleiotropic effects within cells and tissues. However, the complexity and diversity of Myc gene targets has confounded attempts at identifying which of these genes are the critical targets mediating Myc-driven tumorigenesis in vivo. Acute activation of Myc in a reversibly switchable transgenic model of Myc-mediated β cell tumorigenesis induces rapid tumor onset whereas subsequent Myc de-activation triggers equally rapid tumor regression. Thus, sustained Myc activity is required for tumor maintenance. We have used this reversibly switchable kinetic tumor model in combination with high-density oligonucleotide microarrays to develop an unbiased strategy for identifying candidate Myc-regulated genes responsible for maintenance of Myc-dependent tumors. Consistent with known Myc functions, some Myc-regulated genes are involved in cell growth, cycle and proliferation. In addition, however, many Myc-regulated genes are specific to β cells, indicating that a significant component of Myc action is cell-type specific. Finally, we identify a very restricted cadre of genes whose expression is inversely regulated upon Myc activation-induced tumor progression and de-activation-induced tumor regression. By definition, such genes are candidates for tumor maintenance functions. Combining reversibly switchable, transgenic models of tumor formation and regression with genomic profiling offers a novel strategy with which to deconvolute the complexities of oncogenic signaling pathways in vivo
Keywords: in vivo kinetic analyis myc function

Gene expression profiling of in vitro derived induced and natural FOXP3+ regulatory T cells and ex-iTreg cells in the mouse

Induced Treg (iTreg) cells are essential for tolerance and can be used therapeutically, yet their stability in vivo and mechanisms of suppression are unresolved. Here, we used a treatment model of colitis to examine the role of autologous IL-10 in iTreg cell function.  Mice treated with IL-10+/+ iTreg cells in combination with IL-10–/– natural Treg (nTreg) cells survived and gained weight, even though iTreg cells were numerically disadvantaged and comprised just ~20% of all Treg cells in treated mice.   Notably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome which failed to limit their pathogenic potential. The TCR repertoires of iTreg and ex-iTreg cells exhibited almost no overlap, which indicates that the two populations are clonally unrelated and maintained by different selective pressures.  These data demonstrate a potent and critical role for iTreg cell produced IL-10 that can supplant the IL-10 produced by nTreg cells and compensate for the inherent instability of the iTreg population.

Expression data from Adipose Stem Cells (ASC) from morbidly obese and non-obese individuals

The adipose tissue is an endocrine regulator and a risk factor for atherosclerosis and cardiovascular disease when by excessive accumulation induces obesity. Although the adipose tissue is also a reservoir for stem cells (ASC) their function and “stemcellness” has been questioned. Our aim was to investigate the mechanisms by which obesity affects subcutaneous white adipose tissue (WAT) stem cells.
We used microarrays to analyze differences in transcriptomic profiles between the adipose stem cells from morbidly obese and non-obese individuals.

RhoGDIbeta (ARHGDIB) is often expressed in tumor cells. It negatively regulates Rho-GTPases, but may have other functions as well. To analyze its effect on gene expression, RhoGDIbeta was suppressed by RNA interference in MDA-MB-231 breast cancer cells and changes in gene expression monitored by cDNA microarrays.
Keywords: siRNA knock-down analysis

In rodent models, phthalate exposure alters both the fetal and pubertal testis, but the resulting histopathological changes are divergent.  This suggests that the underlying molecular and cellular phthalate mechanism may be age-dependent.  Using genome-wide expression profiling of acutely-exposed rats, the initial molecular response in pubertal rat testis following in vivo phthalate exposure was determined. For this study, postnatal day 28 rats were exposed to a single dose of 1 g/kg mono-(2-ethyl)hexyl phthalate (MEHP) and assayed at 1, 2, 3, 6, and 12 hrs thereafter using Affymetrix Rat Genome 230 2.0 Arrays.
Keywords: Time course, single dose

Identification of Genes Responsive to the Expression of Normal Human CFTR
Keywords: genotype comparison

OTX2 drives medulloblastoma proliferation via direct regulation of cell cycle genes and inhibits differentiation

The transcription factor OTX2 has been implicated as an oncogene in medulloblastoma, which is the most common malignant brain tumor in children. It is highly expressed in most medulloblastomas and amplified in a subset of them. The role of OTX2 in medulloblastoma and its downstream targets are unclear. Therefore, we generated D425 medulloblastoma cells in which we can silence endogenous OTX2 by inducible shRNA. Silencing of OTX2 strongly inhibited cell proliferation and resulted in a neuronal-like differentiation. Expression profiling of time courses after silencing showed a progressive change in gene expression for many cellular processes. Down regulated genes were highly enriched for cell cycle and visual perception genes, while up regulated genes were enriched for genes involved in development and differentiation. This shift in expression profiles is reminiscent to changes described to occur during normal cerebellum development. OTX2 is expressed in proliferating granular progenitor cells, but the expression diminishes when these cells exit the cell cycle and start differentiating. ChIP-on-chip analyses of OTX2 in D425 cells showed that cell cycle and perception genes were direct OTX2 targets, while regulation of most differentiation genes appears to be indirect. These analyses provide the first insight in the molecular network of OTX2, demonstrating that OTX2 is essential in medulloblastoma and directly drives proliferation by regulating the expression of cell cycle genes. Since many of these genes also correlate in expression with OTX2 in primary tumors, they might be potential targets for therapy in medulloblastoma patients.
Keywords: OTX2, medulloblastoma, mRNA profiling

Gene expression profile in melanomas from Pdk1-inactivated BrafV600E::Pten-/- mice

Gene expression profiling was performed to access the changes in gene expression in melanomas from Pdk1-inactivated Brafv600E::Pten-/- mice.  The expression profiles of the BrafV600E::Pten-/-::Pdk1-/- were compared to the BrafV600E::Pten-/-::Pdk+/+ genotypes.  The analysis has identified several important signaling pathways in Pdk1-dependent melanomagenesis.

Cyclin D1 determines estrogen dependent signaling in human breast cancer cell line MCF7

The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein (pRb). It is known that cyclin D1 regulates ERα transactivation using heterologous reporter systems, the significance of this observation to E2 dependent gene activation is unknow. E2 stimulated MCF7 cells treated with cyclin D1 siRNA in order to analyze the genes regulated by estradiol in a cyclin D1 dependent manner. Hormone deprived MCF7 cells were treated with cyclin D1 siRNA or control siRNA and stimulated with E2 or vehicle

Immature cell populations and an erythropoiesis gene expression signature in systemic juvenile idiopathic arthritis: Implications for pathogenesis

Objective. Previous observations suggest that active systemic juvenile idiopathic arthritis (sJIA) is associated with a prominent erythropoiesis gene expression signature. The aim of this study was to determine the association of this signature with peripheral blood mononuclear cell (PBMC) subpopulations and its specificity for sJIA as compared to related conditions.

125 patients with JIA (18 sJIA and 107 non-sJIA) and 29 controls were studied. PBMC were isolated and analyzed for multiple surface antigens by flow cytometry and for gene expression profiles. The proportions of different PBMC subpopulations were compared among sJIA, non-sJIA patients and controls and subsequently correlated with the strength of the erythropoiesis signature. Additional gene expression data from patients with familial hemophagocytic lymphohistiocytosis (FHLH) and from a published sJIA cohort were analyzed to determine if the erythropoiesis signature was present.

Aryl Hydrocarbon Receptor Regulates Distinct Dioxin-Dependent and Dioxin-Independent Gene Batteries

Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expression arrays to contrast mRNA profiles in Ahr-null mice (Ahr–/–) with those in mice with wild-type AHR (Ahr+/+). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 µg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahr–/– mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor alpha as well as two estrogen-receptor-related genes (alpha and gamma) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD.
Keywords: treatment-control and mutant-wildtype

Analysis of gene expression in esophageal squamous cell carcinoma (ESCC)

To characterize gene expression in esophageal squamous cell carcinoma, we examined gene expression in tumor and matched normal adjacent tissue from 17 ESCC patients from a high-risk region of China.

brain expression data from adult mice prenatally exposed to ethanol

Moderate alcohol consumption during pregnancy can result in a heterogeneous range of neurobehavioural and cognitive effects, termed fetal alcohol spectrum disorders (FASD).  We have developed a mouse moder of FASD that involves moderate ethanol exposure throughout gestation achieved by voluntary maternal consumption.  This model results in phenotypes relevant to FASD.  Since ethanol is known to directly affect the expression of genes in the developing brain leading to abnormal cell death, changes to cell proliferation, migration, and differentiation, and potential changes to epigenetic patterning, we hypothesize that this leaves a long-term footprint on the adult brain.  However, the long-term effects of prenatal ethanol exposure on brain gene expression, when behavioural phenotypes are apparent, are unclear.
We used two independent microarray experiments and focused on the genes identified by both to evaluate the genome-wide alterations to the adult brain transcriptome caused by prenatal ethanol exposure via moderate maternal drinking.

Gene expression profiles of fibroblasts and fibroblast-reprogrammed induced pluripotent stem cells (iPSCs) from childhood cerebral adrenoleukodystrophy patients and healthy controls

Although not an affected cell type, skin fibroblasts from individuals with CC-ALD, an early onset X-linked neurological disorder, show defects in very long chain fatty acid (VLCFA) metabolism that provide the basis for clinical diagnostic tests. Skin fibroblasts from CC-ALD patients can be reprogrammed into iPS cells with all the hallmark properties of pluripotency. The iPS cell phenotypes may reflect the tissue-specificity of the lipid metabolic defects found in CC-ALD patients.
We report the gene expression profiles of fibroblasts and fibroblast-reprogrammed iPSCs from childhood cerebral adrenoleukodystrophy patients and healthy controls

Gene expression profiles of fibroblasts from childhood cerebral adrenoleukodystrophy patients and healthy controls

Although not an affected cell type, skin fibroblasts from individuals with childhood cerebral adrenoleukodystrophy (CCALD), an early onset X-linked neurological disorder, show defects in very long chain fatty acid (VLCFA) metabolism that provide the basis for clinical diagnostic tests.
We report the gene expression profiles of fibroblasts from childhood cerebral adrenoleukodystrophy patients and healthy controls

Clontech Atlas Stress array of rat distal colon of rats fed on vitamin E sufficient or deficient diets.

Characterization of differential gene expression in adrenocortical tumors harbouring β-catenin  (CTNNB1) mutations.

Mutations of β-catenin gene (CTNNB1) are frequent in adrenocortical adenomas (AA) and carcinomas (ACC). However, the target genes of CTNNB1 have not yet been identified in adrenocortical tumors.
Our objective was to identify genes de-regulated in adrenocortical tumors harbouring CTNNB1 genetic alterations.

Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction.  However, presence of LDs in heart is also observed in physiological conditions, such as at times when cellular energy needs and energy production from mitochondria fatty acid (FA) β-oxidation are high (fasting).  This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function.  To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific plin5 over-expression (MHC-plin5), a member of the perilipin protein family. Hearts from MHC-plin5 mice expressed at least 4-fold higher levels of plin5 and exhibit a 3.5- fold increase in triglyceride content versus non-transgenic littermate.  Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of PPARα target genes, decreased mitochondria function and left ventricular concentric hypertrophia.  Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative induced genes via NF-E2-related factor 2 anti-oxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and promotes cardiac steatosis without major heart function impairment.

Whole-exome sequencing identifies mutations of BCOR in acute myeloid leukemia with normal karyotype

Among acute myeloid leukemias (AML) with normal karyotype (CN-AML), NPM1 and CEBPA mutations define WHO provisional entities accounting for ~60% of cases, but the remaining ~40% remains poorly characterized. By whole exome-sequencing (WES) of one CN-AML patient lacking mutations in NPM1, CEBPA, FLT3, MLL-PTD and IDH1, we newly identified a clonal somatic mutation in BCOR (BCL6 co-repressor), a gene located in chromosome X. Further analyses showed that BCOR mutations occurred in 11/262 (4.2%) CN-AML cases and represented a substantial fraction (14/82, 17.1%) of CN-AML patients showing the same genetic background as the index patient subjected to WES.
BCOR somatic mutations were: i) disruptive events similar to germline BCOR mutations causing the oculo-cranio-facial-dental (OCFD) genetic syndrome; ii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iii) almost mutually exclusive with NPM1 mutations and frequently associated with DNMT3A and RUNX1 mutations, pointing to a cooperation between these events. Finally, BCOR mutations correlated with poor outcome among a cohort of 160 CN-AML patients (28% versus 66% overall survival at 2 yrs, P=0.024). Our results implicate for the first time BCOR in the pathogenesis of CN-AML without NPM1 mutations.

DUX4 activates germline genes, retroelements and immune-mediators: Implications for facioscapulohumeral dystrophy

Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.

Contribution of Nuclear and Extranuclear PolyQ to Neurological Phenotypes in Mouse Models of Huntington’s Disease

To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization (NLS) or nuclear export sequences (NES) have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for, and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggests that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.
Keywords: Trangenic mouse brain gene expression

Gene Expression Profiling of Familial and Sporadic Cases of Interstitial Pneumonia

Rationale: Idiopathic interstitial pneumonia (IIP) and its’ familial variants are progressive and largely untreatable disorders with poorly understood molecular mechanisms.  Both the genetics and the histologic type of IIP play a role in understanding the etiology and pathogenesis of interstitial lung disease, but transcriptional signatures of these subtypes are unknown.
Objectives: To evaluate gene expression in the lung tissue from patients with usual interstitial pneumonia (UIP) and non-specific interstitial pneumonia (NSIP) that were either familial or non-familial in origin and compare them to gene expression from normal lung parenchyma.
Methods: We profiled RNA from lungs of 16 patients with sporadic IIP, 10 with familial IIP, and 9 normal controls on a whole human genome oligonucleotide microarray.
Results:  Significant transcriptional differences exist in familial and sporadic IIPs.  The genes distinguishing the genetic subtypes belong to the same functional categories as transcripts that distinguish IIP from normal samples. Relevant categories include chemokines and growth factors and their receptors, complement components, genes associated with cell proliferation and death, and genes in the Wnt pathway.

Intestinal gene expression in ENU mutagenesis mouse strains with missense mutations in Muc2 mucin and ER stress

MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier.  MUC2 homo-oligomerizes intracellularly into large secreted polymers which give mucus its viscous properties. The inflammatory bowel disease (IBD) ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, whereas goblet cells and the mucus layer are increased in the other major inflammatory bowel disease, Crohn’s disease.

Methods and Findings
By murine N-ethyl-N-nitrosourea-mutagenesis we identified two distinct non-complementing missense mutations in Muc2 exons encoding N- and C-terminal homo-oligomerization domains causing an ulcerative colitis-like phenotype.  Both strains developed mild spontaneous distal intestinal inflammation, chronic diarrhea, rectal bleeding and prolapse, increased susceptibility to acute and chronic colitis induced by a luminal toxin, aberrant Muc2 biosynthesis, smaller goblet cell thecae (less stored mucin) and a diminished mucus barrier. Enhanced local production of IL-1beta, TNF-alpha and IFN-gamma was seen in the distal colon.  The number of leukocytes within mesenteric lymph nodes was increased five-fold and leukocytes cultured in vitro produced both Th1 and Th2 cytokines (IFN-gamma, TNF-alpha and IL-13). Intestinal permeability was increased and the luminal bacterial flora were more heavily coated with immunoglobulin as occurs in IBD. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis and wound repair. Expression of mutated Muc2 oligomerization domains in vitro demonstrated that aberrant Muc2 oligomerization underlies the ER stress. These models show that mutations in Muc2 oligomerization domains can lead to aberrant assembly of the Muc2 complex leading to ER stress, a depleted mucus barrier and intestinal inflammation.  In ulcerative colitis we demonstrate similar accumulation of non-glycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in non-inflamed intestinal tissue.
The observations that mucin misfolding and ER stress lead directly to intestinal inflammation and that ER stress and goblet cell pathology occur in ulcerative colitis suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of colitis.

LLGL1 depletion effect on pancreatic ductal adenocarcinoma cell line CAPAN2

Identification of gene regulated by LLGL1 in pancreatic ductal adenocarcinoma cell line CAPAN2

Expression data from isolated terminal end buds in mammary glands with or without Ovol2 deletion

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition or EMT). Recent studies suggest that EMT endows differentiated epithelial cells with stem cell traits, posing the interesting question of how epithelial plasticity is properly restricted to ensure epithelial differentiation during tissue morphogenesis. Here we identify zinc-finger transcription factor Ovol2 as a key suppressor of EMT of mammary epithelial cells. Epithelia-specific deletion of Ovol2 completely arrests mammary ductal morphogenesis, and depletes epithelial stem/progenitor cell reservoirs. Further, Ovol2-deficient epithelial cells undergo EMT in vivo to become non-epithelial cell types, and that Ovol2 directly represses key EMT inducers such as Zeb1 and regulates stem/progenitor cell responsiveness to TGF-beta. We also provide evidence for a suppressive role of Ovol2 in breast cancer progression. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity to balance stemness with epithelial differentiation in development and cancer.

This study compared the expression signatures of HL60 cells +/- tretinoin.  This data was then used for another study showing a method for high-throughput gene expression signature analysis.
Keywords: expression analysis

Gene expression in RNP autoantibody+ systemic lupus erythematosus (SLE) patient blood

The goal of this study was to characterize gene expression profiles in RNP autoantibody+ SLE versus healthy blood donors with a focus on select cytokines that may be important in B cell activation and differentiation, including BAFF, IL-21, and IL-33. We utilized Affymetrix microarrays to characterize the global program of gene expression in the SLE patients, and to identify differentially expressed genes in patients compared to healthy controls. We examined a cohort of 79 consecutive patients classified as anti-ribonuclear protein (anti-RNP)+ systemic lupus erythematosus (SLE). All patients provided RNA samples obtained after providing informed consent. There were 73 female and 6 male subjects. Disease duration ranged from 0 to 453 months with a median of 37.5 months. SLE Disease Activity Index (SLEDAI) ranged from 0 to 31 with a median of 6.

Effective Targeting of Quiescent Chronic Myelogenous Leukemia Stem Cells by Histone Deacetylase Inhibitors in Combination with Imatinib Mesylate

We investigated the ability of HDAC inhibitors (HDACi) to target CML stem cells. Treatment with HDACi combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone, and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACi with IM markedly diminished LSC in a transgenic mouse model of CML. The interaction of IM and HDACi inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents a novel and effective strategy to target LSC in CML patients receiving tyrosine kinase inhibitors.

Identification of genes whose expression is modulated by the presence or absence of PTEN

In an effort to identify genes whose expression is regulated by activated PI3K signaling, we performed microarray analysis and subsequent qRT-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program.  Western blots revealed that p53 was stabilized in HCT116 PTEN-/- cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescent-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic but not wild-type PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on PIP3-induced mitogenesis during human cancer pathogenesis.
Keywords: PTEN genotype comparison

Whole genome microarray of  T lymphoma invasion and metastasis (Tiam1) silenced retinoblastoma Y79 cells

We hypothesized that Tiam1 is involved in invassiveness of retinoblastoma. The fuctional role of Tiam1 in cell progression and metastasis was tested by siRNA mediated knockdown of Tiam1 in retinoblastoma Y79 cells. The genes de-regulated in response to Tiam1 knockdown was analysed by cDNA microarray in which most of the actin cytoskeleton regulation proteins and apoptotic proteins were de-regulated. our results prove that Tiam1 modulates actin cytoskeleton and cell invasion in retinoblastoma.

Prediction of left ventricle systolic dysfunction in mice using gene expression profiling

We tested the hypothesis that a set of differentially expressed genes could be used to predict cardiovascular phenotype in mice after prolonged catecholamine stress.

Glioblastoma multiforme is the most common and aggressive form of brain cancer. The use of oncolytic HSV-1 (oHSV) to selectively target brain cancer cells leading to their lytic destruction has shown to be very promising in a preclinical setting, but is lacking efficacy in clinical trials. Cyr61, a secreted extracellular matrix protein which functions to promote angiogenesis, migration, proliferation and tumorigenesis, was found to be upregulated rapidly following oHSV infection. Here we show, using microarray analysis, that Cyr61 expression leads to the induction of several genes with type 1 interferon function. We show that Cyr61 mediated type 1 IFN induction is through its interaction with integrin alpha6beta1 on the cell surface and results in oHSV inhibition, reducing the efficacy of this therapy.
We used microarray to detail the global program of gene expression underlying Cyr61 mediated oncolytic HSV-1 inhibition and identified distinct classes of up-regulated genes during this process.

Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [Expression]

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells.

Gene profiling of the heart myocardium after a single dose administration of Isoproterenol hydrochloride

Gene expression was evaluated in the myocardium of male Wistar rats after a single sc administration of 0.5 mg of isoproterenol, a β-adrenergic agonist that causes acute tachycardia with subsequent myocardial necrosis. Histology of the heart, clinical chemistry and hematology were evaluated at 9 time-points (0.5 hr to 14 d post-injection). Myocardial gene expression was evaluated at 4 time-points (1 hr to 3 d). Contraction bands and loss of cross striation were identified on phosphotungstic acid-hematoxylin-stained sections 0.5 hr post-dosing. Plasma troponin I elevation was detected at 0.5 hr, peaked at 3 hr, and returned to baseline values at 3 d post-dosing. Interleukin 6 (Il6) expression spiked at 1-3 hr and was followed by a short-lived time-dependant dysregulation of its downstream targets. Concurrently and consistent with the kinetics of the histologic findings, many pathways indicative of necrosis/apoptosis (p38 MAPK signaling, NF-kB signaling) and adaptation to hypertension (PPAR signaling) were over-represented at 3 hr. The 1 d and 3 d time-points indicated an adaptative response, with down-regulation of the fatty acid metabolism pathway, up-regulation of the fetal gene program, and superimposed inflammation and repair at 3 d. These results suggest early involvement of Il6 in isoproterenol-induced myocardial necrosis and emphasize the value of early time-points in transcriptomic studies
Keywords: time course study of Isoproternol hydrochloride

Twelve weeks expression data of the antipsychotics Clozapine and Haloperidol in the mouse brain (Affymetrix, GCRMA).

Atypical antipsychotic Clozapine has a superior antipsychotic and antimanic effects compared to other antisphicotics. Its widespread use was limited by the side effects of agranulocytosis, cardiomyopathy and metabolic anomalies such as weight gain, and diabetes. Very little is known about mechanisms by which Clozapine works. The aim of this experiment is to compare the chronic gene expression profile of the atypical antipsychotic Clozapine to the typical antipsychotic drug Haloperidol using gene expression Microarray in order to understand the intercellular mechanism behind the therapeutic and the toxic effects of Clozapine.

The Functional Consequences of Variation in Transcription Factor Binding

One goal of human genetics is to understand how the information for precise and dynamic gene expression programs is encoded in the genome. The interactions of transcription factors (TFs) with DNA regulatory elements clearly play an important role in determining gene expression outputs, yet the regulatory logic underlying functional transcription factor binding is poorly understood. Many studies have focused on characterizing the genomic locations of TF binding, yet it is unclear to what extent TF binding at any specific locus has functional consequences with respect to gene expression output. To evaluate the context of functional TF binding we knocked down 59 TFs and chromatin modifiers in one HapMap lymphoblastoid cell line. We then identified genes whose expression was affected by the knockdowns. We intersected the gene expression data with transcription factor binding data (based on ChIP-seq and DNase-seq) within 10 kb of the transcription start sites of expressed genes. This combination of data allowed us to infer functional TF binding. Using this approach, we found that only a small subset of genes bound by a factor were differentially expressed following the knockdown of that factor, suggesting that most interactions between TF and chromatin do not result in measurable changes in gene expression levels of putative target genes. We found that functional TF binding is enriched in regulatory elements that harbor a large number of TF binding sites, at sites with predicted higher binding affinity, and at sites that are enriched in genomic regions annotated as “active enhancers”.

Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth

Alteration of the PTEN/PI3K pathway is associated with late stage and castrate resistant prostate cancer (CRPC).  However, how PTEN loss involves in CRPC development is not clear.  Here we show that castration-resistant growth is an intrinsic property of Pten-null prostate cancer (CaP) cells, independent of cancer development stage.PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of AR in the epithelium promotes the proliferation of Pten-null cancer cells, at least in part, by down-regulating androgen-responsive gene FKBP5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy

Effects of O-GlcNAc modification on gene expression using O-GlcNAcase deleted Mouse Embryonic Fibroblast cells.

Single O-GlcNAc modification orchestrate by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA alias MGEA5) enzymes, affects signal transduction and gene expression by chromatin modulation. We developed Oga deleted MEF (mouse embryonic fibroblast) cells to investigate effects of O-GlcNAc modification in mice. RNA isolated from Mouse Embryonic Fibroblast cells generated from Oga Knock out (KO) Heterozygous (Het) and wild type (WT) cells and subjected to microarray analysis.
Results provide insight into regulatory pattern of O-GlcNAc modification, and associated signaling pathways

Comparison of gene expression profiles of naïve and in vitro effector CD8+ T cells from wild-type and BATF-/- mice

The transcription factor BATF is required for Th17 and TFH differentiation. Here, we show that BATF also has a fundamental role in regulating effector CD8+ T cell differentiation.  BATF-deficient CD8+ T cells show profound defects in effector expansion and undergo proliferative and metabolic catastrophe early after antigen encounter.  BATF, together with IRF4 and Jun proteins, binds to and promotes early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors, while paradoxically repressing genes encoding effector molecules (IFNg and granzyme B). Thus, BATF amplifies TCR-dependent transcription factor expression and augments inflammatory signal propagation but restrains effector gene expression.  This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.

These data provide scientific information to understand the mechanism of action of lapatinib resistance in HER2-positive patients and to test the combination of HER2-targeted agents and GSK1363089 (foretinib) in the clinic by using an acquired lapatinib-resistant cell line.

Disturbed Hepatic Carbohydrate Management During High Metabolic Demand in Medium-Chain Acyl-CoA Dehydrogenase (MCAD)-deficient Mice

Medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) catalyzes crucial steps in mitochondrial   fatty acid oxidation, a process that is of key relevance for maintenance of energy homeostasis, especially during high metabolic demand. To gain insight into the metabolic consequences   of MCAD deficiency under these conditions, we compared hepatic carbohydrate metabolism in vivo in wild-type and MCAD-/- mice during fasting and during a lipopolysaccharide   (LPS)-induced acute phase response (APR). MCAD-/- mice did not become more hypoglycemic on fasting or during the APR than wild-type mice did. Nevertheless, microarray   analyses revealed increased hepatic peroxisome proliferator-activated receptor gamma coactivator-1a (Pgc-1a) and decreased peroxisome proliferator-activated receptor alpha (Ppar a) and   pyruvate dehydrogenase kinase 4 (Pdk4) expression in MCAD-/- mice in both conditions,suggesting altered control of hepatic glucose metabolism. Quantitative flux measurements revealed   that the de novo synthesis of glucose-6-phosphate (G6P) was not affected on fasting in MCAD-/- mice. During the APR, however, this flux was significantly decreased (-20%) in   MCAD-/- mice compared with wild-type mice. Remarkably, newly formed G6P was preferentially directed toward glycogen in MCAD-/- mice under both conditions. Together with diminished   de novo synthesis of G6P, this led to a decreased hepatic glucose output during the APR in MCAD-/- mice; de novo synthesis of G6P and hepatic glucose output were maintained in   wild-type mice under both conditions. APR-associated hypoglycemia, which was observed in wild-type mice as well as MCAD-/- mice, was mainly due to enhanced peripheral glucose   uptake. Conclusion: Our data demonstrate that MCAD deficiency in mice leads to specific changes in hepatic carbohydrate management on exposure to metabolic stress. This deficiency,   however, does not lead to reduced de novo synthesis of G6P during fasting alone, which may be due to the existence of compensatory mechanisms or limited rate control of MCAD in murine   mitochondrial fatty acid oxidation.

Expression data from epithelial cells during the process of multistep pancreatic carcinogenesis

The host antitumor immunity changes drastically during carcinogenesis. Intraductal papillary-mucinous neoplasm (IPMN) of the pancreas is a precursor lesion of pancreatic cancer and progresses according to adenoma-carcinoma sequence. We found that the host antitumor immune reaction changes from an immune response to immune tolerance between intraductal papillary-mucinous adenoma (IPMA) and intraductal papillary-mucinous carcinoma (IPMC).
In order to determine molecules affecting intraepithelial DC infiltration in IPMNs during multistep carcinogenesis, we examined the gene-expression profiles of entire transcripts of neoplastic cells at different stages.

Ada3 (alteration/deficiency in activation) is a transcriptional adaptor that forms a core structural component of multiple HAT complexes. In order to gain insights into physiological roles of Ada3, we made a conditional knockout mouse for Ada3 which was early embryonic lethal. Deletion of Ada3 in MEFs by using Adenovirus-Cre showed changes in global histone acetylation.
We performed microarray analysis after deletion of Ada3 in these cells to compare gene expression changes caused due to Ada3 loss

Effect of Nrf2 deletion in postnatal lung development and BPD phenotype in newborn mice

Background: Nrf2 is an essential cytoprotective transcription factor. However, association of Nrf2 in organ development and neonatal disease is rarely examined. Hyperoxia exposure to newborn rodents generates pulmonary phenotypes which resemble bronchopulmonary dysplasia (BPD) of prematurity.
Methods: To investigate the role of Nrf2 in lung maturation and BPD pathogenesis, Nrf2-deficient (Nrf2-/-) and wild-type (Nrf2+/+) neonates were exposed to air or hyperoxia (O2). Transcriptome analysis determined Nrf2-directed mechanisms in premature lung. Lung injury was assessed by bronchoalveolar lavage analysis and histopathology.
Results: In Nrf2-/- neonates, basal expression of cell cycle machinery, redox balance, and lipid/carbohydrate metabolism genes were suppressed while immunity genes were overexpressed compared to Nrf2+/+ pups. O2-induced mortality and pulmonary inflammation/injury were significantly higher in Nrf2-/- than in Nrf2+/+. Lung DNA lesion and oxidation were greater in Nrf2-/- than in Nrf2+/+, constitutively and after O2. Nrf2-dependent genes modulated cellular growth/proliferation, defense, immunity, and lipid metabolism against hyperoxia. Bioinformatic elucidation of Nrf2 binding motifs and augmented O2-induced inflammation in genetically deficient neonates validated Gpx2 and Marco as Nrf2 effectors.
Conclusion: Overall, Nrf2 in underdeveloped lungs orchestrated cell cycle, morphogenesis, and immunity as well as cellular defense constitutively and under oxidant stress. Results provide putative molecular mechanisms of Nrf2-directed lung alveolarization and BPD of prematurity.

Overexpression of the AP-2γ  transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy, even in ER positive, ErbB2 negative tumours; markers of a more favourable prognosis. To understand further the role of AP-2γ in breast carcinoma, we have used an RNA interference and gene expression profiling strategy using the MCF-7 cell line as a model for ER positive, ErbB2 negative tumours with AP-2γ overexpression.
Gene expression changes between control and silenced cells implicate AP-2γ  in the control of cell cycle progression and developmental signalling.

Expression data from BRAFV600E A375 melanoma cells treated with vehicle or vemurafenib

Vemurafenib is a BRAF inhibitor with specificity for the most common BRAF mutant encountered in melanomas (BRAFV600E). Vemurafenib suppresses the proliferation of BRAF mutant human melanoma cells by suppressing downstream activation of the MEK/ERK mitogen activated protein kinases.
We used microarrays to examine the transcriptional response of a vemurafenib-sensitive BRAFV600E human melanoma cell line (A375) to vemurafenib in order to further delineate the mechanisms by which BRAFV600E drives cell proliferation and energy metabolism in human melanoma.

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background: ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation, whereas its role in leukemia propagation and maintenance remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. Findings: Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-DOWN set was significantly enriched for genes included in the cell activation, immune response, apoptosis, signal transduction and development and differentiation categories, whereas in the E/R KD-UP set only the PI3K/AKT/mTOR signaling and hematopoietic stem cells categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories stem cell properties, B-cell differentiation, immune response, cell adhesion and DNA damage with RT-qPCR. Conclusion: The results of our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with regular B-cell development by repressing key functions that are necessary under physiological circumstances. E/R may thus constitute also the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.

Gene expression microarray data of bone marrow derived macrophages using GM-CSF(GM-BMMs) co-cultured with E0771 breast tumor cells

E-FABP is dominately expressed in macrophages/dendritic cells. Thus E-FABP may play a siginificant role in their immune response to tumor insult. We treated mouse GM-BMMs of different genotype with E0771 breast cancer cells. Then we investigate the global gene expression in these GM-BMMs in response to tumor treatment.

This study is aimed in identification of gene expression profiles in  cervical cancer and the role of specific genes in cervical carcinogenesis.
Keywords: Gene expression in cervical cancer

3T3-L1 cells treated 24 hours with either 0.1% DMSO, 20uM pioglitazone, 1uM rosiglitazone or 20uM troglitazone

K562 cell line mock treated or for 24 hours with one micromolar imatinib.

Gene expression changes in primary aortic endothelial cells during expression of dominant negative PPAR gamma (V290M).

Ligand-mediated activation of the nuclear hormone receptor PPAR gamma lowers blood pressure and improves glucose tolerance in humans.  Two naturally occurring mutations (P467L, V290M) in the ligand binding domain of PPAR gamma have been described in humans that lead to severe insulin resistance and hypertension.  Experimental evidence suggests that these mutant versions of PPAR gamma act in a dominant negative fashion.  To better understand the molecular mechanisms underlying PPAR gamma action in the vasculature, we determined the global gene expression profile in primary aortic endothelial cells in response to endothelial cell specific expression of a dominant negative isoform of PPAR gamma (V290M).

Differential induction of TLR3-dependent innate immune signaling by closely related parasite species_I

The closely related protozoan parasites Toxoplasma gondii and Neospora caninum display similar life cycles, subcellular ultrastructure, invasion mechanisms, metabolic pathways, and genome organization, but differ in their host range and disease pathogenesis.   Type II (γ) interferon has long been known to be the major mediator of innate and adaptive immunity to Toxoplasma infection, but genome-wide expression profiling of infected host cells indicates that Neospora is a potent activator of the type I (α/β) interferon pathways typically associated with antiviral responses.  Infection of macrophages from mice with targeted deletions in various innate sensing genes demonstrates that host responses to Neospora are dependent on the toll-like receptor Tlr3 and the adapter protein Trif.  Consistent with this observation, RNA from Neospora elicits TLR3-dependent type I interferon responses when targeted to the host endo-lysosomal system.  Although live Toxoplasma fail to induce type I interferon, heat-killed parasites do trigger this response, albeit much weaker than Neospora, and co-infection studies reveal that T. gondii actively suppresses the production of type I interferon.  These findings reveal that eukaryotic pathogens can be potent inducers of type I interferon and that related parasite species interact with this pathway in distinct ways.

Myocardin-like Protein (MKL)-2 Regulates TGF-β Signaling in Embryonic Stem Cells and the Developing Vasculature

Signal transduction from the extracellular matrix to the arterial wall plays a critical role during development of the vasculature.  We now report the discovery of a Myocardin-like Protein (MKL)2/TGF-β signaling pathway that is required for maturation and stabilization of the vasculature.  Mkl2-/- null embryos exhibit profound derangements in the tunica media leading to aneurismal dilation, dissection and hemorrhage.
TGFβ expression, signaling and TGF-β-regulated genes are down-regulated in Mkl2-/- ES cells and in the vasculature of Mkl2-/- embryos.  Transcription of Tgfβ2 is activated via binding of a MKL 2/SRF complex to a conserved CArG element in the Tgfβ2 promoter. In this data set we include expression data from wild type and Mkl2-/- ES cells

Coffin–Lowry Syndrome (CLS) is a syndromic form of mental retardation caused by loss of function mutations in the X-linked RPS6KA3 gene, which encodes Rsk2, a serine/threonine kinase involved in spatial memory. We analyzed hippocampal gene expression profiles  in Rsk2-KO mice to identify changes in molecular pathways.
Total RNA was extracted from hippocampi from 6 KO and 6 WT (littermates) 2-month-old male mice. For each genotype, equivalent amounts of RNA from 2 mice were pooled and processed for hybridization to the genome wide oligonucleotide microarray (Murine 430A 2.0 Affymetrix, 22.000 probe sets). Thus, 3 independent pooled samples were hybridized for each genotype.

Expression data from NRK-52E cells treated with monuron for 6h, 24h and 72h and rats exposed to monuron for 72h.

Monuron (1,1-dimethyl-3-(4-chlorophenyl) urea) is a non-selective phenylurea herbicide, widely used in the developing countries, however concerns have been raised about it toxicity and carcinogenicity. Monuron was evaluated by the National Toxicology Program (1988) and shown to be a male rat-specific renal carcinogen; however the mechanism of carcinogenesis is unknown. In this study we have examined the effect of sub-cytotoxic exposure to monuron (250µM) at 6h, 24h and 72h on the whole-genome expression profile in a rat proximal renal tubule cell line (NRK-52E). In addition we examined the short term exposure of MON in kidney and liver of exposed rats.
We used microarrays to detail the mechanism of toxicity and possibly carcinogenicity of monuron.

Bone morphogenetic protein-7 is a MYC target with pro-survival functions in childhood medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children, among whom overexpression or amplification of MYC oncogenes has been associated with poor clinical outcome. Although the MYC functions during normal development and oncogenesis in various systems have been extensively investigated, the transcriptional targets mediating MYC effects in MB are still elusive. Their identification and roles during MB onset and progression are important and will ultimately suggest novel potential therapeutic targets. cDNA microarray analysis was used to compare the effects of overexpressing and silencing MYC on the transcriptome of a MB-derived cell line. We identified 209 genes with potential relevance to MYC-dependent cellular responses in MB. Among the MYC-responsive genes, we found members of the bone morphogenetic protein (BMP) signaling pathway, which plays a crucial role during the development of the cerebellum. In particular, the cytokine gene BMP7 was identified as a direct target of MYC in MB cells. Similar to the effect induced by BMP7 silencing by siRNA, the use of a small-molecule inhibitor of the BMP/SMAD signaling pathway reduced cell viability in a panel of MB cells. Altogether, our findings indicate that high MYC levels drive BMP7 expression in MB to induce pro-survival and pro-proliferative cellular pathways. This observation suggests that targeting the BMP/SMAD pathway may be a new therapeutic concept for the treatment of childhood MB.

Transcriptional profiling of liver from wild type and PXR-null mice treated with PCN

Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR).  Activation of PXR can lead to increases in liver weight in part through hepatocyte replication similar to a large number of compounds that activate other nuclear receptors such as the peroxisome proliferator-activated receptor alpha and the constitutive activated receptor (CAR).  PXR controls the expression of a large battery of genes involved in xenobiotic metabolism.  Identification of genes that are accurate predictors of PXR activation would be useful in high-throughput screens to assess potential toxicity and drug-drug interactions.  Here, we identified PXR-dependent genes in the mouse liver after exposure to pregnenolone 16alpha-carbinonitrile (PCN), a chemical that is often used as a model PXR agonist.

In this study, we examine the consequences of the loss of two related factors, Onecut1 and Onecut2, during mouse retinal development.

Lesions of chronic idiopathic urticaria (CIU) showed significant up-regulation of 506 genes and reduced expression of 51 genes.

In order to assess the physiological role of Cop1 in vivo we generated mice that do no longer express the protein. Cop1KO mice die at around E10.5 of embryonic development. In order to gain insights into the molecular mechanisms that cause the embryonic death we compared the genome-wide gene expression profile of E9.5 wild-tytpe and Cop1-null embryos. The data do not support a role for Cop1 in the regulation of the p53 pathway in vivo and highlight a role for Cop1 in cardiovascular development and/or angiogenesis. The abstract of the associated publication is as follows:Biochemical data have suggested conflicting roles for the E3 ubiquitin ligase Cop1 in tumourigenesis. Here we present the first in vivo investigation of the role of Cop1 in cancer aetiology. We used an innovative genetic approach to generate an allelic series of Cop1 and show that Cop1 hypomorphic mice spontaneously develop malignancy at a high frequency in their first year of life and are highly susceptible to radiation-induced lymphomagenesis. Biochemically, we show that Cop1 regulates c-Jun oncoprotein stability and modulates c-Jun/AP1 transcriptional activity in vivo. Cop1-deficiency stimulates cell proliferation in a c-Jun-dependent manner. We conclude that Cop1 is a tumour suppressor that antagonizes c-Jun oncogenic activity in vivo.

Analysis of Ubb knockout mouse testes at 7, 4, 21, and 28 dpp.  Ubiquitin (Ub) is an essential protein found in all eukaryotic cells and plays important roles in a variety of cellular functions including germ cell development.  Targeted disruption of the polyubiquitin gene Ubb results in male and female infertility in mice with germ cells arrested at meiotic prophase I.

Liver gene expression in animals with hepatocyte-specific deletion of JAK2

Growth hormone signaling in hepatocytes is fundamentally important. Disruptions in this pathway have led to fatty liver and other metabolic abnormalities. Growth hormone signals through the JAK2/STAT5 pathway. Mice with hepatocyte specific deletion of STAT5 were previously shown to develop fatty liver. Our aim in this study was to determine the effect of deleting JAK2 in hepatocytes on liver gene expression. To do so, we generated animals with hepatocyte specific deletion of JAK2.

Understanding how lung progenitor cells balance
proliferation against differentiation is relevant to clinical
disorders such as bronchopulmonary dysplasia of
premature babies and lung cancer. Previous studies have
established that lung development is severely disrupted in
mouse mutants with reduced levels of the proto-oncogene
Nmyc, but the precise mechanisms involved have not been
explored. We show here that Nmyc expression in the
embryonic lung is normally restricted to a distal population
of undifferentiated epithelial cells, a high proportion of
which are in the S phase of the cell cycle. Overexpression
of NmycEGFP in the epithelium under the control of
surfactant protein C (Sftpc) regulatory elements expands
the domain of S phase cells and upregulates numerous
genes associated with growth and metabolism, as shown by
transcriptional microarray. In addition, there is marked
inhibition of differentiation, coupled with an expanded
domain of expression of Sox9 protein, which is also
normally restricted to the distal epithelial compartment. By
contrast, conditional deletion of Nmyc leads to reduced
proliferation, epithelial differentiation and high levels
of apoptosis in both epithelium and mesenchyme.
Unexpectedly, about 50% of embryos in which only one
copy of Nmyc is deleted die perinatally, with similarly
abnormal lungs. We propose a model in which Nmyc is
essential in the developing lung for maintaining a distal
population of undifferentiated, proliferating progenitor
cells.

Expression data from control and Snai1 knockout embryonic stem cells (ESCs)

Snail1 is a master epithelial-mesenchymal trisition (EMT) factor but its role in ESC maintenance is unknown.
We used microarrays to compare the global gene expression  between control and Snai1 knockout ESCs.

Triplicate samples of RAW 264.7 murine macrophages either untreated, stimulated with 100 ng/ml LPS for 18 hours, or constituitively over-expressing CstF-64  were analyzed by microarray using Affymetrix murine gene chip 430A.
Keywords = RAW 264.7 macrophages

Disease state analysis, genetic modification, Intestinal polyposis, PTEN mutant, Cowden disease mouse model

PTEN-deficient intestinal stem cells initiate intestinal polyposis

Intestinal polyposis, a precancerous neoplasia, results primarily from an abnormal increase in the number of crypts. Crypts contain intestinal stem cells (ISCs). Thus intestinal polyposis provides an ideal condition for studying stem cell involvement in polyp/tumor formation. Using a conditional knock-out mouse model, we found that the tumor suppressor Phosphatase of Tension homolog (PTEN) governs the proliferation rate and number of ISCs and loss of PTEN results in an excess of ISCs. In PTEN mutants, excess ISCs initiate de-novo crypt formation and crypt fission, recapitulating crypt production in fetal/neonatal intestine. Microarray studies were used to profile the changes in gene expression that occurred   when PTEN was knocked out in the intestine.
Keywords: Disease state analysis, genetic modification, Intestinal polyposis, PTEN mutant, Cowden disease mouse model

Comparative microarray between normal and essential thrombocythemia platelets

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17beta-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17beta-HSD3 activity corresponding to approximately 10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17beta-HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.

Hippocampus of HuD overexpressor mice. Perrone-Bizzozero-5R01NS030255-12

Post-transcriptional mechanisms play an important role in the control of gene expression. RNA-binding proteins are key players in the post-transcriptional control of many neural genes and they participate in multiple processes, from RNA splicing and mRNA transport to mRNA stability and translation. Our laboratory has developed the first mouse model overexpressing a RNA-binding protein, the ELAV-like protein HuD, in the CNS under the control of the CaMKinII alpha promoter. Initial behavioral characterization of the mice revealed that they had significant learning deficits together with abnormalities in prepulse inhibition (PPI). At the molecular level, we found that the expression of the growth-associated protein GAP-43, one of the targets of HuD, was increased in the hippocampus of HuD transgenic mice. To characterize these mice further and to evaluate the utility of these animals in understanding human diseases, we propose to use DNA microarray methods.
To test our hypothesis we propose 2 specific aims: 1)To characterize the pattern of gene expression in the hippocampus of HuD overexpressor mice 2)To compare the pattern of gene expression in our mouse model with that in the hippocampus of rats prenatally exposed to alcohol (FAS model) and in post-mortem tissues of patients with schizophrenia
All mice are in C57BL/6 background and are male approximately 60 days old. Initial studies were performed in animals that were not subjected to any experimental manipulation. Animals were bred and sacrificed according to our approved animal protocol.  The brain was rapidly dissected on ice and we isolated the hippocampus, which has the highest expression of the transgene. After dissection both hippocampi were frozen in liquid nitrogen, pooled and stored at -80C until analysis. RNA samples were isolated using RNAeasy Qiagen columns. For our first experiment, we want to examine the pattern of gene expression in the hippocampus of 3 transgenic mice and 3 non-transgenic littermates. RNAs from the 6 hippocampi were of high quality as revealed by the integrity of the 28S and 18S rRNA. We will provide 6 samples containing 10 ug of RNA each in DEPC water at a concentration of about 0.5 ug/ul. Three of the samples (#1, #2 and # 3) are from transgenic mice and three from their non-transgenic littermates (#4, #5 and #6).

Expression data in splenic DC subsets in wild type and Xbp1 deficient mice

Xbp1 is a major transcription factor in the unfolded protein response. To uncover its function in DCs we generated a conditional KO for Xbp1 in dendritic cells. We here compare the expression of mRNAs in two different splenic DC subpopulations, CD8a and CD11b DCs in both WT and KO mice.
Reference: Inositol-requiring enzyme 1-alpha regulates CD8a dendritic cell function via regulated mRNA decay. Osorio et al, Nature Immunology (2014)

neural crest;microarray;retinoic acid;gene expression;cell signaling;cell proliferation;time-course

This series represents murine dorsal neural tube bisected along the midline with one half from each embryo used for control and the other half treated with 10-6M RA dissolved in ethanol for 6, 12, 24 or 48 h. For 6 h exposures, the explants were cultured overnight on fibronectin coated 35mm dishes (Biocoat, Becton Dickinson Labware, Bedford, MA) in DMEM with 10% horse serum in order to allow for sufficient outgrowth of neural crest cells. The RA was added the following morning; RMA Express 0.2 used to initially normalize data; GeneSpring (Silicon Genetics, Inc.) used for subsequent analysis
Samples were analyzed at 6, 12, 24, and 48 hours.

Laser capture microdissection of endothelial and neuronal cells from human dorsolateral prefrontal cortex

We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from patients with schizophrenia and bipolar disorder and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using Affymetrix HG133plus2.0 GeneChips. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology.

Rip-Chip profiling of HnRNP A1 nuclear complexes in HuR WT and HuR KO cells

Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicative of their concerted function in mRNA metabolism.

Genome-wide analysis of transcription factor FoxO1-responsive gene expression in the ventromedial hypothalamus

Analysis of gene expression regulated by FoxO1 in the ventromedial hypothalamus (VMH) of wildtype and knockout mice. Results provide important information of gene expression in the VMH.

The transcription factor FoxO1 contributes to leptin and insulin action, including cells in the brain. However, the neurons mediating these effects and the identity of the molecular targets through which FoxO1 exerts metabolic actions remains to be defined.

Inflammatory Bowel Disease;IBD;interleukin-10;microarray;mice;quantitative trait locus;QTL;susceptibility;parallel sample

Abstract: Interleukin-10-deficient (Il10-/-) mice serve as a model for inflammatory bowel disease (IBD). The severity of colitis strongly depends on the inbred strain carrying the disrupted Il10 gene: C3H/HeJBir (C3) confers disease susceptibility, whereas C57BL/6J (B6) confers resistance. Genome-wide scans with microsatellite markers on segregrating backcross and F2 populations resulted in the detection of ten colitogenic quantitative trait loci (QTL). The aim of this study was to reduce the large number of candidate genes within the QTL intervals by identifying those genes which are located within the candidate gene intervals and which are differentially expressed in the colon of IBD-susceptible and -resistant strains. Using this combination of QTL mapping and microarray analysis, we identified 16 genes which were differentially expressed between B6- and C3-Il10-/- mice and were located within the candidate gene intervals. Three of these genes (Pla2g2a, Gbp1, Cd14) showed prominent differences in expression levels between B6- and C3-Il10-/- as well as between B6 and C3 wildtype mice and were considered to be major candidate genes. Pla2g2a and Gbp1 are known to be polymorphic between C3 and B6 mice. Expression data for Cd14 were confirmed by real-time RT PCR using specified pathogen free and germfree Il10-/- mice. In conclusion, the large number of candidate genes was reduced to three major candidates by using a combination of QTL mapping and microarray analysis. All three genes play an important role in inflammatory processes and immune response.
Material & Methods:
Two independent microarray experiments were performed using SPF mice: 19 for the first experiment (4 C3 WT [GSM39296], 5 C3-Il10-/- [GSM39297], 5 B6 WT [GSM39290], 5 B6-Il10-/- [GSM39291]) and 18 for the second (4 C3 WT [GSM39295], 4 C3-Il10-/- [GSM39294], 5 B6 WT [GSM39288], 5 B6-Il10-/- [GSM39289]). The mice were euthanized by cervical dislocation. Following a ventral midline incision, the colon (including the rectum, but not the anus) was removed and flushed with PBS. Each colon was opened longitudinally with a pair of scissors, further dissected and homogenized using a micropistill in 1.5 ml peqGOLD Trifast FL. RNA was isolated according to the manufacturer´s protocol, and quality was checked using RNA Nano LabChip® technology (Agilent, Böblingen, Germany). For each of the two array experiments, equal amounts (7 micro gram) of RNA were pooled from the four or five animals of each mouse strain, resulting in a total of eight samples.
For biotin-labeled target synthesis starting from 3-5 µg of total RNA, reactions were performed using standard protocols supplied by the manufacturer (Affymetrix; Santa Clara, CA). Briefly, 3-5 µg total RNA were converted to dsDNA using 100 pmol of a T7T23V primer (Eurogentec; Seraing, Belgium) containing a T7 promoter. The cDNA was then used directly in an in-vitro transcription reaction in the presence of biotinylated nucleotides.
The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 µg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MG U74Av2 for 16 hours.
Data selection and transformation procedures. For normalization, all array experiments were scaled to a target intensity of 150, otherwise using the default values of the Microarray suite 5.0 (MAS 5.0). Filtering of the results was done as follows: Genes were considered as strong regulated if their fold change was greater than or equal 2 or less than or equal -2, the statistical parameter for a significant change was less than 0.01 (change p-Value for changes called Increased) or greater than 0.99 (change p-Value for changes called Decreased). Additionally, the signal difference of a certain gene was greater than 200. Genes were considered as weak regulated if their fold change was greater than or equal 1.5 or less than or equal -1.5, the statistical parameter for a significant change was less than 0.001 or greater than 0.999 and the signal difference of a certain gene was greater than 40.

Mus musculus gene expression profiling for Wt, SKM-Tg, C and SKM-KO in Affymetrix 430PM strip arrays

We analyzed the functional role of DOR (Diabetes and Obesity Regulated gene) (also named Tp53inp2) in skeletal muscle. We show that DOR has a direct impact on skeletal muscle mass in vivo. Thus, using different transgenic mouse models, we demonstrate that while muscle-specific DOR gain-of-function results in reduced muscle mass, loss-of-function causes muscle hypertrophy. DOR has been described as a protein with two different functions, i.e., a nuclear coactivator and an autophagy regulator (Baumgartner et. al., PLoS One, 2007; Francis et. al., Curr Biol, 2010; Mauvezin et. al., EMBO Rep, 2010; Nowak et. al., Mol Biol Cell, 2009). This is why we decided to analyze which of these two functions could explain the phenotype observed in our mice models. In this regard, we performed a transcriptomic analysis using microarrays looking for genes differentially expressed in the quadriceps muscle of WT and SKM-Tg mice as well as in C and SKM-KO animals. Surprisingly, only a reduced number of genes were dysregulated upon DOR manipulation and most of the genes underwent mild changes in expression. These data strongly suggest that DOR does not operate as a nuclear co-factor in mouse skeletal muscle under the conditions subjected to study. In contrast, DOR enhances basal autophagy in skeletal muscle and promotes muscle wasting when autophagy is a contributor to muscle loss.
To determine the functional role of DOR in skeletal muscle, we generated transgenic mice (SKM-Tg) overexpressing DOR specifically in skeletal muscle under the Myosin-Light Chain 1 promoter/enhancer. The open reading frame of DOR was introduced in an EcoRI site in the MDAF2 vector, which contains a 1.5 kb fragment of the MLC1 promoter and 0.9 kb fragment of the MLC1/3 gene containing a 3' muscle enhancer element (Rosenthal et. al., PNAS, 1989; Otaegui et. al., FASEB J, 2003). The fragment obtained after the digestion of this construct with BssHII was the one used to generate both transgenic mouse lines. Nontransgenic littermates were used as controls for the transgenic animals (Wt). In addition, a muscle-specific DOR knock-out mouse line (SKM-KO) was also generated by crossing homozygous DOR loxP/loxP mice with a mouse strain expressing Cre recombinase under the control of the Myosin-Light Chain 1 promoter (Bothe et. al., Genesis, 2000). Deletion of exons 3 and 4 driven by Cre recombinase caused the ablation of DOR expression. Non-expressing Cre DOR loxP/loxP littermates were used as controls for knockout animals (C). Four-month-old male mice were used in all experiments. Mice were in a C57BL/6J pure genetic background.

We used Affymetix HG Focus GeneChip to measure the expression levels of HIV seronegative and seropositive individuals in human PBMCs in vivo. GSM39104 -GSM39115 are HIV seronegative samples; GSM39116-GSM39137 are HIV seropositive but drug naive samples; GSM39138-GSM39147 are HIV seropositive samples used to test serostatus biomarker; GSM39148-GSM39170 are HIV seropositive individuals whose CD4 cells decrease over time; GSM39171-GSM39187 are HIV seropositive individuals whose CD4 cells increase over time; GSM39188-GSM39190 are HIV seropositive samples used to test CD4 cell increase/decrease over time.

effect of over-expression LIGHT on T cells for the liver gene expression
We used microarrays to detail the global programme of gene expression underlying WT and LIGHT Tg liver

This series studied the patterns of gene expression among different types of gastric tumors. Gastric tumors and adjacent non-tumor normal tissues were obtained from 50 gastric cancer patients of diverse stages (IA, IB, II, IIIA, IIIB, and IV), tumor types (intestinal, diffuse, or mixed), and degree of tumor differentiation (moderately or poorly differentiated).
Keywords: Cancer patient samples

Evidence for immune response, axonal dysfunction and reduced endocytosis preceding Lewy body pathology in the substantia nigra in Parkinson’s disease

Subjects with incidental Lewy body disease (iLBD) may represent the premotor stage of Parkinson’s disease (PD). To identify molecular mechanisms underlying neuronal dysfunction and alpha--synuclein pathology in the premotor phase of PD, we investigated the transcriptome of post-mortem substantia nigra (SN) of iLBD, PD donors and age-matched controls with Braak alpha--synuclein stage ranging from 0-6. In Braak alpha--synuclein stages 1 and 2, we observed deregulation of pathways linked to axonal degeneration, unfolded protein response (UPR), immune response and endocytosis, including axonal guidance signaling, protein kinase A signaling, mTOR signaling, EIF2 signaling and clathrin-mediated endocytosis. In Braak stages 3 and 4, we observed a deregulation in pathways involved in protein translation and cell survival, including mTOR and EIF2 signaling. In Braak stages 5 and 6, we observed deregulation of pathways such as dopaminergic signaling, axonal guidance signaling and thrombin signaling. Throughout the progression of PD pathology, we observed a deregulation of mTOR, EIF2 and regulation of eIF4 and p70S6K signaling in the SN. This implicates that molecular mechanisms related to UPR, axonal dysfunction, endocytosis and immune response are an early event in PD pathology, and may hold the key to altering the disease progression in PD.

p/CIP binds to many nuclear receptors and plays a major role in hormone dependent transcription of genes. Recently, p/CIP was shown to affect mouse stem cell pluripotency.
Microarray gene expression analysis was conducted to assess the role of p/CIP in mouse embryonic stem cells.

Microarray expression profiling study of Runx1-null and wild type luminal mammary epithelial cells

RUNX1 encodes a RUNX family transcription factor (TF) and was recently identified as a novel mutated gene in human luminal breast cancers. We found that Runx1 is expressed in all subpopulations of murine mammary epithelial cells (MECs) except the secretory alveolar luminal cells. Conditional knockout of Runx1 in MECs by MMTV-Cre led to a decrease in luminal MECs, largely due to a profound reduction in the estrogen receptor (ER)-positive mature luminal subpopulation, a phenotype that could be rescued by loss of either Trp53 or Rb1. Mechanistically RUNX1 represses Elf5, a master regulatory TF gene for alveolar cells, and activates Foxa1, a key mature luminal TF gene involved in the ER program. Collectively, our data identified a key regulator of the ER+ luminal lineage whose disruption may contribute to development of ER+ luminal breast cancer when under the background of either TP53 or RB1 loss.

Non-telomeric role for Rap1 in regulating metabolism and protecting against obesity

The mammalian telomere-binding protein Rap1 was found to have additional non-telomeric functions, acting as a transcriptional cofactor and a regulator of the NF-kB pathway. Here, we assess the effect of disrupting mouse Rap1 in vivo, and report on its unanticipated role in metabolic regulation and body weight homeostasis. Rap1 inhibition causes dysregulation in hepatic as well as adipose function. In addition, using a separation-of-function allele, we show that the metabolic function of Rap1 is independent of its recruitment to TTAGGG binding elements found at telomeres, and at other interstitial loci.
We have utilized microarrays to outline gene expression changes resulting from Rap1-deficiency when compared to the wild-type controls.

Expression data from 18h-fasted WT versus KLF15-null mice: liver and quadriceps skeletal muscle

We used microarray analysis to identify differences in gene expression levels, in liver and in quadriceps skeletal muscle, between 18h (overnight) fasted WT control and Kruppel-like factor 15 (KLF15)-null mice.
Keywords: comparative expression analysis, fasted state

Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating H3K4me2 methylation (Affymetrix)

Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is a novel H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2 binding sites, and a consequent dysregulation of target gene transcription. Furthermore, characterization of LSD2 complex revealed that LSD2 forms active complexes with euchromatic histone methyltransferases EHMT1/2 and NSD3 as well as cellular factors involved in active transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation post initiation.
We used microarray analysis to identify the subset of genes that are differentially expression after depletion of endogenous LSD2/KDM1b/AOF1.

The cleavage of sphingoid base phosphates by sphingosine-1-phosphate (S1P) lyase to produce phosphoethanolamine and a fatty aldehyde is the final degradative step in the sphingolipid metabolic pathway. We have studied mice with an inactive S1P lyase gene and have found that, in addition to the expected increase of sphingoid base phosphates, other sphingolipids (including sphingosine, ceramide, and sphingomyelin) were substantially elevated in the serum and /or liver of these mice. This latter increase is consistent with a reutilization of the sphingosine backbone for sphingolipid synthesis due to its inability to exit the sphingolipid metabolic pathway. Furthermore, the S1P lyase deficiency resulted in changes in the levels of serum and liver lipids not directly within the sphingolipid pathway, including phospholipids, triacyglycerol, diacylglycerol, and cholesterol. Even though lipids in serum and lipid storage were elevated in liver, adiposity was reduced in the S1P lyase-deficient mice. Microarray analysis of lipid metabolism genes in liver showed that the S1P lyase deficiency caused widespread changes in their expression pattern. These results demonstrate that S1P lyase is a key regulator of the levels of multiple sphingolipid substrates and reveal functional links between the sphingolipid metabolic pathway and other lipid metabolic pathways that may be mediated by shared lipid substrates and changes in gene expression programs. The disturbance of lipid homeostasis by altered sphingolipid levels may be relevant to metabolic diseases.

Increased Expression of Angiogenic Genes in the Brains of Mouse Meg3-null Embryos

MEG3 (Maternally Expressed Gene 3) is a non-coding RNA that is highly expressed in the normal human brain and pituitary.  Expression of MEG3 is lost in gonadotroph-derived clinically non-functioning pituitary adenomas.  Meg3 knock-out mice were generated to identify targets and potential functions of this gene in embryonic development and tumorigenesis.  Gene expression profiles were compared in the brains of Meg3-null embryos and wild-type litter-mate controls using microarray analysis.  Microarray data were analyzed with GeneSifter which uses Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) classifications to identify signaling cascades and functional categories of interest within the data set.  Differences were found in signaling pathways and ontologies related to angiogenesis between wild-type and knock-out embryos.  Quantitative RT-PCR and histological staining showed increased expression of some VEGF pathway genes and increased cortical microvessel density in the knock-out embryos.  These results are consistent with reported increases in VEGF signaling observed in human clinically non-functioning pituitary adenomas.  In conclusion, Meg3 may play an important role in control of vascularization in the brain and may function as a tumor suppressor by preventing angiogenesis.

Microarray analysis of mdx mice expressing high levels of utrophin: therapeutic implications for DMD

Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disorder caused by dystrophin deficiency. Previous work suggested that increased expression of the dystrophin-related protein utrophin in the mdx mouse model of DMD can prevent dystrophic pathophysiology.   Physiological tests showed that the transgenic mouse muscle functioned in a way similar to normal muscle.  More recently, it has become possible to analyse disease pathways using microarrays, a sensitive method to evaluate the efficacy of a therapeutic approach.  We thus examined the gene expression profile of mdx mouse muscle compared to normal mouse muscle and compared the data with that obtained from the transgenic line expressing utrophin.  The data confirm that the expression of utrophin in the mdx mouse muscle results in a gene expression profile virtually identical to that seen for the normal mouse.  This study confirms that a strategy to up-regulate utrophin is likely to be effective in preventing the disease.
Keywords: Global gene expression profile

Innate immune response of murine nasal-associated lymphoid tissue (NALT) to Streptococcus pyogenes infection.

Streptococcus pyogenes is a major causative agent of tonsillitis or pharyngitis in children, which can lead to more invasive infections and noninfectious sequellae. S. pyogenes can persist in tonsils, while one-third of children treated with antibiotics continue to shed streptococci and have recurrent infections. Mouse nasal-associated lymphoid tissue (NALT) is functionally analogous to human oropharangeal lymphoid tissues. The innate immune responses of naïve cells from a mucosal site to S. pyogenes is not well described; therefore, we infected C57BL/6 mice intranasally with 108 CFU S. pyogenes. Transcriptional responses by NALT after S. pyogenes infection were analyzed by Affymetrix microarray and quantitative RT-PCR.  Wild-type S. pyogenes induces transcription of both type I and IFN-gamma-responsive genes, pro-inflammatory genes, and acute phase response plasma proteins within 24h after infection.   Invasion of NALT and the induction of the interferon response were not dependent on expression of anti-phagocytic M1 protein.  However, infection with an attenuated, less invasive mutant indicated that a robust innate response by NALT is significantly influenced by intra-NALT bacterial load.  Granulocytic populations of NALT, cervical lymph nodes and spleen were discriminated by characteristic surface and intracellular markers.  Intranasal infection induces systemic release of neutrophils and a substantial influx of neutrophils into NALT at 24h, which decline by 48h after infection.  Macrophages do not significantly increase in S. pyogenes-infected NALT.  Intranasal infection of IFN-gamma -/- (GKO) C57BL/6 mice did not lead to systemic dissemination of wild type S. pyogenes, despite reduced expression of IFN-gamma-responsive mRNAs in NALT.   Infected GKO mice had an unregulated influx of neutrophils into NALT compared to immunocompetant mice and mice treated with an anti-IFN-gamma antibody more rapidly cleared S. pyogenes from NALT.  Thus, IFN-gamma-induced responses have a suppressive influence on early clearance of this pathogen from NALT.

Loss of Hepatocyte-Nuclear-Factor-1α Impacts on Adult Mouse Intestinal Epithelial Cell Growth and Cell Lineages Differentiation

Although hepatocyte-nuclear-factor-1α (Hnf1α) is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions.  The aim of this study was to assess the consequences of abrogating Hnf1α on the maintenance of adult small intestinal epithelial functions.

Methodology/Principal Findings
An Hnf1α knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals.  Changes in global gene expression were also analyzed. Mice lacking Hnf1α displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life.   This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1α mutant mice.  The intestinal epithelium of Hnf1α null mice displayed a reduction of the enteroendocrine cell population.  An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway.

Conclusions/Significance
Together, these results unravel a functional role for Hnf1α in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Parp-2 is required for efficient erythropoiesis in mice by limiting replicative stress in erythroid progenitors

Erythropoiesis is a tightly regulated process in which multipotential hematopoietic stem cells give rise to mature red blood cells. Here, we show that Parp-2  deficiency in mice leads to chronic anemia, despite increased EPO plasma levels, providing a novel role for Parp-2 in erythropoiesis. Loss of Parp-2 causes shortened red blood cells lifespan and impaired differentiation of erythroid cells. Transcriptomic analyses revealed the activation of the p53-dependent-DNA damage response pathways on Parp-2-deficient erythroblasts, triggering the expression of genes involved in cell cycle checkpoints and apoptosis. Accordingly, Parp-2-deficient erythroblasts shows G2/M cell cycle arrest and increased apoptosis.

Expression data from GNMT and MAT1A knockout models that develop all the stages of non-alcoholic fatty liver disease including hepatocellular carcinoma [MAT1A_15months]

Liver global gene expression patterns of 15-month-old MAT1A knockout mice histopathologically determined to have hepatocellular carcinoma (HCC). 5 samples are of tumoral tissue and 5 samples are of peritumoral tissue. All these have their respective wild type patterns. These were analyzed to define signatures to study the pathogenesis of NAFLD-derived HCC, explore which subtypes of cancers can be investigated using mouse models and define a signature of HCC differential survival that can be used to characterize HCC subtypes of different survival derived from mixed etiologies.

Transcriptional profiling of human cancer cell lines upon ZMPSTE24 silencing

Defining the aging-cancer relationship is a challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate many features of aging. However, their short lifespan and cell-intrinsic and -extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. To circumvent these limitations we have generated Zmpste24 mosaic mice. Interestingly, these mice develop normally - revealing cell-extrinsic mechanisms are preeminent in progeria- and display decreased incidence of infiltrating oral carcinomas. Moreover, ZMPSTE24 knock-down reduces human cancer cell invasiveness. Our results disclose the ZMPSTE24-prelamin A system as an example of antagonistic pleiotropy on cancer and aging, support the potential of cell-based and systemic therapies for progeria, and highlight ZMPSTE24 as a new anticancer target.

AIRE hypoexpression and transcriptome profiles correlate with thymic impairment in Down syndrome

Down syndrome (DS) patients frequently develop organ-specific autoimmune disorders, particularly endocrinopathies and coeliac disease, as well as an increased susceptibility to mucosal candidiasis. These clinical features resemble APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy), a monogenic condition due to mutations in the AIRE gene, located on 21q22.3 and already described as down-regulated in 21 trisomy. Here we investigated AIRE expression and global gene expression profiles in surgically removed thymuses from 14 DS infants and children with congenital heart defects and from 42 age-matched individuals with cardiac defect as an isolated malformation. Immunohistochemistry revealed significantly reduced AIRE expression in DS thymuses (70.48±49.59 positive cells/mm2 in DS X 154.70±61.16 in controls, p<0.0001). qPCR confirmed the lower expression of AIRE in DS thymuses. Global thymic RNA profiles from DS patients and controls revealed 407 genes significantly hypoexpressed in DS. Network transcriptional analysis showed that hypoexpressed genes are related to biological processes such as antigen processing and presentation of endogenous antigen (ERAP2, CD1D), negative (PRDX2) and positive (CD3D, CD74) thymic T-cell selection and homeostasis of number of cells (PRDX2). Altogether these findings may explain the high prevalence of autoimmune phenomena in DS patients. Moreover, our data are in accordance with previous findings of thymic abnormal development in DS patients, characterized by lymphocyte depletion, diminution of the cortex, and loss of corticomedullary demarcation. Global gene profiles indicate that in DS patients, the trisomic imbalance probably leads to thymic hypofunction. In conclusion, lower AIRE expression and the impairment of other crucial pathways for central tolerance could well explain the high prevalence of organ-specific autoimmune disorders in DS.

Hes6 expression is controlled by c-Myc and the AR to promote E2F1 activity and poor outcome in castrate-resistant prostate cancer (xenografts)

Hes6 is a transcription co-factor that is associated with stem cell characteristics in neural tissue, but its role in cancer remains uncertain. Here we show that Hes6 is controlled by c-Myc and the AR and can drive castration resistance in xenografts of the androgen-dependent LNCaP prostate cancer cell line model. Hes6 activates a cell cycle enhancing transcriptional network that maintains tumour growth in the absence of circulating androgen but with maintained nuclear AR. We demonstrate interaction between E2F1, the AR and Hes6 and show the co-dependency of these factors in the castration-resistant setting. In the clinical setting, we have discovered a Hes6-associated signature that predicts poor outcome in prostate cancer, which could be pharmacologically targeted.

Transcriptional effects of chronic heroin and methamphetamine treatment in the mouse striatum

To identify molecular effects of chronic drug treatment, heroin and methamphetamine treated animals were compared with saline treated animals at multiple time-points using microarray technology. Gene expression profile was assessed 14 h after the last dose of 1, 3, 6 or 12 days drug treatment and after 13, 15, 18 or 24 days of withdrawal.

Angiogenesis inhibitors ameliorates inflammatory infiltrate, fibrosis and portal pressure in cirrhotic rats

Background and aims: There are considerable evidences demonstrating that angiogenesis and chronic inflammation are mutually dependent. However, although cirrhosis progression is characterized with a chronic hepatic inflammatory process, this connection is not sufficiently explored as a therapeutic strategy. Therefore, this study was aimed to assess the potential benefits of targeting angiogenesis in cirrhotic livers to modulate inflammation and fibrosis. For this purpose, we evaluate the therapeutic utility of angiogenesis inhibitors. Methods: The in vivo effects of angiogenesis inhibitors were monitored in liver of cirrhotic rats by measuring angiogenesis, inflammatory infiltrate, fibrosis, a-smooth muscle actin (a-SMA) accumulation, differential gene expression (by microarrays), and portal pressure. Results: Cirrhosis progression was associated with a significant enhancement of vascular density and expression of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1, angiopoietin-2 and placental growth factor (PlGF) in cirrhotic livers. The newly formed hepatic vasculature expressed vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Interestingly, the expression of these adhesion molecules correlated well with local inflammatory infiltrate. Livers of cirrhotic rats treated with angiogenesis inhibitors presented a significant decrease in hepatic vascular density, inflammatory infiltrate, a-SMA abundance, collagen expression and portal pressure. Conclusion: Angiogenesis inhibitors may offer a potential novel therapy for cirrhosis due to its multiple mechanisms of action against angiogenesis, inflammation and fibrosis in cirrhotic livers.
Keywords: angiogenesis, cirrhosis, liver, Affymetrix, fibrosis

The primary aim of this study was to evaluate the changes in hepatocyte gene expression under short-term hypoxic conditions in wild type and HIF-1a null cultures.  To this end, hypoxia treated cultures were subjected to incubation with 1% O2/5% CO2/94% N2 at 37° C for eight hours prior to RNA isolation.  Duplicate normoxic controls were established from separate animals wherein cultures were untreated and treated with Adbgal.  Biological triplicates of wild type and HIF-1a null cultures were placed under hypoxic conditions and subsequently processed for microarray analysis.  A total of 10 microarray hybridizations were performed.

Aquaporin-11 (AQP11), a new member of the aquaporin family, is localized in the endoplasmic reticulum (ER).  Aqp11–/– mice neonatally suffer from polycystic kidneys derived from the proximal tubule.  Its onset is proceeded by the vacuolization of ER.  However, the mechanism for the formation of vacuoles and the development of cysts remain to be clarified.  Here, we show that Aqp11–/– mice and polycystic kidney disease animals share a common pathogenic mechanism of cyst formation.
Keywords: Geneexpression profile of Aqp11 knokout mouse kidney

Gene expression profiling of Hodgkin lymphoma cell line KMH2: Comparison of CIITA-BX648577 knockdown cultures with non-silencing controls

Description of differentially expressed genes between KMH2 CIITA-BX648577 knockdown cultures and non-silencing controls

Expression profile of osteosarcoma cells in which Sox2 maintains cancer stem cells

Sox2 is required to maintain osteosarcoma cell tumor initiation.Knockdown of Sox2 leads tpo loss of tumorigenic properties. To examine gene expression changes upon Sox2 knockdown,  we performed microarray analysis on mouse osteosarcoma cells expressing scrambled or Sox2shRNA.  We found that genes  upregulated upon Sox2 knockdown included osteoblast diffrentiation genes and genes down regulated included cell cycle and RNA processing genes as well as YAP-TEAD target genes.
The Hippo pathway has a profound tumor suppressive role in cancer by restraining the strong growth-promoting function of YAP. We have previously shown that the stem cell transcription factor Sox2 maintains the tumorigenicity of osteosarcoma cancer stem cells (CSCs). In this report, we describe that Sox2 maintains stemness by antagonizing the Hippo pathway via direct repression of Hippo activators, Nf2 (Merlin) and WWC1 (Kibra), thereby leading to exaggerated YAP function. YAP is potently oncogenic in osteosarcoma and its depletion sharply reduces the tumorigenic CSC fraction.  Low Nf2, low WWC1, and high YAP expression mark the CSC fraction of the tumor population, while the more differentiated fraction has high Nf2, high WWC1 and reduced YAP expression.  This Sox2-Hippo axis is conserved and also operates in other Sox2-dependent cancers such as glioblastomas. We propose that disruption of YAP transcriptional activity reduces CSCs and could be a therapeutic strategy for Sox2- dependent tumors.

The regional specificity and timing of gene activation following chemotherapy, and how this relates to subsequent mucositis development is currently unknown.  The aim of the study was therefore to determine the early time course of gene expression changes along the gastrointestinal tract (GIT) of the DA rat following irinotecan treatment, so as to provide an insight into the genetic component of mucositis.
We have found that changes in gene expression following irinotecan occur by 1 hour, and persist for at least 72 h after treatment.  Overall changes in gene expression are similar along the GIT, however there is temporal variability between regions.

The transcription factor Otx2 regulates choroid plexus development and function

The choroid plexuses (ChPs) are the main regulators of cerebrospinal fluid (CSF) composition and thereby also control the composition of a principal source of signaling molecules that is in direct contact with neural stem cells in the developing brain. The regulators of ChP development mediating the acquisition of a fate that differs from the neighboring neuroepithelial cells are poorly understood. Here, we demonstrate in mice a crucial role for the transcription factor Otx2 in the development and maintenance of ChP cells. Deletion of Otx2 by the Otx2-CreERT2 driver line at E9 resulted in a lack of all ChPs, whereas deletion by the Gdf7-Cre driver line affected predominately the hindbrain ChP, which was reduced in size, primarily owing to an increase in apoptosis upon Otx2 deletion. Strikingly, Otx2 was still required for the maintenance of hindbrain ChP cells at later stages when Otx2 deletion was induced at E15, demonstrating a central role of Otx2 in ChP development and maintenance. Moreover, the predominant defects in the hindbrain ChP mediated by Gdf7-Cre deletion of Otx2 revealed its key role in regulating early CSF composition, which was altered in protein content, including the levels of Wnt4 and the Wnt modulator Tgm2. Accordingly, proliferation and Wnt signaling levels were increased in the distant cerebral cortex, suggesting a role of the hindbrain ChP in regulating CSF composition, including key signaling molecules. Thus, Otx2 acts as a master regulator of ChP development, thereby influencing one of the principal sources of signaling in the developing brain, the CSF.

The aim of this study was to investigate the effects of hypoxia on human macrophages
Keywords: Effect of treatment

Excerpt from a larger study which characterized the transcriptional effects of a spinal cord contusion injury in rats.  This is the data from the almost chronic contusion state (35 days) at the injury site (Thoracic 8) - where we saw significant changes in several areas, including cholesterol metabolism genes.  Other spinal cord areas (rostral, caudal) and time-points (3 hours, 24 hours, 7 days and 35 days) were analyzed as well and are discussed in our paper and at www.crpf.org/microarray.
Keywords = Spinal Cord Injury

Gene expression profiling of Activin-like kinase 2 knockout in mice

Implantation of an embryo in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the pregnant uterus, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing uterine-specific Alk2 conditional knockout (cKO) mice. In the absence of ALK2, embryos can invade the uterine epithelium and stroma, but stromal cells cannot undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein β (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of C/EBPβ to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (HESC) and discovered that ablation of ALK2 alters HESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing HESC confirmed that BMP signaling protein, SMAD1, directly regulates expression of CEBPB by binding a distinct regulatory sequence in the CEBPB promoter; C/EBPβ, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate embryo implantation in rodents and primates and, for the first time, uncovers a linear pathwayof BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

Re-analysis by microarray using cDNA target of samples from psoriasis patients enrolled in an etanercept trial

cDNA and cRNA hybridization technologies have different, probe-specific sensitivities. We used samples from an etanercept trial (GSE11903) to explore in a real-life setting the uniqueness of each platform.

Series of samples studying effect of knocking out RUNX1 (a.k.a. AML1) in mouse embryos at 8.5 and 12 days.  Wildtype and knockout animals at the two time points were generated and all conditions done in duplicate.

The LEDGF transcript from the PSIP1 gene was knocked down in Jurkat cells using RNAi technology. The resulting Jurkat-derived cell line (Jurkat-siJK2) was compared to a control cell line (wild type Jurkat) using microarray analysis. Genes identified as being modulated by LEDGF were preferential targets of HIV integration.
Keywords: effects of gene knockdown

A dominant negative form of cJun affects genes that have opposing effects on lipid homeostasis in mice

cJun is a transcription factor activated by phosphorylation by SAPK/JNK MAP kinase pathway that has been linked to atherosclerosis. Adenovirus mediated gene transfer of a dominant negative form of cJun in C57BL/6 mice increased greatly the apolipoprotein E (ApoE) mRNA and plasma apoE levels and induced dyslipidmia, characterized by increased plasma cholesterol, triglyceride and VLDL levels and accumulation of discoidal HDL particles. Unexpectedly, infection of ApoE-/- mice with adenovirus expressing dn-cJun reduced by 50% plasma cholesterol, suggesting that the dn-cJun affected other genes that control plamsa cholesterol. To determine the molecular pathways implicated in this process we performed whole genome expression profiling using total RNA from the liver of infected ApoE-/- mice.
Keywords: disease

Experimental asthma was induced in BALB/c mice by sensitization and challenge with the allergen ovalbumin. Control groups received PBS. To investigate the innate immune component of experimental asthma, we also analyzed recombinase activating gene (RAG) deficient mice following exposure to ovalbumin and control PBS
Keywords: case control

The undifferentiated phenotype in neuroblastoma depends on Cyclin D1 and CDK4 activity

Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4  knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype.
Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4

Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrial-targeted nuclear genes in concert with reduced signaling via PPARα/PGC-1α and other transcriptional regulators. In cultured myocytes Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO, but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity.

Gene expression analysis of the effect of activating ATF6 in the hearts of transgenic mice upon treatment with tamoxifen.

Identification of MCIP1 as an ATF6-inducible ER Stress Response Gene in the Heart by Gene Expression Profiling

We recently found that the endoplasmic reticulum (ER) stress response (ERSR) is activated in surviving cardiac myocytes in a mouse model of in vivo myocardial infarction.  ATF6 is an ER stress-activated transcription factor that induces ERSR genes, some of which encode proteins that may protect against ischemic damage.  However, few ERSR genes have been identified in the heart, and there have been no gene expression profiling studies of ATF6-inducible genes, in vivo.  We previously generated transgenic (TG) mice that express tamoxifen-activated ATF6, ATF6-MER, in the heart; ATF6-MER conferred tamoxifen-dependent ATF6 activation and protection from ischemic damage.  To understand of the mechanism of ATF6-mediated cardioprotection, gene expression profiling of ATF6-MER TG mouse hearts was performed.  Activated ATF6 changed expression levels of 1,162 genes in the heart; of the 775 ATF6-inducible genes, only 23 are known ERSR genes.  One of the genes not expected to be induced by ATF6 is modulatory calcinuerin-interacting protein-1 (MCIP1).  MCIP1 is induced in a calcineurin/NFAT-dependent manner during myocardial hypertrophy and it can feedback inhibit cardiomyocyte growth.  We found that MCIP1 expression in cultured cardiomyocytes was increased by the prototypical ER stresser, tunicamycin (TM), or by simulated ischemia.  Moreover, infecting cardiomyocytes with adenovirus encoding activated ATF6 induced MCIP1 expression and inhibited myocyte growth in response to the alpha 1-adrenergic agonist, phenylephrine.  These results suggest that MCIP1 can be induced in the heart by ER stresses, such as ischemia.  Moreover, b integrating hypertrophy and ER stress, MCIP-modulated myocyte growth may help rejuvenate nascent ER protein folding, which could contribute to protection from ischemic damage.
Keywords: Gene expression analysis of the effect of activating ATF6 in the hearts of transgenic mice upon treatment with tamoxifen.

The effect of TRPM2-AS depletion in human prostate carcinoma PC3 cell line

There is overwhelming evidence indicating that human cancer is a genetic disease caused by sequential accumulation of mutations in oncogenes and tumor suppressor genes. However, it is also increasingly apparent that cancer depends not only on mutations in these coding genes, but also on alterations in the large class of the non-coding RNAs (ncRNAs). Here, we report that one such long ncRNA, TRPM2-AS, an antisense transcript in respect to TRPM2, a gene encoding for a ion channel activated by oxidative stress, is significantly overexpressed in prostate cancer. High expression levels of either TRPM2-AS or of a related gene signature are linked to poor clinical outcome, with the related gene signature working also independently of the patient's Gleason score. Moreover, TRPM2-AS knock-out leads to apoptosis of prostate cancer cells, with transcriptional profiling indicating an unbearable increase of cellular stress in dying cells, coupled to the down-regulation of several already known oncological targets. Considering that TRPM2-AS transcripts are barely detectable in healthy cells, these data strongly suggest that it, beside being a novel prognostic marker, is also a suitable therapeutic target in prostate cancer and establish a strong rationale for developing agents targeting either its expression or the expression of molecules in its pathway.

Schizophrenia is a debilitating neurological disorder for which no cure exists. Few defining characteristics of schizophrenic neurons have been identified and the molecular mechanisms responsible for schizophrenia are not well understood, in part due to the lack of patient material for study. Human induced pluripotent stem cells (hiPSCs) offer a new strategy for studying schizophrenia. We have created the first cell-based human model of a complex genetic psychiatric disease by generating hiPSCs from schizophrenic patients and subsequently differentiating these cells to hiPSC-derived neurons in vitro. Schizophrenic hiPSC-derived neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of schizophrenic hiPSC-derived neurons identified altered expression of many components of the cAMP and WNT signaling pathways. Key cellular and molecular elements of the schizophrenic phenotype were ameliorated following treatment of schizophrenic hiPSC-derived neurons with the antipsychotic loxapine.

Microarray profiling of primary neurons transduced with shRNA for multiple ASD-implicated genes

Austism spectrum disorder (ASD) is a heterogeneous behavioral disease most commonly characterized by severe impairment of social engagement and the presence of repetitive activities. The molecular etiology of ASD is still largely unknown despite a strong genetic component.  Part of the difficulty in turning genetics into disease mechanisms and potentially new therapeutics is the sheer number and diversity of the genes that have been associated with ASD and ASD symptoms.  The goal of this work is to use shRNA-generated models of genetic defects proposed as causative for ASD to identify the common pathways that might explain how they produce a common clinical outcome.  Transcript levels of Mecp2, Mef2a, Mef2d, Fmr1, Nlgn1, Nlgn3, Pten, and Shank3 were knocked-down in mouse primary neuron cultures using shRNA/lentivirus constructs. Whole genome expression analysis was conducted for each of the knock-down cultures as well as a mock-transduced culture and a culture exposed to a lentivirus expressing luciferase.  Gene set enrichment and a causal reasoning engine were employed to indentify pathway level perturbations generated by the transcript knock-down. Quantitation of the shRNA targets confirmed the successful knock-down at the transcript and protein levels of at least 75% for each of the genes.  After subtracting out potential artifacts caused by transfection and viral infection, gene set enrichment and causal reasoning engine analysis showed that a significant number of gene expression changes mapped to pathways associated with neurogenesis, long-term potentiation, and synaptic activity.  This work demonstrates that despite the complex genetic nature of ASD, there are common molecular mechanisms that connect many of the best established autism candidate genes.  By identifying the key regulatory checkpoints in the interlinking transcriptional networks underlying autism, we are better able to discover the ideal points of intervention that provide the broadest efficacy across the diverse population of autism patients.

Gene expression of 4, 5, and 6 days differentiated Flk1+ WT ES cells, and of 6 days differentiated Flk1+ Runx1-/- and Tal-1-/- ES cells

In order to identify genes that are activated in differentiating WT ESCs, but are missing in Tal-1-/- and Runx1-/- ESCs, and which might be involved in the generation of definitive hematopoietic progenitors and their specification thereafter, we performed microarray analyses on purified Flk-1+ cells, differentiated from these ESCs for 4, 5, and 6 days “in vitro”.

Gene expression changes in mouse embryonic fibroblasts (MEF cells) deficient for Ing1 and Gadd45a

ING1b and GADD45a are nuclear proteins involved in the regulation of cell growth, apoptosis and DNA repair. We found that ING1b and GADD45a physically and functionally interact in the epigenetic regulation of specific target genes. In order to study this interaction further, we analysed the transcriptional changes in MEF cells from single and double Ing1/Gadd45 knockout mice via microarray profiling.

Inflammation, adenoma and cancer: objective classification of colon biopsy specimens with gene expression signature

Background and Aims: Gene expression analysis of colon biopsies using high-density oligonucleotide microarray can contribute to the understanding of local pathophysiological alterations and to functional classification of precancerous adenoma, different stage colorectal carcinomas (CRC) and inflammatory bowel diseases (IBD).

Results: Significant overexpression of collagen IV, lipocalin-2, caveolin-1, calumenin genes, and significant dowregulation of aquaporin-8, amnionless homolog, prostaglandin D2 receptor genes were detected in CRC patients compared to normal. Adenoma samples were characterized by upregulated CD44 antigen, met proto-oncogene and downregulated chemokine ligand-12, ADAM-like decysin-1 and ATP-binding casette A8 discriminatory genes. In IBD samples significantly increased lipocalin-2, interferon induced transmembrane protein 1 and 3 mRNA levels, decreased zinc finger protein 91 and transient receptor potential cation channel M6 mRNA levels were found. Ulcerative colitis and Crohn’s disease can be distinguished  according to the top five genes: cyclin-G2; tripartite motif-containing-31; TNFR shedding aminopeptidase regulator-1; C-type lectin superfamily member-14 and AMICA. 88.3-97.8% of  the cases was correctly classified according to discriminatory genes.

Conclusions: Our whole genomic microarray analysis of biopsy samples provides discriminative signatures, and an insight into pathophysiological background of colonic diseases. The results afford a data warehouse which can be further mined for in-depth pathway analyses.

Hsp70 and a Novel Axis of Type 1 Interferon-Dependent Antiviral Immunity in the Measles Virus-Infected Brain

The major inducible 70 kDa heat shock protein (hsp70) is host protective in a mouse model of measles virus (MeV) brain infection. Transgenic constitutive expression of hsp70 in neurons, the primary target of MeV infection, abrogates neurovirulence in neonatal H-2d congenic C57BL/6 mice. A significant level of protection is retained after depletion of T lymphocytes, implicating innate immune mechanisms. Focus of the present work was to elucidate the basis for hsp70-dependent innate immunity using this model. Transcriptome analysis of brains from transgenic (TG) and non-transgenic (NT) mice 5 days after infection identified type 1 interferon (IFN) signaling and macrophage activation/antigen presentation as the main differences linked to survival. The pivotal role for type 1 IFN in hsp70-mediated protection was demonstrated in mice with a genetically disrupted type 1 IFN receptor (IFNAR-/-), where IFNAR-/- eliminated the difference in survival between TG and NT mice. Brain macrophages, not neurons, are the predominant source of type 1 IFN in the virus-infected brain, and in vitro studies provided a mechanistic basis by which MeV-infected neurons can induce IFN-β in uninfected microglia in an hsp70-dependent manner. MeV infection induced extracellular release of hsp70 from mouse neuronal cells that constitutively express hsp70, and extracellular hsp70 induced IFN-β transcription in mouse microglial cells through Toll-like receptors 2 and 4. Collectively, results support a novel axis of type 1 IFN-dependent antiviral immunity in the virus-infected brain that is driven by hsp70.

A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation

A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression.  We report the identification and biologic characterization of an LPS-like molecule extracted from the cyanobacterium Oscillatoria Planktothrix FP1 (CyP).
Keywords: different ligands

Antenatal steroid (AS) adversely affects placental function: A study of molecular effects using micro-array analysis

Antenatal steroids (AS) are used to augment fetal lung maturity and there is a major concern that they impair fetal growth. If delivery is delayed after using AS, placental function and hence fetal growth may be compromised even further. We treated three pairs of pregnant C57/BL mice with AS and studied changes in placental gene expression by microarray analysis, these results were later confirmed by semi quantitative RT-PCR, in situ hybridization and Oligo ApopTaq assay. AS treated placentas were hydropic, friable and pale. Placental weight was decreased (78.8±16.7 mg compared to 81.3±17 mg, p<0.05) and so was fetal weight (914 38 mg compared to 1160 85 mg, p<0.05). There was more than 99% similarity between the three gene chip data sets. AS led to down-regulation of 1212 genes and up-regulation of 1382 genes. AS led to decrease in expression of genes involved in cell division like cyclin A2, B1, D2, CDK2, CDK4 and M phase protein kinase along with growth promoting genes like BMP4 and IGF-BP4. Tryptophan hydroxylase gene a key regulator of serotonin production was upegulated by >15 fold. AS treatment caused deleterious effects on placenta by decreasing expression of growth related genes and increased apoptosis of trophoblast cells.
Keywords: comparitive gene expression

transcription factor PlagL2 regulates steps in chylomicron metabolism

Enterocytes assemble dietary lipids into chylomicron particles that are taken up by intestinal lacteal vessels and peripheral tissues. Although chylomicrons are known to assemble in part within membrane secretory pathways, the modifications required for efficient vascular uptake are unknown. We report that the transcription factor Pleomorphic adenoma gene-like 2 (PLAGL2) is essential for this aspect of dietary lipid metabolism. PlagL2-/- mice die from post-natal wasting owing to failure of fat absorption. Lipids modified in the absence of PlagL2 exit from enterocytes but fail to enter interstitial lacteal vessels. Dysregulation of enterocyte genes closely linked to intracellular membrane transport identified candidate regulators of critical steps in chylomicron assembly. PlagL2 thus regulates essential and poorly understood aspects of dietary lipid absorption and its deficiency represents an authentic animal model with implications for amelioration of obesity or the metabolic syndrome.
Keywords: gene expression profiling analysis

Transcription factors, Sp3, knockout mice, cardiac malformations, E12.5

Gene expression profiling of E12.5 wildtype- and Sp3 null hearts

Mice lacking the zinc finger transcription factor Specificity protein 3 (Sp3) die prenatally in the C57Bl/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at E10.5, and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analysed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of Cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. ChIP analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development, and suggest that it has a crucial role in myocardial differentiation. (
Keywords: Transcription factors, Sp3, knockout mice, cardiac malformations, E12.5

The aim of this project is to identify distinct temporal patterns of RNA expression in the skeletal muscle of rats following a bolus dose of the corticosteroid methylprednisolone.  51 RG_U34A chips were used over 17 time points.

LDL receptor deficiency, high fat diet, atherosclerosis, liver;repeat sample

Examination of gene expression profiles from liver of C57BL/6 mice and LDL receptor deficient mice fed on either a low fat diet or a high fat Western-style diet for 12 weeks.  Three replicates of each condition analyzed.
Keywords = LDL receptor deficiency, high fat diet, atherosclerosis, liver

Regulatory mechanisms of TSG-6 (TNF-alfa-induced protein-6: Tnfip6)-deficient and wild-type synovial fibroblasts

BALB/c mice are susceptible to proteoglycan (PG) aggrecan-induced arthritis (PGIA), and the absence of TSG-6 further increases susceptibility and local inflammatory reactions, including neutrophil invasion into the joints. To gain insight into the mechanisms of TSG-6 action, synovial fibroblasts were isolated from wild-type and TSG-6-KO mice, cultured and exposed to various agents affecting either the TSG-6 expression and/or modify the intracellular function of TSG-6.
In the present microarray studies, we have identified differences in gene expression by fibroblasts isolated from wild-type or TSG-6-KO mice

Analysis of gene expression in mammary epithelial cells transduced with either hTERT, empty LXSN vector or empty BABE vector.

The paraventricular hypothalamic nucleus (PVH) is a key site for integrating neuroendocrine, autonomic, and behavioral adjustments to diverse homeostatic challenges, including "physiological" (e.g., infection or hemorrhage) and "emotional" [e.g., restraint (RST) or footshock] stresses. Both types of challenges ultimately converge to activate common response systems represented in PVH, including the hypothalamo-pituitary-adrenal axis and the sympathoadrenal system. Oligonucleotide microarrays (U74A; Affymetrix, Santa Clara, CA) were used to compare and contrast gene expression profiles in the PVH elicited at 1 and 3 hr after acute exposure to representative physiological [intraperitoneal injection of 10 microg lipopolysaccharide (LPS)] and emotional (30 min RST) stressors. In general, the two challenges recruited relatively few genes in common, with the degree of overlap varying across functional classes of genes. The greatest degree of commonality was seen among signaling molecules and neuropeptides, whereas transcription factors upregulated by RST and LPS were largely distinct. Unexpectedly, RST induced a number of immune-related molecules, which were not regulated by LPS. Hybridization histochemical analyses localized a subset of responsive transcripts to the PVH and/or immediately adjoining regions. Immunerelated molecules in particular distributed broadly to vascular and other barrier-associated cell types. These global transcriptional profiles inform the search for early (transcription factors) and late (target genes) mechanisms in the modulation of PVH, and generalized CNS, responses to categorically distinct stressors.

It is currently unknown how extensively the double-stranded RNA binding protein Staufen (Stau)1 is utilized by mammalian cells to regulate gene expression.  To date, Stau1 binding to the 3’ untranslated region (3’UTR) of ARF1 mRNA has been shown to target ARF1 mRNA for Stau1-mediated mRNA decay (SMD).  ARF1 SMD depends on translation and recruitment of the nonsense-mediated mRNA decay factor Upf1 to the ARF1 3’UTR by Stau1.  Here, we use microarray analyses to examine changes in the abundance of cellular mRNAs that occur when Stau1 is depleted.  Results indicate that 1.1% and 1.0% of the 11,569 HeLa-cell transcripts that were analyzed are, respectively, upregulated and downregulated at least two-fold in three independently performed experiments.  Additionally, we localize the Stau1 binding site to the 3’UTR of four mRNAs that we define as natural SMD targets.  Together, these and substantiating results suggest that Stau1 influences the expression of a wide variety of physiologic transcripts and metabolic pathways.
Keywords: Staufen1-mediated mRNA decay; Stau1 downregulation by siRNA.

Palmitoyl protein thioesterase-1 knockout mice hofma-affy-mouse-370000

Infantile neuronal ceroid lipofuscinosis (aka infantile Batten disease) is a severe neurodegenerative disorder of early childhood characterized by cortical atrophy, blindness and seizures, leading to premature death in the first decade. The disorder is caused by deficiency in a soluble lysosomal enzyme, palmitoyl protein thioesterase-1 (PPT1). PPT1 knockout mice faithfully reproduce the features of the human disease, with motor deterioration, blindness, seizures, and death before 10 months of age. The progression of events leading from enzyme deficiency to organ failure is poorly defined. The neuronal ceroid lipofuscinoses are of particular interest because of the similarities between the accumulated material and lipofuscin that is associated with normal aging.
We will determine changes in gene expression that occur during the course of neurodegeneration in PPT1 knockout as compared to control mice.
Gene expression profiling of brain tissue from PPT1 knockout mice as compared to normal controls will provide insights into the mechanism of neurodegeneration. Comparison of this profile with gene expression profiles associated with normal aging may provide insights into the contribution of lipofuscin to aging and neurodegeneration.
PPT1 knockout mice on a C57BL/6 background will be sacrificed under CO2 and whole brains removed and frozen under liquid nitrogen.  Three mice will be used for each time point. Data points will be collected at 3 months, 5 months and 8 months of age. Normal age- and sex-matched C57BL/6 mice will be used as controls.

Transcriptional profiles were compared in microdissected lateral walls of the inner ears from Errb mutant mice and wild type littermate controls.  The goal is to identify transcriptional targets of Errb and candidate genes for inner ear diseases.
Keywords: genetic mutant analysis

A phase II neoadjuvant trial of anastrozole (A), fulvestrant (F) and gefitinib (I - iressa) in patients with newly diagnosed estrogen receptor positive breast cancer

Endocrine therapy in patients with breast cancer can be limited by the problem of resistance. Preclinical studies suggest that complete blockade of the estrogen receptor (ER) combined with inhibition of the epidermal growth factor receptor (EGFR) can overcome endocrine resistance. We tested this hypothesis in a phase II neoadjuvant trial of anastrozole and fulvestrant combined with gefitinib in postmenopausal women with newly diagnosed ER-positive breast cancer. After a baseline tumor core biopsy, patients were randomized to receive anastrozole and fulvestrant (AF) or anastrozole, fulvestrant, and gefitinib (AFG) for 3 weeks.  After a second biopsy at 3 weeks, all patients received AFG for 4 months and surgery was done if the tumor was operable. The primary endpoint was best clinical response by RECIST criteria and secondary endpoints were toxicity and change in biomarkers. The study closed after 15 patients were enrolled because of slow accrual. Median patient age was 67 years and median clinical tumor size was 7 cm. Four patients had metastatic disease present. Three patients withdrew before response was assessed. In the remaining twelve patients, there were two complete clinical responses (17%), three partial responses (25%), five had stable disease (41%), and two (17%) had progressive disease. Most common adverse events were rash in four patients, diarrhea in four, joint symptoms in three, and abnormal liver function tests in three. There were no grade 4 toxicities and all toxicities were reversible. At 3 weeks, cell proliferation as measured by Ki-67 was significantly reduced in the AFG group (p value= 0.01) with a parallel reduction in the expression of the Cyclin D1 (p value=0.02). RNA microarray data showed a corresponding decrease in the expression of cell cycle genes. These results suggest that AFG was an effective neoadjuvant therapy and consistently reduced proliferation in ER-positive tumors.

Transcriptomic Analysis of Autism Brain Reveals Convergent Molecular Pathology [array data]

Autism spectrum disorder (ASD) is a common, highly heritable neuro-developmental condition characterized by marked genetic heterogeneity. Thus, a fundamental question is whether autism represents an etiologically heterogeneous disorder in which the myriad genetic or environmental risk factors perturb common underlying molecular pathways in the brain. Here, we demonstrate consistent differences in transcriptome organization between autistic and normal brain by gene co-expression network analysis. Remarkably, regional patterns of gene expression that typically distinguish frontal and temporal cortex are significantly attenuated in the ASD brain, suggesting abnormalities in cortical patterning. We further identify discrete modules of co-expressed genes associated with autism: a neuronal module enriched for known autism susceptibility genes, including the neuronal specific splicing factor A2BP1/FOX1, and a module enriched for immune genes and glial markers. Using high-throughput RNA-sequencing we demonstrate dysregulated splicing of A2BP1-dependent alternative exons in ASD brain. Moreover, using a published autism GWAS dataset, we show that the neuronal module is enriched for genetically associated variants, providing independent support for the causal involvement of these genes in autism. In contrast, the immune-glial module showed no enrichment for autism GWAS signals, indicating a non-genetic etiology for this process. Collectively, our results provide strong evidence for convergent molecular abnormalities in ASD, and implicate transcriptional and splicing dysregulation as underlying mechanisms of neuronal dysfunction in this disorder.

Contribution of the STAT1alpha and STAT1beta isoforms to IFN-gamma mediated innate immunity

The transcription factor STAT1 is essential for interferon- (IFN) mediated protective immunity in humans and mice. Two splice isoforms of STAT1, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. Dimers of STAT1β are therefore considered transcriptionally inactive and potential competitive inhibitors of STAT1α. Contrasting this view, generation and analysis of mice deficient for either STAT1α or STAT1β demonstrated transcriptional activity of the STAT1β isoform and its enhancement of innate immunity. Gene expression profiling in primary cells revealed overlapping, but also non-redundant and gene-specific activities of STAT1α and STAT1β in response to IFNγ. Consistently, both isoforms mediated protective, IFNγ-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiency. In contrast, STAT1α and STAT1β were largely redundant for transcriptional responses to IFNα/β and for IFNα/β-dependent antiviral activity. Collectively, our data shed new light on how STAT1 isoforms contribute to antimicrobial immunity.

Anaplastic thyroid carcinoma cell lines (HTH83 and TL3) infected with LAMC2 shRNA

Laminin-5 gamma-2 (LAMC2) is highly expressed in anaplastic thyroid carcinoma and associated with tumor progression, migration and invasion by modulating signaling of EGFR LAMC2 was highly expressed in ATC samples and cell lines compared to normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed migration, invasion and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells, reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered expression of genes associated with migration, invasion, proliferation and survival. Immunoprecipitation studies showed that LAMC2 was bound to EGFR in ATC cells. Silencing of LAMC2 partially blocked EGF-mediated activation of EGFR and its downstream pathway.
Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy having no effective treatment. Laminin subunit gamma-2 (LAMC2) is an epithelial basement membrane protein involved in cell migration and tumour invasion and might represent an ideal target for the development of novel therapeutic approaches for ATC. LAMC2 was highly expressed in ATC samples and cell lines compared to normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed migration, invasion and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells, reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered expression of genes associated with migration, invasion, proliferation and survival. Immunoprecipitation studies showed that LAMC2 was bound to EGFR in ATC cells. Silencing of LAMC2 partially blocked EGF-mediated activation of EGFR and its downstream pathway. LAMC2 was highly expressed in ATC samples and cell lines compared to normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed migration, invasion and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells, reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered expression of genes associated with migration, invasion, proliferation and survival. Immunoprecipitation studies showed that LAMC2 was bound to EGFR in ATC cells. Silencing of LAMC2 partially blocked EGF-mediated activation of EGFR and its downstream pathway.

Specific changes of liver transcriptome in the early stages of copper accumulation in the mouse model of wilson disease

Wilson disease (WD) is a severe metabolic disorder caused by genetic inactivation of copper-transporting ATPase ATP7B.  In WD, copper accumulates in several tissues, particularly in the liver, inducing marked time-dependent pathological changes.  To identify initial events in the copper-dependent development of liver pathology we utilized the Atp7b-/- mice, an animal model for WD.  Analysis of mRNA from livers of control and Atp7b-/- 6 weeks-old mice using oligonucleotide arrays revealed specific changes of the transcriptome at this stage of copper accumulation.  Few messages (29 up-regulated and 46 down-regulated) change their abundance more than 2-fold pointing to the specific effect of copper on gene expression/mRNA stability.  The gene ontology analysis revealed copper effects on distinct metabolic pathways.

The E2A transcription factors promote the development of thymus-seeding cells but it remains unknown whether these proteins play a role in T lymphocyte lineage specification or commitment. By examining E2A-dependent genes in developing T cells, we will address whether these proteins are involved in these processes.

Expression data from wild type versus HDAC knock out mouse embryonic stem cells

Histone deacetylase 1 (HDAC1) is an enzyme that promotes deacetylation of acetylated lysine residues in histones and other proteins. Histone acetylation is often associated with gene activation and expression. Los of HDAC1 leads to severe problems in development and proliferation. Moreover, it seems to be the major histone deacetylase in mouse embryonic stem cells.
We used microarrays to identify putative HDAC1 target genes.

To characterize gene response in RPE65-/- mouse model of Leber’s congenital amaurosis during progression of the disease, we analyzed differential gene expression in retinae early in the development of the disease, namely before and at the onset of photoreceptor cell death in knock-out mice of 2, 4 and 6 months of age.
Keywords: knock out response over time

Genome-wide expression profiling of the retinoschisin deficient retina in C57CL/6 mice.

mRNA expression data were collected from patients with brain tumor to improve diagnostic of gliomas on molecular level.
Keywords: survey

Parathyroid hormone-related protein (PTHrP) is overexpressed in many cancer types. This secretory protein can induce hypercalcaemia of malignancy by mimicking the action of calcium-regulating parathyroid hormone (PTH). PTHrP may also be involved in the regulation of migration, invasion, proliferation and apoptosis. It can either stimulate signal cascades, such as the cAMP pathway, or act on not yet defined targets in the nucleus. In this study, we analyzed the effect of PTHrP-specific siRNA on gene expression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were either transfected with the PTHrP-specific siRNA (siPTHrP) or a control siRNA (siLuc). Differences in gene expression pattern in siPTHrP- vs. siLuc-treated cells were measured by microarray analysis. More than 200 differentially expressed genes were found. Some of these genes have important functions in carcinogenesis.

Gene Expression in White Adipose Tissue of KSR2-/- mice compared to wild type mice

The goal of this study was to define the gene expression changes that take place in the white adipose tissue of KSR2-/- mice at 6 months of age.

Neurotrophin NT3 promotes ovarian primordial to primary follicle transition

Neurotrophins are growth factors that are known to have a role in promoting cell survival and differentiation. The focus of the current study is to examine the role of neurotrophins in regulating ovarian primordial follicle development. Ovaries from 4-day old rats were placed into organ culture and cultured for 10 days in the absence or presence of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF), or nerve growth factor (NGF). Treatment of ovaries with NT3 resulted in a significant (P<0.01) increase in primordial follicle development (i.e. primordial to primary follicle transition). Treatment with BDNF at high doses of 100–250 ng/ml also significantly (P<0.01) increased primordial follicle development, but NGF had no effect. Immunohistochemical studies determined that NT3 was present in granulosa cells, interstitial tissue, and in the oocytes of primordial and primary follicles. The NT3 receptor NTRK3 was present in oocytes at all stages of development. Analysis of ovaries that contain predominantly primordial follicles demonstrated the transcripts for NT3, NTRK3, NGF, and the BDNF/neurotrophin-4 (NT4) receptor NTRK2 are expressed, while BDNF, NT4, and the NGF receptor NTRK1 are not detectable. Inhibition of the NTRK3 receptor with the tyrophostin AG 879 resulted in oocyte death and a significant (P<0.01) reduction in follicle pool size. Inhibition of the NTRK receptors with K252a slowed primordial to primary follicle transition. A microarray analysis demonstrated that a small number of genes were differentially expressed after NT3 treatment. Observations indicate that the neurotrophin NT3, acting through the NTRK3 receptor in oocytes, promotes the primordial to primary follicle transition.  Reproduction (2009) 138, pp. 697-707

We used microarrays to determine genes expressed differentially between control and NT3 (neurotrophin-3) treated P4 ovary.

Nonlesional atopic dermatitis skin is characterized by broad terminal differentiation defects and variable immune abnormalities

Atopic dermatitis (AD) is a common inflammatory skin disease with a T(H)2 and T22 immune polarity. Despite recent data showing a genetic predisposition to epidermal barrier defects in some patients, a fundamental debate still exists regarding the role of barrier abnormalities versus immune responses in initiating the disease. An extensive study of nonlesional AD (ANL) skin is necessary to explore whether there is an intrinsic predisposition to barrier abnormalities, background immune activation, or both in patients with AD. We sought to characterize ANL skin by determining whether epidermal differentiation and immune abnormalities that characterize lesional AD (AL) skin are also reflected in ANL skin.  We performed genomic and histologic profiling of both ANL and AL skin lesions (n = 12 each) compared with normal human skin (n = 10). We found that ANL skin is clearly distinct from normal skin with respect to terminal differentiation and some immune abnormalities and that it has a cutaneous expansion of T cells. We also showed that ANL skin has a variable immune phenotype, which is largely determined by disease extent and severity. Whereas broad terminal differentiation abnormalities were largely similar between involved and uninvolved AD skin, perhaps accounting for the background skin phenotype, increased expression of immune-related genes was among the most obvious differences between AL and ANL skin, potentially reflecting the clinical disease phenotype. Our study implies that systemic immune activation might play a role in alteration of the normal epidermal phenotype, as suggested by the high correlation in expression of immune genes in ANL skin with the disease severity index.

Inflammation; sepsis; innate immunity; T cells; MHC antigen; zinc

Validation of Genome-wide Expression patterns in Pediatric Septic Shock

Rationale:  We previously generated genome-wide expression data in children with septic shock, based on whole blood-derive RNA, having the potential to lead the field into novel areas of investigation.
Objective:  Herein we seek to validate our data through a bioinformatic approach centered on a validation patient cohort.
Methods:  Microarray- and bioinformatics-centered analyses involving our previous data as a training data set (n = 42) and a new, validation cohort (n = 30) as the test data set.
Measurements and Main Results:  Class prediction modeling using the training data set and the previously reported genome-wide expression signature of pediatric septic shock correctly identified 93 to 100% of septic shock patients in the test data set, depending on the class prediction algorithm and the gene selection method. Subjecting the test data set to an identical filtering strategy as that used for the training data set, demonstrated 72% concordance between the two gene lists. Subjecting the test data set to a purely statistical filtering strategy, with highly stringent correction for multiple comparisons, demonstrated less than 50% concordance with the previous gene filtering strategy. However, functional analysis of this statistics-based gene list demonstrated similar functional annotations and signaling pathways as that seen in the learning data set. In particular, we validated that pediatric septic shock is characterized by large scale repression of genes related to zinc homeostasis and lymphocyte function.
Conclusions:  These data demonstrate that the previously reported genome-wide expression signature of pediatric septic shock is applicable to a validation cohort of patients.

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity amongst individuals afflicted with vascular diseases. We obtained BOEC (blood outgrowth endothelial cells) from 20 subjects with sickle cell anemia (age 4-19) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke due to, respectively, having or not having occlusive disease at the Circle of Willis (CoW).
Gene expression profiling identified no significant single gene differences between the two groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were pre-determined to survey each of nine biological systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches

HIF-1 is an important transcription factor for immune responses to bacterial infection. We wanted to analyze the HIF-1 dependent gene expression upon S. aureus infection and analyzed the gene expression of HepG2 nt and HepG2 HIF-1-/- cells four hours upon infection using affymetrix human gene 1.0 st. gene arrays.

Total RNA was prepared from the entire small intestines of 40 day old Control and CFTR null mice (2 males and 1 female of each genotype), congenic on the black6 background, using TRIzol reagent. Mice were fed Peptamen from age 10 days to prevent intestinal obstruction.

breast cancer;chemotherapy;gene expression;microarray;stress response;time-course

Cell-type specific responses to chemotherapeutics in breast cancer

Recent studies have identified clinical subtypes of breast tumors that arise from different cell types and have different outcomes in response to chemotherapy.  Tumors derived from basal epithelium have a poorer prognosis than tumors derived from luminal epithelium.  To gain insight into differences underlying this disparity, we treated cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and those derived from luminal epithelium (MCF-7 and ZR-75-1) with two chemotherapeutics commonly used in the treatment of breast cancer.  Treatment doses for doxorubicin (DOX) and 5-fluorouracil (5FU) were selected to cause comparable cytotoxicity across all four cell lines.  The predominant gene expression in each of the four cell lines was a general stress response, but distinct gene expression patterns were observed depending upon cell type.  Both cell types up-regulated DNA damage response genes such as p21waf1, but the response in the luminal cells was much more dramatic and included many p53-regulated genes.  Luminal cell lines down-regulated a large number of cell cycle regulators and other genes involved in cellular proliferation, while basal cell lines down-regulated a smaller set of genes, many of which are involved in cellular differentiation. These results were compared to gene expression data from tumor samples collected before and after treatment with DOX or 5FU/mitomycin C.  Similarities between the in vitro and in vivo responses validate this model for studying gene expression responses to chemotherapy in these cell types.  Understanding cell-type specific responses to chemotherapeutics will help in tailoring treatment to patients based upon tumor characteristics.

Overexpression of dn-p21ras as a model system for severe dilated cardiomyopathy

Mice heterozygous for dn-p21 littermates were sacrificed at 10 weeks of age. Ventricles were isolated and snap-frozen in liquid nitrogen.  Use 3 month old FVB Females as controls.  [GSM2334, GSM2335, GSM2336]
Read more at http://cardiogenomics.med.harvard.edu/groups/proj1/pages/ras_home.html

Inhibiting tankyrases sensitizes KRAS mutant cancer cells to MEK inhibitors by FGFR2 feedback signaling

Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis and mitosis, offering attractive targets for anti-cancer treatment. Using unbiased combination screening in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors in KRAS mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and anti-tumor activity both in vitro and in vivo than effects observed by previously reported MEK inhibitor combinations. Altogether, our results show how a novel combination of TNKS and MEK inhibitors can be highly effective in targeting KRAS mutant cancers by suppressing a newly discovered resistance mechanism.
This experiment is designed to detect genes differentially expressed in the combination treatment compared to others

Total RNA was prepared using TRIzol reagent from the pancreata of eight week old male mice.  The genotypes were Control: gastrin+/-, CFTR+/+; and CF: gastrin+/-, CFTR-/-.  All mice were on 95% black6, 5% 129Sv background.  Mice were fed Peptamen from age 10 days to prevent intestinal obstruction.

estrogen, estrogen receptor knockout, gene expression, mouse aorta

Estrogen receptors alpha and beta mediation of gene expression in mouse vascular tissue

Estrogen plays an important role in the regulation of vascular tone and in the pathophysiology of cardiovascular disease. Physiological effects of estrogen are mediated through estrogen receptors alpha (ERalpha) and beta (ERbeta), which are both expressed in vascular smooth muscle and endothelial cells. However, the molecular pathways mediating estrogen effects in blood vessels are not well defined. We have performed gene expression profiling in the mouse aorta to identify comprehensive gene sets the expression of which is regulated by long-term (1 wk) estrogen treatment. The ER subtype dependence of the alterations in gene expression was characterized by parallel gene expression profiling experiments in ERalpha-deficient [ERalpha knockout (ERalphaKO)] and ERbeta-deficient (ERbetaKO) mice.
Importantly, these data revealed that ERalpha- and ERbeta-dependent pathways regulate distinct and largely nonoverlapping sets of genes. Whereas ERalpha is essential for most of the estrogen-mediated increase in gene expression in wild-type aortas, ERbeta mediates the large majority of estrogen-mediated decreases in gene expression.  Biological functions of the estrogen-regulated genes include extracellular matrix synthesis, in addition to electron transport in the mitochondrion and reactive oxygen species pathways. Of note, the estrogen/ERbeta pathway mediates down-regulation of mRNAs for nuclear-encoded subunits in each of the major complexes of the mitochondrial respiratory chain. Several estrogen-regulated genes also encode transcription factors. Overall, these findings provide a foundation for understanding the molecular basis for estrogen effects on vasculature gene expression.

Fibroblast growth factor-23 (FGF23), a circulating protein produced in bone, causes renal inorganic phosphate (Pi) wasting by down-regulation of sodium phosphate co-transporter 2a (Npt2a). The mechanism behind this action is unknown. We have previously generated transgenic mice (TG) expressing human wild-type FGF23 under the control of the α1 (I) collagen promoter. In this study we performed a large scale gene expression study of kidneys from TG mice and wild-type littermates. Several genes that play a role in Pi regulation had decreased expression levels, such as Npt2a, but also Pdzk1 which is a scaffolding protein known to interact with NPT2a. Importantly, the Klotho gene, a suggested crucial co-factor for FGF23 receptor binding and activation, was the most affected decreased gene. However, other genes proposed to regulate Pi levels, such as secreted Frizzled Related Protein 4 (sFRP4), Na+/H+ exchanger regulatory factor 1 (NHERF1) and the FGF-receptors 1-4, revealed no changes. Interestingly, expression levels of inflammatory response genes were increased and histological analysis revealed tubular nephropathy in the TG mice kidneys. In conclusion, FGF23 TG mice have altered kidney gene expression levels of several genes thought to be part of Pi homeostasis and an increase in inflammatory response genes, data supported by histological analysis. These findings may lead to further understanding of how FGF23 mediates its actions on renal Pi regulation.

Genome wide gene expression profiles of distal colon from 5% DSS-treated mice after administration of various siRNA against TNFa.

Differential gene expression assessed in siTNF-OMe-P treated animals showed significant correlation between improved colon integrity and clinical parameters of colitis with reduced TLR activation, tissue regeneration and reduced pro-inflammatory cytokines, as compared to all treatment groups.
This data shows the changes in gene expression profile associated with effective silencing of TNFa by a chemically-modified siRNA in the colon of colitic mice.

Gene expression profile of normal v early MDS v non-MDS anemia bone marrow CD34 cells

Early, low risk IPSS (International Prognostic Scoring System) myelodysplasia (MDS) is a heterogeneous disorder where the molecular and cellular haematopoietic defects are poorly understood. To gain insight into this condition, we analyzed gene expression profiles of marrow CD34+ progenitor cells from normal karyotype, low blast count MDS patients, age-matched controls and patients with non-MDS anaemia. The aim of the study was to further understanding of the cellular defect in MDS and to identify biomarkers of disease
Keywords: Disease v normal

Ubiquinol-induced gene expression signatures are translated into reduced erythropoiesis and LDL cholesterol levels in humans

Studies in vitro and in mice indicate a role for Coenzyme Q10 (CoQ10) in gene expression. To determine this function in relationship to physiological readouts, a 2-week supplementation study with the reduced form of CoQ10 (ubiquinol, Q10H2, 150 mg/d) was performed in 53 healthy males. Mean CoQ10 plasma levels increased 4.8-fold after supplementation. Transcriptomic and bioinformatic approaches identified a gene-gene interaction network in CD14-positive monocytes, which functions in inflammation, cell differentiation and PPAR-signaling. These Q10H2-induced gene expression signatures were also described previously in liver tissues of SAMP1 mice. Biochemical as well as NMR-based analyses showed a reduction of LDL cholesterol plasma levels after Q10H2 supplementation. This effect was especially pronounced in atherogenic small dense LDL particles (19-21 nm, 1.045 g/l). In agreement with gene expression signatures, Q10H2 reduces the number of erythrocytes but increases the concentration of reticulocytes. In conclusion, Q10H2 induces characteristic gene expression patterns, which are translated into reduced LDL cholesterol levels and erythropoiesis in humans.

Knock-down of BCL6 expression in human Diffuse Large B-Cell Lymphoma cell lines

This dataset was used to benchmark the Virtual Inference of Protein-activity by Regulon Readout algorithm (VIPER). Despite recent advances in molecular profiling, proteome-wide assessment of protein activity in individual samples remains a highly elusive target. In stark contrast, protein activity quantitation is increasingly critical to the dissection of key regulatory processes and to the elucidation of biologically relevant mechanisms. Importantly, its value extends to the study of drug activity, as most small molecules inhibit activity of their cognate protein substrates without affecting the protein’s or associated mRNA’s abundance.
VIPER leverages the increasingly accurate and context specific knowledge of regulatory networks. Specifically, it uses the expression of the transcriptional targets most directly regulated by a given protein in an individual sample as a reporter for the computational inference of its activity.

Analysis of microdissected invasive lobular and ductal breast carcinomas in relation to normal ductal and lobular cells

The aim of our study was to identify gene expression profiles of ductal and lobular carcinomas in relation to normal ductal and lobular cells. We examined ten mastectomy specimens from postmenopausal breast cancer patients. Ductal and lobular tumor and normal cells were microdissected from cryosections. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. GCOS pairwise comparison algorithm and rank products have identified multiple genes that are differentially expressed in comparisons between ductal and lobular tumor and normal cell types. The results suggest that these genes are involved in epithelial-mesenchymal transition, TGFbeta and Wnt signaling. These changes are present in both tumor types but appear to be more prominent in lobular carcinomas.

CD47 Inhibits Self-renewal and Reprogramming by Regulating c-Myc and Other Stem Cell Transcription Factors

Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells and enables these cells to spontaneously reprogram to form multipotent embryoid bodies.  c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- or thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.

Microarray analysis of differentiation of human glioblastoma stem cells

Glioblastoma multiforme is one of the most devastating cancers and presents unique challenges to therapy due to its aggressive behaviour. Cancer stem cells have been described to be the only cell population with tumorogenic capacity in glioblastoma. Therefore, effective therapeutic strategies targeting these cells may be beneficial. We have established different cultures of glioblastoma stem cells (GSCs) derived from surgical specimens and found that, after induction of differentiation, NFκB was activated, which allows intermediate tumor precursor cells to remain cycling. We also showed that blockade of NFκB signaling in differentiating GSCs by different genetic strategies or treatment with small molecule inhibitors, promoted replication arrest, progression to a mature phenotype, mainly neuronal cells, and senescence. This effect was partly mediated by downregulation of the NFκB target gene cyclin D1. Furthermore, intravenous treatment of immunodeficient mice bearing human GSC-derived tumors with a novel small-molecule inhibitor of the NFκB pathway induced senescence of tumor cells but no ultraestructural alterations of the brain parenchymal cells were detected. These findings reveal that activation of NFκB may keep differentiating GSCs from acquiring a mature postmitotic phenotype, thus allowing cell proliferation, and support the rationale for therapeutic strategies aimed at promoting premature senescence in GSCs undergoing differentiation.

All-trans retinoic acid effect on the squamous cell carcinoma cell line ME180: time course

All-trans retinoic acid (ATRA) has been shown to have anti-proliferative effects, particularly in the context of cancer.  However, the effects of ATRA on gene and microRNA expression in solid tumors have not been investigated.  In this study, we performed gene expression and microRNA analysis of the squamous cell carcinoma cell line, ME180, following treatment with 10 micromolar all-trans retinoic acid (ATRA) for 1, 3, and 6 hours.  Results provide insight into the temporal regulation of genes and microRNAs by retinoids.

Screening for Epigenetically Masked Genes in Colorectal Cancer using 5-aza-2’-deoxycytidine treatment, Microarray and Gene Expression Profile

Unearthing of silenced genes in colorectal cancer (CRC) is of great importance. We employed oligonucleotide microarray to find changes in global gene expression of five CRC cell lines. These were analyzed before and after treatment with the 5-aza-2'-Deoxycitidine. Expression of the responding genes was integrated with gene expression profiling generated by microarray analysis of matched colorectal tissue samples. Selected candidates were subjected to methylation-specific PCR (MSP) and real-time quantitative reverse transcription-PCR using CRC cell lines and paired tumor and normal samples from CRC patients. Sixty eight genes were re-expressed after 5-aza-2'-Deoxycitidine treatment and over-expressed in normal colorectal mucosa, including genes that were known to be methylated in CRC. After applying study selection criteria, we identified 16 potential genes. Two candidates were selected (ASPP1 and SCARA5). Among 15 CRC cell lines, methylation was identified in SCARA5 (20%). The methylation status of SCARA5 was subsequently investigated in 23 paired colorectal tissue samples; methylation was detected in 17%, respectively. Observed promoter methylation showed a tendency towards methylation in tumor-derived samples, in SCARA5 gene. Significant down expression of SCARA5 mRNA was observed in CRC cell lines and tumor tissues compared to adjacent normal tissues (P < 0.001 and P = 0.001, respectively). The use of genome-wide screening led to the identification of a group of candidate genes. Among them, SCARA5 was methylated and markedly down-regulated in CRC. SCARA5 gene may have a role in CRC tumorigenesis.

Divergent genome wide transcriptional profiles from immune cell subsets isolated from SLE patients with different ancestral backgrounds

Background/Purpose:  Systemic lupus erythematosus (SLE) is a complex multi-system autoimmune disease of uncertain etiology. Patients from different ancestral backgrounds demonstrate differences in clinical manifestations and autoantibody profiles.  In this study we examined genome-wide transcriptional patterns in major immune cell subsets across different ancestral backgrounds.

Methods:  Peripheral blood was collected from 21 African-American (AA) and 21 European-American (EA) SLE patients, 5 AA controls, and 5 EA controls. CD4+ T-cells, CD8+ T-cells, monocytes and B cells were purified by flow sorting. Each cell subset from each subject was run on an Illumina HumanHT-12 V4 expression BeadChip array (n=208 arrays). Differentially expressed genes (DEGs) were determined by comparing cases and controls of the same ancestral background.

Results: The overlap in DEG lists between different cell types from the same ancestral background was very modest (<1%).  Typically between 5-10% of DEGs were shared when comparing the same cell type between different ancestral backgrounds (for ex. CD20 AA vs. CD20 EA).  Quantitative measurement of global IFN-stimulated gene (ISG) expression revealed that AA subjects demonstrated more concordance across all studied cell types. Two subgroups of patients were identified based on the ISG expression profiles. One subgroup showed higher ISGs expression in all cell types, and the other subgroup had higherISG expression only in T and B lymphocytes but not in monocytes.  The correlation of ISG expression with medication data revealed that only the B cells had lower ISG expression in patients taking immunosuppressants, while ISG expression in the other cell types did not differ based upon medication use.

Conclusion: We find striking differences in gene expression between different immune cell subsets and between ancestral backgrounds in SLE patients.  The IFN signature is diverse, with different transcripts represented in different cell populations, and signature-positive cell subsets differed in EA vs. AA patients. We also find that treatment with the immunosuppressive agents correlates with the down-regulation of B cell ISG expression, and this was not observed in other cell types.

Expression data from Down syndrome and non-Down syndrome pediatric acute lymphoblastic leukemia cases

Gene expression profiling (GEP) can reveal characteristic signatures associated with distinct biologic subtypes of acute lymphoblastic leukemia (ALL).
We performed GEP on Down syndrome (DS) and comparison non-Down syndrome (NDS) ALL cases to identify biologic differences between these groups.

Expression of HCT116 cells transfected with CD44 siRNAs relative to that of HCT116 cells transfected with a control siRNA

To examine whether depletion of CD44 might affect antioxidant gene expression in cancer cells, HCT116 cells were transfected with CD44 or control siRNAs and subjected to cDNA microarray analysis. Although the CD44 siRNA was found to deplete the cells of CD44 mRNA, the expression of antioxidant genes was largely unaffected.

Transcriptional signatures of Itk-deficiency using CD3+, CD4+ and CD8+ T-cells

The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

Janus-Like Opposing Roles of CD47 in Autoimmune Brain Inflammation in Human and Mice

Gene transcripts and proteins expressed during disease pathogenesis identify targets for therapy. We performed microarray analysis of histologically characterized multiple sclerosis (MS) brain lesions in comparison with control brain samples to identify differentially expressed molecules. We identified CD47 as a target of interest and studied its biology in MS and EAE.

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

overexpression of different antioxidant response element activators

A genomic screen for activators of the antioxidant response element

The antioxidant response element (ARE) is a cis-acting regulatory enhancer element found in the 5’ flanking region of many phase II detoxification enzymes.  Upregulation of ARE-dependent target genes is known to have neuroprotective effects; yet, the mechanism of activation is largely unknown.  By screening an arrayed collection of approximately 15,000 full-length expression cDNAs in the human neuroblastoma cell line IMR-32 with an ARE-luciferase reporter, we have identified several cDNAs not previously associated with ARE activation.  A subset of cDNAs, including sequestosome 1 (SQSTM1) and dipeptidylpeptidase III (DPP3), activated the ARE in primary mouse-derived cortical neurons.  Overexpression of SQSTM1 and DPP3 in IMR-32 cells stimulated NRF2 nuclear translocation and led to increased levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a protein which is transcriptionally regulated by the ARE.  When transfected into IMR-32 neuroblastoma cells that were depleted of transcription factor NRF2 by RNA interference, SQSTM1 and DPP3 were unable to activate the ARE or induce NQO1 expression, indicating that the ARE activation upon ectopic expression of these cDNAs is mediated by NRF2.  Studies with pharmacological inhibitors indicated that 1-phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) signaling are also essential for activity.  Lastly, overexpression of these cDNAs conferred partial resistance to hydrogen peroxide induced toxicity, consistent with the induction of antioxidant and phase II detoxification enzymes which can protect from oxidative stress.  This work and other such studies may provide mechanisms for activating the ARE in the absence of general oxidative stress, and a novel therapeutic approach to degenerative diseases and aging.
Keywords: overexpression of different antioxidant response element activators

Expression data of HL-60 leukemia cells after treatment with Curcumin and TCDD

Curcumin has antileukemic potential that is mediated by dfifferent pathways. Because we could not confirm that the Arylhydrocarbonreceptor, a ligand activated transcription factor, is involved in curcumins antileukemic effects, we performed microarray experiments to have an overall screening of the pathways affected by curcumin in HL-60 cells.
We identified an involvement of the vitamin D receptor in the effects of curcumin on HL-60 cells by comparing our results with other experiments.

Expression data of ATM, ATR, CHK1, CHK2, and 53BP1 shRNA knockdown and control shRNA in MCF-10A cells

Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information.

Hematopoietic stem cells, myeloid progenitors, oxidative stress, ROS, Affymetrix Mouse Genome 430 2.0

FoxO are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress

To investigate the role of FoxO transcription factors as mediators of hematopoietic stem cell resistance to oxidative stress.
Keywords: Hematopoietic stem cells, myeloid progenitors, oxidative stress, ROS, Affymetrix Mouse Genome 430 2.0

Gene Expression Profiling of Rat Hippocampus Following Exposure to the Acetylcholinesterase Inhibitor Soman

Soman (O-Pinacolyl methylphosphonofluoridate) is a potent neurotoxicant.  Acute exposure to soman causes profound inhibition of the critical enzyme acetylcholinesterase, resulting in excessive levels of the neurotransmitter acetylcholine.  Excessive acetylcholine levels cause convulsions, seizures, and respiratory distress.  The initial cholinergic crisis can be overcome by rapid anti-cholinergic therapeutic intervention, resulting in increased survival.  However, conventional treatments do not protect the brain from seizure-related damage, and thus neurodegeneration of soman-sensitive areas of the brain is a potential post-exposure outcome.  We performed gene expression profiling of rat hippocampus following soman exposure to gain greater insight into the molecular pathogenesis of soman-induced neurodegeneration.

Expression data from murine hematopoietic stem cells isolated from mice treated with control oil or estradiol

The division rate of hematopoietic stem cells (HSCs) are promoted by estradiol. To identify the mechanism by which estradiol regulates HSCs, we performed gene expresssion profiling of HSCs isolated from mice of both sexes treated with either control vehicle (oil) or estradiol for one week.

Gene expression response to implanted drug (paclitaxel)-eluting or bare metal stents in denuded human LIMA arteries

[original title] Gene expression response to the implantation of drug (paclitaxel)-eluting or bare metal stents in denuded human LIMA arteries.

Different clinical outcomes have been observed for paclitaxel-eluting and bare metal cardiovascular stents. The aim of this project was to identify genes that might be associated with the observed clinical outcomes.

Acute myeloid leukemia (AML) is one of the most common and deadly forms of hematopoietic malignancies.  We hypothesized that microarray studies could identify previously unrecognized expression changes that only occur only in AML blasts. We were particularly interested in those genes with increased expression in AML, believing that these genes may be potential therapeutic targets.
Keywords: AML vs Normal hematopoietic cells

Microarray analysis to examine the relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (CPR) null mice.

Whole tissues corresponding to the proximal and distal small intestine and to the colon were dissected from each rat, washed with ice-cold saline solution and immediately frozen in liquid nitrogen. Rats were either control or treated with Amoxicillin (100mg/kg/day). Small intestine was divided in two identical length considered as the proximal and distal part. In each intestinal part 1 cm of tissue was taken in the middle of this part to perform the RNA extraction and then the GeneArray analysis.

Extremely variable clinic and genetic features characterize Mitochondrial Encephalomyopathy Disorders (MED). Pathogenic mitochondrial DNA (mtDNA) defects can be divided into large-scale rearrangements and single point mutations. Clinical manifestations become evident when a threshold percentage of the total mtDNA is mutated. In some MED, the "mutant load" in an affected tissue is directly related to the severity of the phenotype. However, the clinical phenotype is not simply a direct consequence of the relative abundance of mutated mtDNA. Other factors, such as nuclear background, can contribute to the disease process, resulting in a wide range of phenotypes caused by the same mutation. Using Affymetrix oligonucleotide cDNA microarrays (HG-U133A), we studied the gene expression profile of muscle tissue biopsies obtained from 12 MED patients (4 common 4977-bp deleted mtDNA and 8 A3243G: 4 PEO and 4 MELAS phenotypes) compared with age-matched healthy individuals.
Keywords = mtDNA mutation

Gene expression profile in HUVECs before and after Angiopoietin stimulation

Angiopoietin-Tie2 sytem has been inplicated in both vascular quiescence and angiogenesis. It is unclear how these two opposing signals are regulated from the same receptor-mediated intracellular signal transduction. We have noticed that Tie2 localization upon Angiopoietin stimulation depends upon the presence or absence of cell-cell contacts.
Therefore, to identify the downstream signaling of Tie2 upon Angiopoietin stimulation, we performed DNA microarray analyais using RNAs obtained from confluent human umbirical vein endothelial cells (HUVECs) or sparse HUVECs stimulated with after Angiopoietin-1. There is striking difference on gene expression profile between confluent and sparse HUVECs.

Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced inter-patient heterogeneity. To characterize RA at the molecular level and to uncover key pathomechanisms, we performed whole-genome gene expression analyses. Synovial tissues from rheumatoid arthritis patients were compared to those from osteoarthritis patients and to normal donors.

Expression data from LNCap cell line treated with COP1 (RFWD2), ETV1, and JUN siRNAs

The proto-oncogenes ETV1, ETV4, and ETV5 encode members of the E26 transformation-specific (ETS) transcription factor family, which includes the most frequently rearranged and overexpressed genes in prostate cancer.  Despite being critical regulators of development, little is known about their post-translational regulation.  Here we identify the ubiquitin ligase COnstitutive Photomorphogenic-1 (COP1, also called RFWD2) as a tumor suppressor that negatively regulates ETV1, ETV4, and ETV5.  ETV1, which is the member mutated more frequently in prostate cancer, was degraded after being ubiquitinated by COP1.  Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs (degrons) and was 50-fold more stable than wild-type ETV1.  Almost all patient translocations eliminate these ETV1 degrons, implying that translocations rendering ETV1 insensitive to COP1 confer a significant selective advantage to prostate epithelial cells.  Indeed, COP1 deficiency in mouse prostate elevated ETV1 levels and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia.  The combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas.  Finally, relatively rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein expression, and abnormally elevated ETV1 protein while lacking a translocation event.  These findings identify COP1 as a bona fide tumor suppressor whose down-regulation promotes prostatic epithelial cell proliferation and tumorigenesis.

Gene Expression Profiles of MYC+ and MYC- mouse Germinal Center B cells

Germinal centers (GC) arise within B cell follicles upon antigenic challenge. In the dark zones (DZ) of GCs, B cells proliferate and hypermutate their immunoglobulin genes, and mutants with increased affinity are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells, or re-enter the DZ for further refinement. However, the molecular circuits governing GC positive selection are not known. Here, we show that the GC reaction requires the biphasic regulation of c-MYC expression, involving its transient induction during early GC commitment, its repression by BCL6 in DZ B cells, and its re-induction in a subpopulation of positively selected LZ B cells destined to DZ re-entry. Accordingly, acute disruption of MYC function in vivo leads to GC collapse, indicating an essential role in GC physiology. These results have implications for our understanding of GC selection and the role of MYC deregulation in B cell lymphomas.
We used microarrays to determine the global gene expression programs that distinguish MYC+ GC B cells from their MYC- negative counterparts.

The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity leading to the development of a steroid independent tumour.  The p160 steroid coactivator protein SRC-1, through interactions with developmental proteins and other non-steroidal transcription factors drives this tumour adaptability.  Here, using discovery studies we identify ADAM22, a non-protease member of the ADAMs family, as a direct target of SRC-1, independent of estrogen receptor(ER). Molecular, cellular, in vivo and clinical studies confirmed SRC-1 as a regulator of ADAM22 and established a role for ADAM22 in endocrine resistant tumour progression.  ADAM22 has the potential to act as a therapeutic drug target and a companion predictive biomarker in the treatment of endocrine resistant breast cancer.

Nfix is a novel regulator of murine hematopoietic stem and progenitor cell survival

Hematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates.  Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPC) of murine adult bone marrow.  Although shRNA mediated knockdown of Nfix expression in Lineage-Sca-1+c-Kit+ HSPC had no effect on in vitro cell growth or viability, Nfix-depleted HSPC displayed a significant loss of colony forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity.  Analysis of recipient mice 4-20 days post-transplant revealed that Nfix-depleted HSPC establish in the bone marrow but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPC reveals that loss of Nfix expression in HSPC is concomitant with a decrease in the expression of multiple genes known to be important for HSPC survival, such as Erg, Mecom, Mpl and Prdm16.  These data reveal that Nfix is a novel regulator of HSPC survival post-transplantation and establish, for the first time, a role for Nfi genes in the regulation of this cellular compartment.

The lipocalin Apolipoprotein D (ApoD), known to protect the nervous system against oxidative stress (OS) in model organisms, is up-regulated early in the mouse brain in response to the ROS generator paraquat (PQ). However, the processes triggered by this up-regulation have not been explored.

The AP-1 Transcription Factor c-Jun Prevents Stress-Imposed Maladaptive Remodeling of the Heart

Systemic hypertension increases cardiac workload and subsequently induces signaling networks in heart that underlie myocyte growth (hypertrophic response) through expansion of sarcomeres with the aim to increase contractility. However, conditions of increased workload can induce both adaptive and maladaptive growth of heart muscle. Previous studies implicate two members of the AP-1 transcription factor family, junD and fra-1, in regulation of heart growth during hypertrophic response. In this study, we investigate the function of the AP-1 transcription factors, c-jun and c-fos, in heart growth. Using pressure overload-induced cardiac hypertrophy in mice and targeted deletion of Jun or Fos in cardiomyocytes, we show that c-jun is required for adaptive cardiac hyphertrophy, while c-fos is dispensable in this context. c-jun promotes expression of sarcomere proteins and suppresses expression of extracellular matrix proteins. Capacity of cardiac muscle to contract depends on organization of principal thick and thin filaments, myosin and actin, within the sarcomere. In line with decreased expression of sarcomere-associated proteins, Jun-deficient cardiomyocytes present disarrangement of filaments in sarcomeres and actin cytoskeleton disorganization. Moreover, Jun-deficient hearts subjected to pressure overload display pronounced fibrosis and increased myocyte apoptosis finally resulting in dilated cardiomyopathy. In conclusion, c-jun but not c-fos is required to induce a transcriptional program aimed at adapting heart growth upon increased workload.
Microarrays were used to identify specific genes that might be globally affected in the absence of c-jun in cardiomyocytes.

Microglia are resident CNS immune cells that are active sensors in healthy brain and versatile effectors under pathological conditions. Cerebral ischemia induces a robust neuroinflammatory response that includes marked changes in the gene expression and phenotypic profile of a variety of endogenous CNS cell types (astrocytes, neurons, microglia) as well as an influx of leukocytic cells (neutrophils, macrophages, T-cells) from the periphery. Many molecules and conditions can trigger a transformation of “resting” (or surveying) microglia to an “activated” (alerted/reactive) state. Here we review recent developments in the literature that relate to microglial activation in the experimental setting of in vitro and in vivo ischemia. We also present new data from our own laboratory demonstrating the direct effects of in vitro ischemic conditions on the microglial phenotype and genomic profile. Emphasis is placed on the role of specific molecular signaling systems such as hypoxia inducible factor-1 (HIF-1) and toll-like receptor-4 (TLR4) in regulating the microglial response in this setting. We then review histological and recent novel radiological data that confirms a key role for microglial activation in the setting of ischemic stroke in humans. We discuss recent progress in the pharmacological and molecular targeting of microglia in acute ischemic stroke. Finally, we explore how recent studies on ischemic preconditioning have increased interest in preemptively targeting microglial activation in order to reduce stroke severity.

A Regulatory Pathway Involving Notch1/β-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate

The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation.  Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

Microarray of cardiac biventricle from PGC-1a-/-bf/f/MerCre mice

The following abstract from the submitted manuscript describes the major findings of this work.
The metabolic development of high energy-utilizing organs such as the heart involves mitochondrial proliferation at birth followed by a maturation process during the postnatal period. Conditional gene targeting was used in mice to explore the role of the PPARgamma coactivator 1 (PGC-1) coactivators during postnatal development and in adult heart. Marked mitochondrial derangements were observed in hearts of PGC-1a/b-deficient mice during the postnatal period, including fragmentation and elongation associated with the development of a lethal cardiomyopathy. The expression of multiple genes involved in mitochondrial fusion and fission was downregulated in hearts of PGC-1a/b-deficient mice. PGC-la was shown to activate transcription of the mitofusin 1 (Mfn1) gene by coactivating the estrogen-related receptor a (ERRa) upon a highly conserved element.  Surprisingly, PGC-1a/b deficiency did not alter cardiac function or general mitochondrial density and myocyte distribution in adult heart. However, transcriptional profiling and mitochondrial function studies demonstrated that the PGC-1 coactivators are required for full respiratory capacity and high level expression of nuclear- and mitochondrial-encoded genes involved in mitochondrial energy transduction and oxidative phosphorylation pathways in adult heart. These results unveil distinct developmental stage-specific transcriptional programs involved in the maturation and maintenance of mitochondria.

Human leukemia cell line RS4.11 was treated with GSK-3 inhibitor SB216763 for 20 hours.  Gene expression profiling was performed to analyze genes affected by GSK-3 inhibition.

Jag1-dependent gene expression in human endometrial stromal cells

The goal is to investigate gene regulation in endometrial stromal cells expressing the Notch ligand Jag1.

The aim of this study was to investigate whether the differences in memory decline associated with aging are a result of differences in gene expression.  We first categorized age-unimpaired and age-impaired rats based on their performance in the Morris Water Maze and then isolated messenger RNA from the CA1 hippocampal region of each animal to interrogate Affymetrix microarrays.  Microarray analysis (p<0.005) identified a set of 50 genes that were transcribed differently in age-unimpaired animals that had successfully learned a spatial task compared to aged learning-impaired animals and a variety of groups designed to control for all non-learning aspects of exposure to the water maze paradigm.
Keywords: behavior comparison, age comparison

Distinct Gene Expression Profiles in Adult Mouse Heart Following Targeted  MAP Kinase Activation

Three major MAP kinase signaling cascades, ERK, p38 and JNK, play significant roles in the development of cardiac hypertrophy and heart failure in response to external stress and neural/hormonal stimuli.  In order to study the specific function of each MAP kinase branch in adult heart, we have generated three transgenic mouse models with cardiac specific and temporally regulated expression of activated mutants of Ras, MKK3 and MKK7, which are selective upstream activators for ERK, p38 and JNK, respectively.  Gene expression profiles in transgenic adult hearts were determined using cDNA microarrays at both early (4-7 days) and late (2-4 weeks) time points following transgene induction.  From this study, we revealed common changes in gene expression among the three models, particularly involving extracellular matrix remodeling.  However, distinct expression patterns characteristic for each pathway were also identified in cell signaling, growth and physiology.  In addition, genes with dynamic expression differences between early vs. late stages illustrated primary vs. secondary changes upon MAP kinase activation in adult hearts. These results provide an overview to both short term and long term effects of MAP kinase activation in heart and support some common as well as unique roles for each MAP kinase cascade in the development of heart failure.
Keywords: MAP Kinase induction comparison, time course

WM164 BRAF mutant Melanoma cells were treated with 500nM PLX4032 for 16 days and their gene expression was compared to parent cells kept in DMSO.

Mediator complex has been known as pivotal regulator of RNA polymerase II. Mediator complex has two CDK subunits in vertebrates, named CDK8 and CDK19.  To elucidate functional difference between CDK8 and CDK19 in human cell, we employ siRNA mediate knockdown assay using HeLa S3 cell line. According to this assay these CDKs possess highly redundancy in HeLa S3 cell transcription regulation mechanism but in several genes, each CDK shows gene specific regulatory function.

Gene expression profile of the Cmah gene depletion on mouse liver, lung, kidney and heart

N-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases. To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (liver, lung, kidney, and heart) from a control mouse and a Cmah-null mouse.

Pre-LVAD and explanted ischemic and nonischemic cardiomyopathy and nonfailing hearts all normalized with RMA

Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation locus. Pitx2 haploinsufficient mice are prone to pacing induced atrial fibrillation indicating that reduced Pitx2 promotes an arrhythmogenic substrate within the atrium. Here, we inactivated Pitx2 in postnatal heart and discovered that unstressed adult Pitx2 mutant mice had sinus node dysfunction with impaired atrial conduction, an arrhythmia closely associated with atrial fibrillation.
A genome-wide search for Pitx2 transcriptional targets using ChIP-sequencing and RNA expression profiling shows that Pitx2 represses target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators many of which have been implicated in human atrial fibrillation by genome wide association studies.

Our present study reveals significant decelerating effects on senescence processes in middle-aged SAMP1 mice supplemented for 6 or 14 months with the reduced form (QH2, 500 mg/ kg BW/ day) of coenzyme Q10 (CoQ10). To unravel molecular mechanisms of these CoQ10 effects, a genome-wide transcript profiling in liver, heart, brain and kidney of SAMP1 mice supplemented with the reduced (QH2) or oxidized form of CoQ10 (Q10) was performed. Liver seems to be the main target tissue of CoQ10 intervention, followed by kidney, heart and brain. Stringent evaluation of the resulting data revealed that QH2 has a stronger impact on gene expression than Q10, which was primarily due to differences in the bioavailability. Indeed, we found that QH2 supplementation was more effective than Q10 to increase levels of CoQ10 in the liver of SAMP1 mice (54.92-fold and 30.36-fold, respectively). To identify functional and regulatory connections of the “top 50” (p < 0.05) up- and down-regulated QH2-sensitive transcripts in liver (fold changes ranging from 21.24 to -6.12), text mining analysis (Genomatix BiblioSphere, GFG level B3) was used. Hereby, we identified 11 QH2-sensitive genes which are regulated by PPAR-α and are primarily involved in cholesterol synthesis (e.g. HMGCS1, HMGCL, HMGCR), fat assimilation (FABP5), lipoprotein metabolism (PLTP) and inflammation (STAT-1). Thus, we provide evidence that QH2 is involved in the reduction of fat and cholesterol synthesis via modulation of the PPAR-α signalling pathway. These data may explain, at least in part, the observed effects on decelerated age-dependent degeneration processes in QH2-supplemented SAMP1 mice.

Gene expression profiling of ATRA-differentiated wild-type and TG2 knockout NB4 cells

Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNAi-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions and their silencing lead to reduced adhesive, migratory and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, 3, 22, 24 and cytokines IL1B and IL8 involved in the development of differentiation syndrome (DS) are expressed at significantly lower levels in TG2-KD NB4  cells than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of DS.

Expression data from CD71+ cells from the bone marrow of WT, CD70TG, IFNg-/- and CD70TG*IFNg-/- mice.

CD70TG mice are a model for sterile chronic immune activation and develop Anemia of Inflammation, which is dependent on the production of Ifng by effector CD4 and CD8 T cells.
We used microarrays to identify Ifng-dependent differentially expressed genes that could account for the erythropoietic defect.

The effect of resection of mouse livers on survival serve as a model for the effect of resection of human livers, which are performed for both the purposes of removing sick tissue and for transplanting healthy tissue. When less than 70% of a mouse liver is resected, the mouse usually lives. However, when more than 70% is resected, there is an increased probability that the mouse will die. We have found that deletion of Ager (Advanced Glycation End products receptor) (Rage) increases the chance of mouse survival relavtive to WT. Conversely, the deletion of Myd88 decreases the chances of survival, as does the simultaneous deletion of both Ager and Myd88. To understand these genotype/phenotype relations, we performed gene expression measurements.

Arx is a paired-box homeodomain transcription factor and the vertebrate ortholog to the Drosophila aristaless (al) gene.  Mutations in Arx are associated with a variety of human diseases, including X-linked infantile spasm syndrome (OMIM: 308350), X-linked myoclonic epilepsy with mental retardation and spasticity (OMIM: 300432), X-linked lissencephaly with ambiguous genitalia (OMIM: 300215), X-linked mental retardation 54 (OMIM: 300419), and agenesis of the corpus callosum with abnormal genitalia (OMIM: 300004).  Arx-deficient mice exhibit a complex, pleiotrophic phenotype, including decreased proliferation of neuroepithelial cells of the cortex, dysgenesis of the thalamus and olfactory bulbs, and abnormal nonradial migration of GABAergic interneurons.  It has been suggested that deficits in interneuron specification, migration, or function lead to loss of inhibitory neurotransmission, which then fails to control excitatory activity and leads to epilepsy or spasticities.  Given that Arx mutations are associated with developmental disorders in which epilepsy and spasticity predominate and that Arx-deficient mice exhibit deficits in interneuron migration, understanding the function of Arx in interneuron migration will prove crucial to understanding the pathology underlying interneuronopathies.  Yet, downstream transcriptional targets of Arx, to date, remain unidentified.
The aim of this project is to identify bona fide transcriptional targets for the Arx, a transcription factor required for normal migration of interneurons from the ganglionic eminences to the cortex, and to investigate the functions of these genes in the Arx-dependent pathway regulating nonradial neuronal migration.
We hypothesize that the genes regulated by the Arx transcription factor will play a critical role in the nonradial migration of interneurons and that the results of this study will provide novel insights into the molecular mechanisms of nonradial neuronal migration, in particular, and possibly the molecular and biochemical pathogenesis underlying epilepsy, mental retardation, infantile spasm syndromes, and other so-called interneuronopathies;
We have recently generated a transgenic mouse with a floxed Arx allele (Arxflox).  We have generated conditional knockouts in which Arx is removed specifically from the brain by mating Arxflox mice with transgenic mice expressing Cre behind the neural tube-specific transcriptional regulatory elements of the POU domain, class 3, transcription factor 4 promoter.  Preliminary analyses of these mice suggest that conditional knockout mice recapitulate the nonradial migration defects associated with conventional knockout mice.  We will compare the gene expression profiles of ganglion eminences (GEs; the anatomical source for nonradially migrating interneurons) from male Arxflox mice that express Pou3f-Cre to those from male mice without Arxflox allele.  Animals will be prepared and sacrificed following our institutional protocol.  Tissue will be rapidly dissected from E14.5 (the temporal peak of interneuron migration) GEs (MGE and LGE from both left and right hemispheres).  Preliminary experiments suggest that the amounts of RNA that can be isolated from a pair of GEs is in the range of 2000-3500 ng, which should be sufficient for microarray analysis following linear amplification of RNA.  The GEs from each animal will be combined, snap frozen in liquid nitrogen, and stored at -80 C until RNA is extracted.  Total RNA will be extracted using Trizol followed by RNA purification with the RNeasy cleanup kit.  We will be providing total RNA samples from four wildtype and four transgenic animals (true biological replicates) from three separate litters to mitigate any expression differences resulting from mouse to mouse or litter to litter variation.

Immature, ovariectomized C57BL/6 mice were treated with sesame oil (vehicle) or with 0.1 mg/kg 17-alpha-ethynylestradiol (EE) for the length of time indicated. Mice received one dose, except for the 3x24hr samples, which received 3 consecutive daily doses and were sacrificed 24hr after the final dose. Two replicates of each sample are provided. In addition, one time=0 sample is included.
Keywords = uterus estrogen time mouse

Gene expression data from oncogene transformed murine prostate epithelial cell lines

Primary prostate epithelial cell cultures were established from the VENTRAL prostates of FVB mice .  Cells were transduced with retroviral expression vectors encoding a single distinct oncogene (c-Myc, Ha-Ras (V-12), v-Src and NeuT, an activating mutant of ErbB2).   Individual colonies of oncogene-transduced cells were selected and characterized.  These cell lines demonstrate contact-independent growth characteristics in soft agar and in immune competent mice.
To further characterize the molecular genetic signaling pathways regulated by specific oncogenes in prostate epithelial cells

Peripheral blood cells expression data form 7 patients with severe pdm(H1N1) influensa and 7 gender and age matched healthy controls

Using PAXgene tubes, peripheral blood samples were collected  from seven patients >18 years with documented pdm(H1N1) influenza, bilateral chest infiltrates, and in need of ventilation support. Significant co-morbidity was exclusion criterion. Expression profiles were compared with 7 age matched controls. Using  a false discovery rate < 5% and  an absolute fold change > 2, 370 genes  were differentially expressed in case and controls.
A second sample was collected after ca 6 days in the 7 patients and temporal changes in expression profiles investigated. Over expressed putative mediators of inflammatory lung dammage were measured on the protein level.

Comparative transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) isolated from patients in secondary prevention of CVD taking placebo, grape extract or resveratrol-enriched grape extract

We used Affymetrix microarrays to investigate gene expression changes in PBMCs isolated from male patients ongoing secondary prevention of CVD to determine significant modulatory effects that may have been induced by the intake of an initial dose of 8 mg of resveratrol-enriched grape extract for 6 months and then, 16 mg for a further 6 months.

Monocyte gene expression profiling in familial combined hyperlipidemia and its modification by atorvastatin treatment

Introduction: The genetic origin of familial combined hyperlipidemia (FCH) is not well understood. We used microarray profiling of peripheral blood monocytes to search  novel genes and pathways involved in FCH. Methods: Fasting plasma for determination of lipid profiles, inflammatory molecules, and adipokines was obtained and peripheral blood monocytes were isolated from male FCH patients basally and after 4 weeks of atorvastatin treatment. Sex-, age- and adiposity-matched controls were also studied. Gene expression profile was analyzed using Affymetrix Human Genome U133A 2.0 GeneChip arrays. Results: Analysis of gene expression by cDNA microarrays showed that 82 genes were differentially expressed in FCH monocytes compared to controls. Atorvastatin treatment modified the expression of 87 genes. Changes in the expression of some genes, confirmed by real time RT-PCR, (CD36, leucine-rich repeats and immunoglobulin-like domains-1, tissue factor pathway inhibitor 2, myeloid cell nuclear differentiation antigen tumor necrosis factor receptor superfamily, member 25 and CD96) may be related to a proinflammatory environment in FCH monocytes, which is partially reversed by atorvastatin. Higher plasma levels of triglycerides and free fatty acids and lower levels of adiponectin in FCH patients could also trigger changes in gene expression that atorvastatin cannot modify. Conclusions: Our results demonstrate clear differences in gene expression in FCH monocytes compared with those of matched healthy controls, some of which are influenced by atorvastatin treatment.
Keywords: comparative study differential gene expression

Effect of methylphenidate on gene expression profiles of Attention Deficit Hyperactivity Disorder patients and unaffected controls.

Attention deficit hyperactivity disorder (ADHD) is a common psychiatric condition of children with a prevalence of 5-10% worldwide. Up to 30% of adults with a history of childhood ADHD maintain symptoms in later life; these adult ADHD patients  are severely impaired in social and professional life due to persistence of ADHD core symptoms like impulsivity, attention deficit and hyperactivity as well as frequently observed co-morbidities like alcohol and drug abuse, major depression, bipolar and personality disorders.
Pharmaceutical treatment options include methylphenidate (MPH), which is amongst others an inhibitor of the dopamine transporter and therefore increases dopamine levels in the brain. However, not all ADHD patients are MPH responders with clinical features to distinguish responders and non-responders being not at hand so far. Likewise, neurobiological reasons for drug response are still elusive.

A number of IFN-induced proteins are believed to be responsible for the antiviral state induced by IFNs. Our microarray analysis of IFN-regulated genes in MEFs from wild-type mice identified 124 probe sets that were differentially regulated upon IFN treatment. This group consisted of many known ISGs such as Ifit1, Gbp2, mx1, Isg15, Stat1, nmi, mx2, If204, Adar, Irf1, and protein kinase R.

Expression data of CHK1 shRNA knockdown and control shRNA in U2OS cells

Pluripotency-associated transcription factor Foxd3 is required for maintaining pluripotent cells. However, the molecular mechanisms underlying its function are largely unknown. Here, we report that Foxd3 maintains the ESC identity through counteracting differentiation induction of Calcienurin-NFAT signaling.
We used microarrays to detail the global programme of gene expression underlying overexpression of Foxd3 and NFATc3 in ESCs or deletion of Foxd3 in ESCs

Multiscale Gene Networks Dissect the Complexity of Breast Cancer

Despite the enormous amounts of molecular, cellular, and clinical data that are increasingly available for many different types of cancer, it remains a challenge to integrate different dimensions of data to construct mechanistic models that can robustly distinguish key driver genes from passenger genes, predict tumor progression, and tailor therapies optimally for individual patients. We present an integrative biology approach to constructing and analyzing multiscale regulatory networks of breast cancer. We systematically uncover not only known and novel gene subnetworks (modules) linked to breast cancer, but also their key drivers, the majority of which are not transcription factors or signaling molecules. A number of independent lines of evidence support that the predicted key drivers play central roles in breast cancer biology. We predict and experimentally validate ARF1 as a key driver of breast tumor phenotypes, and then demonstrate the ARF1-controlled subnetwork is a novel regulator of intra- and inter- cellular vesicle dynamics involved in epithelial-mesenchymal transition (EMT).

Neurofibromatosis 1 (NF1) is a genetic, neurocutaneous syndrome caused by a mutation in the gene encoding neurofibromin. Individuals with NF1 develop growths that define the NF1 phenotype, including Lisch nodules, cafe-au-lait spots, and neurofibromas. However, more than 50% of individuals with NF1 have cognitive deficits, such as learning disorders and attention deficit/hyperactive disorder that are neuroanatomically unrelated to the neurofibromas.

The purpose of this study is to determine the molecular basis of the neurodevelopmental changes that result from the primary gene mutation using a mouse model of NF. These mice are heterozygous for the Nf1 gene (NF+/-), and develop learning and memory difficulties that mimic humans, but do not develop tumors.

By applying the technique of gene expression profiling to both NF+/- and control mice, we expect get a "genetic fingerprint" of 12,000 RNA molecules expressed in different parts of the brain during development. Comparison of the "genetic fingerprints" in these mice during development may help us identify key molecular changes that eventually result in cognitive deficits. Such an understanding of the molecular changes triggered by the primary gene mutation may eventually lead to treatments that prevent the cognitive deficits in NF1.

Collection of data from ´mRNA obtained from the spinal cord of hree human fetuses suffering from Lethal Congenital Contracture Syndrome and two age-matched human fetuses aborted to unrelated causes.

Expression data from human colon cancer cell line HCT116 with NFX1-91 knockdown and control cells

NFX1-91, a novel E6 cellular downstream target, functions as a transcriptional regulator and is involved in repressing hTERT expression.  Other functions and downstream targets regulated by NFX1-91 were not well understood. We used microarrays to determine gene expression deregulated when NFX1-91 was knocked down.

Effect of somatostatin knockout on sexually dimorphic hepatic gene expression

The liver is one of the most sexually dimorphic organs as measured by gene expression differences. About 80% of the sexually dimorphic genes are known to be regulated by growth hormone (GH). Somatostatin (SST) inhibits the release of GH.
We generated a SST-knockout mouse and analyzed the hepatic gene expression changes in both sexes.

Gene expression for 47 human breast tumor cases;
(* normalized by GCRMA for global expression analysis)

Comparing the mRNA expression profiles of c-Myb deficient and c-Myb sufficient Tcra-/- DP thymocytes.
Results provide insight into the role of c-Myb in the regulation of survival and differentiation during the pre-selection DP stage where c-Myb expression is abundant during T cell development.

The long noncoding RNA PCAT-1 promotes prostate cancer cell proliferation through post-transcriptional stabilization of the cMyc oncogene

Transcriptional profiling of Du145-PCAT1 and RWPE-PCAT1 cells treated with either non-targeting siRNA or two independent siRNAs targeting cMyc. The goal was to determine the role of cMyc in mediating PCAT-1 transcriptional regulation.

Understanding the nature of the various glucose-derived signals for insulin secretion (both triggering and amplifying) is essential for gaining insight into the functional failure of the beta-cells in diabetes and the development of drugs for correcting this problem. The beta-cells uniquely couple changes in cellular metabolism to electrical activity and thus insulin release. In mice, beta-cell specific deletion of the von Hippel-Lindau (VHL) tumor suppressor protein leads to the activation of a HIF transcription program that includes genes involved in glycolysis, suppression of mitochondrial activity and lactate production. This reprogramming of cellular metabolism results in abnormal insulin secretion properties.

Global gene expression analysis comparing wild type and beta catenin deficient BxPC3 pancreatic adenocarcinoma cells.

Goal:obtaing an overview of gene expression changes in BxPC3 cells rendered deficient in beta-catenin protein due to a zinc-finger nuclease mediated disruption of the beta-catenin gene (CTNNB1).

We studied adipose tissue from wild type mice, kinin B1 receptor knockout mice (B1KO), and B1KO mice with rescued expression of kinin B1 receptor selectively in fat.
We used microarrays to detail the gene expression profile of the epididymal fat of these mice.

Genes regulated after transient knock-down of Pirin in WM-266 cells

Pirin (PIR) is a putative transcriptional regulator abundantly expressed in melanocytes and in a subset of primary and metastatic melanomas. Ablation of PIR in the melanoma cell lines results in induction of a senescence-like phenotype.

The experiment was to compare leukemic T cells from thymic lymphomas from homozygote mice for the IkL/L hypomorphic mutation and non-transformed thymocytes, either of WT or IkL/L genotype. The aim was to identify a gene expression signature specific to the IkL/L tumors.
Keywords = thymic lymphoma Ikaros

Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs).
We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9.  Loss of expression Sox9 has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process.  Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.

Expression data from A673 cell line treated with compounds at several doses and time points

An increasing number of cancer-associated mutations have been identified. Unfortunately, little therapy today exploits these tumor-specific genetic lesions. Often, the resulting oncoproteins have been intractable to easy manipulation with current small molecule screening approaches. To overcome this impasse, we developed an expression-based approach to small molecule library screening. We applied this platform to the discovery of modulators of the activity of EWS/FLI, the Ewing sarcoma associated oncoprotein. Cytarabine (ARA-C) was identified as the top hit in a small molecule library screen. ARA-C modulates EWS/FLI by decreasing EWS/FLI protein level and has striking effects on cellular viability and transformation in in vitro and in vivo models of Ewing sarcoma. With poor outcomes for patients with relapsed Ewing sarcoma and the well established safety profile of ARA-C, clinical trials testing ARA-C in Ewing sarcoma are warranted. Expression data was created for A673 cells treated with ARA-C and two other compounds used to treat Ewing sarcoma (Puromycin and Doxorubicin) at two doses (EC50 and 2xEC50) and three time points (24 hours, 3 days, and 5 days).
Keywords: time course and dose response

Essential roles of the histone methyltransferase ESET in the epigenetic control of neural progenitor cells during development

In the developing brain, neural progenitor cells (NPCs) switch the differentiation competency via changing gene expression profiles that are governed partly by epigenetic control such as histone modification, although the precise mechanism is unknown. Here we found that ESET/Setdb1/KMT1E, a histone H3 Lys-9 (H3K9) methyltransferase, was highly expressed at early stages of brain development but down-regulated over time, and that ablation of ESET led to decreased H3K9 trimethylation and misregulation of genes, resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed, and non-neural gene expression was activated. Furthermore, early neurogenesis was most severely impaired, while astrocyte formation was enhanced. We conclude that there is an epigenetic role of ESET in temporal and tissue-specific gene regulation in the developing brain.
We used microarrays to identify genes up- or down- regulated when ESET is ablated in the dorsal telencephalon.

Expression profile between mast cells from diabetic prone and diabetic resistant rat strains

Abstract
Human type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the lymphopenic and spontaneously diabetic BioBreeding (BB) DRlyp/lyp rat. Previously, pre-onset expression profiling of whole DRlyp/lyp pancreatic lymph nodes (PLN) revealed innate immune activity, specifically that of mast cells and eosinophils. Furthermore, we observed that pancreatic islets of DRlyp/lyp rats as well as those of diabetes-inducible BB DR+/+ rats potentially recruit innate cells through eotaxin expression. Here we determine that lifelong eotaxin expression begins before 40 days of life and localized specifically to β cells.  In this report, we find PLN mast cells more abundant in DRlyp/lyp compared to related BB DR+/+ rats (2.1 +/-0.9% versus 0.9+/-0.4% of total cells, p<0.0001). DRlyp/lyp PLN mast cell gene expression profiling revealed an activated population and included significant overrepresentation of transcripts for mast cell protease 1, cationic trypsinogen, carboxypeptidase A, IL-5, and phospholipase Cγ. In the DR+/+ rat, which develops T1DM upon depletion of TREG cells, mast cells displayed gene expression consistent with the negative regulation of degranulation, including significant overrepresentation of transcripts encoding tyrosine phosphatase SHP-1, lipid phosphatase SHIP, and E3 ubiquitin ligase c-Cbl. To recapitulate negative mast cell regulation observed in the DR+/+, we treated DRlyp/lyp rats with the mast cell “stabilizer” cromolyn which significantly (p<0.05) delayed T1DM onset. These findings are consistent with a growing body of evidence in humans and animal models where a role for mast cells in the initiation and progression of autoimmune disease is emerging.

Background. Nuclear factor I-A (NFI-A), a phylogenetically conserved transcription/replication protein, plays a crucial role in mouse brain development. Previous studies showed that disruption of the Nfia gene in mice leads to perinatal lethality, corpus callosum agenesis, and hydrocephalus.
Results. To identify potential NFI-A target genes involved in the observed tissue malformations, we analyzed gene expression in brains from NFI-A-deficient and wild-type littermate control mice at the mRNA level using oligonucleotide microarrays. In young postnatal animals (P16), 356 genes were detected as differentially regulated, whereas at the late embryonic stage (E18), only 5 dysregulated genes were found. An in silico analysis identified phylogenetically conserved NFI binding sites in at least 70 of the differentially regulated genes. Moreover, assignment of gene function showed that marker genes for immature neural cells and neural precursors were expressed at elevated levels in young postnatal NFI-A mutants. By contrast, marker genes for differentiated neural cells were down-regulated at this stage. In particular, genes relevant for oligodendrocyte differentiation were affected.
Conclusions. Our results suggest that a delay in early postnatal development, especially oligodendrocyte maturation, is exhibited by NFI-A knock-out mice, and at least partly accounts for their phenotype. The identification of potential NFI-A target genes by our study should both help to elucidate NFI-A-dependent transcriptional pathways and contribute to a better understanding of this period of brain formation, especially with regard to the function of NFI-A.

Material and method
Animals
NFI-A-deficient mice were bred and maintained as described [20]. Animals were sacrificed by CO2 at embryonic stage E18.5 (E18) or postnatal day 16 (P16). Brains were dissected and snap frozen in liquid nitrogen.
The respective KO and WT animals used for analysis at E18 and P16 were littermates. Only male mice were used. All of the animals were F1 hybrids of C57Bl/6 and 129S6 animals. Within these matings, males had a B6 and females had a 129S6 background. In a large scale test, 38.5% of the offspring survive until P30 using this kind of mating. In purebred B6, we observed 0% survival at P1 (R.M.G., unpublished observations).
All animal use was performed under the approved protocol BCH05082N (RMG) of the University at Buffalo Institutional Animal Care and Use Committee in the UB Laboratory Animal Facility, an AAALAC licensed facility.

RNA preparation
Total RNA was extracted from the entire brain of NF-I-A-deficient and wild-type control mice (n=3 each) using TRIzol (Invitrogen) according to the manufacturer’s instructions. Further purification of RNA was performed with the RNeasy Mini Kit (Qiagen). Total RNA concentration was determined using a spectrophotometer at 260 nm and 280 nm wavelength. To check RNA integrity, total RNA was separated in a 1% agarose gel containing formaldehyde, and the intensity ratio of 28S and 18S ribosomal RNA bands was assessed after ethidium bromide staining.

Microarray hybridization and signal detection
Procedures for cDNA synthesis, labeling and hybridization were carried out according to the manufacturer's protocol (Affymetrix). All experiments were performed using Affymetrix mouse genome Genechip U74A version 2. Briefly, 15mg of total RNA were used for first strand cDNA synthesis with an HPLC-purified T7-(dT)24 primer. Synthesis of biotin-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (Affymetrix). For hybridization, 15 µg of fragmented cRNA were incubated with the chip in 200 µl of hybridization solution in Hybridization Oven 640 (Affymetrix) at 45oC for 16 hours. Genechips were washed and stained with streptavidin-phycoerythrin using the microfluidic workstation™ (Affymetrix) and scanned with a laser scanner (Agilent Technologies).

Microarray quantification and statistical analysis
Quality controls were performed using Affymetrix Microarray Suite 5.0 (MAS 5.0) software. Data and statistical analysis and data visualization were performed with GeneSpring™ (Agilent Technologies). Expression values were extracted from the CEL file, and imported to GeneSpring using Robust Multi-array Average (RMA) for further analysis [24]. Two criteria were used to select for significant changes in gene expression. First, probe sets with less than 1.2 fold change comparing the KO to WT (control) were removed in order to reduce the number of genes undergoing statistical analysis. Second, genes were then tested statistically for significant changes in all three wild-type or NFI-A-deficient samples, using Student's t-test. Finally, genes were grouped based on their biological function using Affymetrix NetAffx, and NCBI Locuslink.

Gene expression profile of liver tissue in carbon tetrachloride (CCl4)-treated mouse treated with erlotinib

Gene-expression profiles of liver tissue of cabon tetrachloride (CCl4)-treated mouse and the effect of erlotinib

Expression data from WT, E2f8 KO, Rb KO and Rb;E2f8 DKO spleen Ter19+CD71high sorted cells

To understand molecular mechanisms underlying the synergy of Rb loss and E2F8 loss, we used gene expression profiling to assess molecular changes in Mx1-Cre-mediated knockout (KO) mice using RNA isolated from sorted Ter119+CD71high Erythroblasts.
The top four upregulated and enriched pathways in the DKO mice included DNA replication, DNA damage response, DNA repair, and RNA processing. Interestingly, the majority of upregulated genes in the enriched sections of the four shared pathways contain E2f binding site(s) in their promoters, suggesting that Rb and E2F8 largely repress those genes through the E2f binding sites. Collectively, our data suggest that Rb and E2F8 co-regulate E2F-responsive genes.

A key role for Mbd3 in 5-hydroxymethylcytosine-dependent gene regulation in embryonic stem cells

To identify functional interactions among six chromatin regulators with key roles in embryonic stem cells, we examined gene expression changes in mouse ES cells with single and pair-wise combinations of knockdowns for those factors, along with control (EGFP) KDs.

Rotavirus infection is the single most important cause of severe diarrhea in young children worldwide. We used Affymetrix Human U95Av2 high density oligonucleotide arrays to compare gene expression profiles in peripheral blood mononuclear cells (PBMC) of 10 children with acute rotavirus diarrhea and 8 age-matched healthy children. We also examined patterns of gene expression in 5 convalescent-phase PBMC samples from rotavirus patients. For data analysis, we imported .cel files generated by Affymetrix MAS5.0 into Genetraffic 3.1 software (Iobion) and performed robust multi-chip analysis. We considered a gene in patients differentially expressed if its level of expression was at least 1.5-fold higher or lower than the baseline (arithmetic mean) of the corresponding gene in 8 controls and if its pattern of elevated or repressed expression was observed in at least 7 of the 10 patients. Using these criteria, we identified ~1% up- and ~2% down-regulated genes in acute-phase PBMC of patients. Up-regulated genes included those involved in the differentiation, maturation, activation, and survival of B cells, as well as an array of genes with function in inflammatory and antiviral activities. We observed a pattern of repressed expression of a number of genes involved in the various stages of T-cell development and activation. On the basis of these results, we conclude that rotavirus infection induces robust inflammatory response and B-cell activation but represses T-cell response.

Gene Expression and Biological Processes Influenced By Deletion of Stat3 in pulmonary Alveolar Type II Cells

Deletion of Stat3 induced genes influencing protein metabolism, transport, chemotaxis and apoptosis and decreased the expression of genes mediating lipid synthesis and metabolism. Srebf1 and 2, key regulators of fatty acid and steroid biosynthesis, were decreased in Stat3D/D mice. Stat3 influenced both pro- and anti-apoptotic pathways, regulating and maintaining the balance between a subset of pro- and anti-apoptotic genes that determine cell death or survival. Akt, a known target of Stat3, participates in many Stat3 mediated pathways including Jak-Stat signaling, apoptosis, the MAPK signaling, cholesterol and fatty acid biosynthesis. Deletion of Stat3 from type II epithelial cells altered the expression of genes regulating diverse cellular processes, including cell growth and apoptosis, lipid biosynthesis and metabolism. Stat3 regulates cell formation through a complex regulatory network that likely enhances alveolar epithelial cell survival and surfactant/lipid synthesis, necessary for the protection of the lung during injury.

Wildtype vs mutant comparison as a method for studying contributors to disease processes.

Expression data from wildtype and alb/cre liver-conditional Pdss2 knockout mutant mice

Utilizing M. musculus as a model of mitochondrial dysfunction provides insight into cellular adaptations which occur as a consequence of genetic alterations causative of human disease.  We characterized genome-wide expression profiles of liver-conditional knockout mice for Pdss2 compared with loxP controls.
Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways utilizing gene set enrichment analysis.

Genomic Collaboration of Estrogen Receptor-α and ERK2 in Regulating Gene and Proliferation Programs

The nuclear hormone receptor, estrogen receptor-alpha (ERα), and MAP kinases both play key roles in hormone-dependent cancers, yet their interplay and the integration of their signaling inputs remain poorly understood. In these studies, we document that estrogen-occupied ERα activates and interacts with ERK2, a downstream effector in the MAPK pathway, resulting in ERK2 and ERα colocalization at chromatin binding sites across the genome of breast cancer cells.
KEYWORDS: siRNA knock-down, ligand treatment

Advances in genomic signatures have begun to dissect breast cancer heterogeneity, and application of these signatures will allow the prediction of which pathways are important in tumor development.  Here we used genomic signatures to predict involvement of specific E2F transcription factors in Myc-induced tumors.  We genetically tested this prediction by interbreeding Myc transgenics with mice lacking various activator E2F alleles.  Tumor latency decreased in the E2F1 mutant background and significantly increased in both the E2F2 and E2F3 mutants.  Investigating the mechanism behind these changes revealed a reduction in apoptosis in the E2F1 knockout strain.  E2F2 and E2F3 mutant backgrounds alleviated Myc effects on the mammary gland, reducing the susceptible tumor target population.   Gene expression data from tumors revealed that the E2F2 knockout background resulted in fewer tumors with EMT, corresponding with a reduction in probability of Ras activation.  In human breast cancer we found that a low probability of E2F2 pathway activation was associated with increased relapse-free survival time.  Together these data illustrate the predictive utility of genomic signatures in deciphering the heterogeneity within breast cancer and illustrate the unique genetic requirements for individual E2Fs in mediating tumorigenesis in both mouse models and human breast cancer.

A series of gene expression measurements of normal myometrium and uterine fibroids with mutated or wild-type fumarate hydratase (FH) gene.

The effect of FH mutations on fibroid expression profile, normal myometrium vs. FH-mutant fibroids.

A series of gene expression measurements of uterine fibroids with mutated fumarate hydratase (FH) gene and normal myometrium.

Global developmental gene expression and pathway analysis of normal brain development and mouse models of human neuronal migration defects

Proper cortical development relies on the balance of neuronal migration and proliferation. We investigated the gene expression differences of mouse knock-outs for Lissencephaly in humans.
Our analysis suggests that gene expression and pathway analysis in mouse models of a similar disorder or within a common pathway can be used to define novel candidates for related human diseases.

To study the possible fibrotic role of FIZZ2, bleomycin was used to induce pulmonary fibrosis in wild type and FIZZZ2 knockout mice, lungs were then harvested and processed for RNA isolation.
We used microarrays to detail the global gene expression regulated by FIZZ2 during fibrotic process.

In this study we compared the effects of ALK inhibitor on the gene expression, activation of cell signaling pathways, and functional properties of cells derived from a patient with Anaplastic Large Cell Lymphoma. we used microarrays to map the genome-wide gene expression patterns in ALK+TCL cells in response to ALK inhibition.

Determine the effect and specificity of HDAC2 siRNA compared to SAHA inhibition of HDAC2 in hepatocellular carcinoma (HCC)

Hepatic regeneration gene expression signature predicts late recurrence of hepatocellular carcinoma

While we and others have uncovered and validated several genomic predictors for metastatic recurrences, a molecular or genomic predictor that can reliably identify high-risk patients for late de novo recurrence has not been firmly established. We analyzed previously reported gene expression data from human livers that underwent partial hepatectomy or transplantation, which were representative physiological conditions that trigger liver regeneration signals. We generated gene expression data from tumor and matched non-tumor surrounding tissues of 72 hepatocellular carcinoma (HCC) patients who underwent surgical resection as the primary treatment. We used these gene expression data to develop a new prognostification model for recurrence of HCC after surgery.

Type of experiment: Comparison of liver samples between sham (control) and 20% TBSA (total burn surface area) burn rats collected at 1h, 4h, 8h, and 24h.

Experimental factors:  Burn injury and time

Experiment design: All experimental liver samples collected after 1h, 4h, 8h, and 24h after burn injury were compared to a common reference (sham control treated as 0h time point)

Bio source: Sprague-Drawley male rats weighing 150-200 g supplied through Charles river laboratories, MA (www.criver.com)

Bio material manipulations: Rats (n=3 for each scald-burn and sham-burn time point) were individually housed in a temperature-controlled (25oC) and light-controlled room (12h light-dark cycle) and allowed to adjust to their new surroundings for at least 5 days prior to the experiment.  Water and rat chow were provided ad libitum to the animals.  On the day of the treatment, the animals were randomly divided into two groups, burned and sham-burned.  The burn injury consisted of a full-skin-thickness scald burn of the dorsum, calculated to be ~ 20% of the rat’s total body surface area (TBSA), induced by immersing the designated area in boiling water for 10 s [Yamaguchi, 1997 #416].  Rats were resuscitated with an intra-peritoneal injection of sterile saline solution (1.5 mL/Kg body weight/% TBSA) immediately after burn.  At each time point, three animals belonging to each group was sacrificed, the serum collected, and stored at –80oC.

Hybridization extract: Total RNA was isolated from approximately 50 mg of pooled liver tissue using the Nucleospin II RNA isolation kit from Clontech (Palo Alto, CA), as per the manufacturer’s instructions.

Labeling protocol: Labeling was performed as instructed by Affymetrix, Inc (www.affymetrix.com). In brief, 10 μg of total RNA was used for the double-stranded cDNA synthesis using the T7-oligo (dT) promoter primer kit (Affymetrix Inc.) and superscript RT II enzyme (Invitrogen). 1 μg of cDNA was labeled through invitro transcription reaction using T7 DNA polymerase in the presence of biotin labeled ribonucleotides. The cRNA was cleaned up using the Qiagen RNeasy columns following manufactures instructions. The cRNA (20 μg) was fragmented using the fragmentation buffer supplied as part of the Gene Chip Sample Cleanup Module (Affymetrix).

Image analysis and raw data description: The images were analyzed using MAS V.0 software provided by Affymetrix, Inc. The raw data output consists of several metrices: Signal, Detection, Detection p-value, Stat pairs, Stat pairs used. Signal is a measure of the abundance of the transcript. Detection is call that indicates whether the transcript is detected (P, present), undetected (A, absent), or at the limit of detection (M, marginal). Detection p-value is a measure of the significance of the detection call. Stat pairs, is the number of probe pairs for a particular probe set on an array. Stat pairs used, is the number of probe pairs per probe set used in the analysis.

Normalized and summarized data: All chips were scaled to a target intensity of 500 to account for differences between the replicate chips and their hybridization efficiencies using Affymetrix GENECHIP MAS V.0 software.  The entire dataset is available at gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE802. Three filters were serially used to obtain the list of annotated genes that demonstrated differential expression in intensity between sham and burn injured animals.  In the first filter, only genes that were flagged as ‘present’ by the MAS V.0 analysis software in at least one of the time points in all replicate chips were considered for further analysis.  This step eliminated all genes for which the expression data was not reproducible between the replicate chips.  The genes that passed this criterion were subjected to a second filter where ANOVA was performed to test each gene independently for a statistical difference in expression between groups (0, 1, 4, 8 and 24 h). The output of the analysis is the probability (p value) that a difference in expression can be observed by chance i.e., probability of getting a false positive.  While the occurrence of false positives can be controlled by choosing a higher significance level (0.01 or 0.001) it also concomitantly increases the false negatives.  Therefore, in this study we have chosen to work with ANOVA p-value cut-off 0.05.  To reduce the occurrence of the false positives, we have used the false discovery rate (FDR) method (33) which adjusts the ANOVA p-value (cut-off 0.05) while taking the dependencies between the genes into consideration.  The 695 genes that demonstrated a significant ANOVA p-value were then selected and their expression values averaged.  The third filter selected 339 genes that were either up-regulated or down-regulated by at least 2-fold between any one time point (1, 4, 8 or 24h) and the 0h time point (Sham control).  To determine the temporal profiles in the data, gene expression values were hierarchically clustered using the dChip software (www.dChip.org). The expression values for a gene across all samples were standardized by setting the mean to 0 and standard deviation to 1.  The software then builds a hierarchical cluster tree based on centroid-linkage method using Pearson correlation coefficient as the distance metric.  A set of genes were assigned to cluster groups empirically based on visual inspection of their temporal expression patterns.

Expression data from human skin biposies (lesional and uninvolved) from psoriatic patients

Biopsies from uninvolved and from lesional skin of 13 patients with plaque-type psoriasis. Based on paired samples, 179 genes were more than 2-fold differentially expressed in lesional skin.
Keywords: Comparison of uninvolved and lesional skin

Validated Gene Expression Signatures of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing lung disease that is difficult to diagnose and follows an unpredictable clinical course. The object of this study was to develop a predictive gene signature model of IPF from whole lung tissue. We collected whole lung samples from 11 IPF patients undergoing diagnostic surgical biopsy or transplantation. Whenever possible, samples were obtained from different lobes. Normals consisted of healthy organs donated for transplantation. We measured gene expression on microarrays. Data were analyzed by hierarchical clustering and Principal Component Analysis. By this approach, we found that gene expression was similar in the upper and lower lobes of individuals with IPF. We also found that biopsied and explanted specimens contained different patterns of gene expression; therefore, we analyzed biopsies and explants separately. Signatures were derived by fitting top genes to a Bayesian probit regression model. We developed a 153-gene signature that discriminates IPF biopsies from normal. We also developed a 70-gene signature that discriminates IPF explants from normal. Both signatures were validated on an independent cohort. The IPF Biopsy signature correctly diagnosed 76% of the validation cases (p < 0.01), while IPF Explant correctly diagnosed 78% (p < 0.001). Examination of differentially expressed genes revealed partial overlap between IPF Biopsy and IPF Explant and almost no overlap with previously reported IPF gene lists. However, several overlapping genes may provide a basis for developing therapeutic targets.

PML regulates PPAR signaling and fatty acid oxidation in breast cancer

Cancer cells exhibit an aberrant metabolism which facilitates more efficient production of biomass, and hence tumor growth and progression. The genetic cues modulating this metabolic switch remain largely undetermined, however. Here we identify a metabolic function for PML which reveals an unexpected role for this bona-fide tumor suppressor in pro-survival activity in breast cancer. We find that PML acts as both a negative regulator of PGC1A acetylation and as a potent activator of peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid oxidation (FAO). We further show that as FAO PML promotes ATP production, inhibits anoikis, and allows luminal filling in 3D basement membrane breast culture models. Furthermore, analysis of breast cancer biopsies reveals that PML is over-expressed in a subset of breast cancers, and is enriched in triple-negative cases. Indeed, PML expression in breast cancer correlates strikingly with reduced time to recurrence, a gene signature of poor prognosis and activated PPAR signaling. These findings have important therapeutic implications, as PML and its key role in FAO metabolism are amenable to pharmacological suppression as a mode of cancer prevention and treatment.

The Cohesin complex has recently been described to regulate gene expression. We wanted to determine the gene expression profile specific in mouse ES cells after depletion of the Cohesin subunit Rad21.
We used microarrays to detail the global programme of gene expression underlying depletion of Rad21 and identified distinct early development related genes up-regulated and many pluripotency related genes downregulated.

Expression profiling of GATA-1 wild type and mutant fetal megakaryocytes

Children with Down syndrome (DS) are predisposed to acute megakaryoblastic leukemia (AMKL, or AML M7) and a related myeloid disorder, referred to as transient myeloproliferative disorder (TMD), or transient leukemia (TL). Recently, acquired mutations in the erythroid/megakaryocytic lineage-specific transcription factor GATA-1 have been identified in megakaryoblasts from virtually all DS patients with AMKL (DS-AMKL), as well as in nearly all DS neonates with TMD. These mutations prevent synthesis of full-length GATA-1, but lead to synthesis of a truncated GATA-1 protein (GATA-1s) that lacks the N-terminal transactivation domain.
To determine the in vivo requirement of GATA-1 N-terminal domain in hematopoiesis and to model DS megakaryocytic leukemia initiated by GATA-1 mutation, we generated a GATA-1 knockin allele with a truncation at this domain (GATA-1deltaN). ES cells bearing this mutant allele generated a unique type of large Thrombopoietin (Tpo)-dependent megakaryocytes-containing colonies, when differentiated in vitro. Fetal livers from mice carrying this mutant allele contained elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. In in vitro hematopoietic colony assays, mutant cells from yolk sacs and mid-gestation fetal livers gave rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). Consistent with that, when mutant fetal liver progenitors were cultured in liquid medium in the presence of Tpo, the derived megakaryocytes displayed marked hyperproliferation and grew for longer period of time than WTs.

Specific aims:
To understand the molecular basis for the megakaryocytic hyperproliferation phenotype of the GATA-1deltaN mutants, we plan to profile gene expression patterns of fetal liver-derived primary megakaryocytes and megakaryocytic progenitors from the GATA-1deltaN mutants and compare them with WTs, as well as GATA-1deltaNeodeltaHS mutants (GATA-1– megakaryocytes).

Experimental design:
E12.5 fetal livers were harvested from either GATA-1 WT and mutant embryos. Single cell suspensions were made from these livers and the cells were cultured in liquid medium in the presence of Tpo for 3 days. On day 3, primary megakaryocytes were enriched by a BSA gradient, and further enriched by anti-CD41 antibody and magnetic microbeads. Megakaryocytic progenitors were collected by FACS sorting for small cells in the same culture that express low level of CD41. Total RNAs were then prepared from these two populations and submitted for microarray profiling.

Conclusions:
When examining genes that are normally upregulated upon megakaryocyte differentiation (many of this class are megakaryocyte-specific genes), we found in GATA-1deltaN megakaryocytes, the majority of these genes are upregulated, although not to the full extent as seen in WTs. In contrast, this class of genes is largely not upregulated in GATA-1deltaNeodeltaHS megakaryocytes, which are blocked at an immature stage of differentiation. Genes that are downregulated during megakaryocyte differentiation include those that are normally repressed by GATA-1. Among genes that are not properly downregulated in GATA-1deltaN megakaryocytes, five transcription factors (Myc, Myb, GATA-2, PU.1, Ikaros) are of particular interest. Inadequate GATA-1s-mediated repression of transcription factors required for proliferation of hematopoietic progenitors (Myc, Myb and GATA-2) and for specifying lineage choices other than megakaryocyte/erythrocyte (PU.1 and Ikaros) may contribute to hyperproliferation of GATA-1deltaN megakaryocytes.

CR mimetics;microarray profiling;drug discovery;gene-expression biomarkers;other

Mice and diets.  One-month-old male mice of the long-lived B6C3F1 strain were purchased from Harlan (Indianapolis, IN) and maintained as described after weaning (15).  Mice were housed in groups of 4 per cage and fed a non-purified diet, PMI Nutrition International Product #5001 (Purina Mills, Richmond, IN).  At 5 months of age, the mice were individually housed, and randomly assigned to one of two groups, control (CON) or long-term CR (LTCR).  CON mice were fed 93 kcal per week of a defined control diet (AIN-93M, Diet No. F05312, BIO-SERV).  LTCR mice were fed 52.2 kcal per week of a defined CR diet (AIN-93M 40% Restricted, Diet No. F05314, BIO-SERV).  The LTCR mice consumed approximately 40% fewer calories than mice in the CON mice.  The CR diet was enriched so that the CR mice consumed approximately the same amount of protein, vitamins, and minerals per gram body weight as the control mice.  Experimental design.  At 20 months of age, cohorts of mice in the CON group were randomly assigned to one of 7 experimental groups (Fig. 1).  A CON group continued to be fed 93 kcal per week of control diet for 8 weeks.  A CR8 group was fed 77 kcal per week of CR diet for 2 weeks, followed by 52.2 kcal per week of CR diet for 6 weeks.  The remaining dietary groups were fed the control diets containing, respectively, metformin (MET; Sigma, St. Louis, MO), glipizide (GLIP; Sigma), GLIP plus MET (GM), rosiglitazone (ROS; Avandia, SmithKline Beecham) or soy isoflavone extract (SOY; NOVASOY 400, Life Extension Foundation) for 8 weeks at the dosages indicated in Table 1.  The drugs were mixed with powdered control diet and cold-pressed into one gram pellets (BIO-SERV Inc.).  At 0900, all mice were fed 2/7 of the weekly allotment of food on Monday and Wednesday and 3/7 on Friday.  The mice had free access to acidified tap water.  They were fasted for 48 hours and killed by cervical dislocation at 22 months of age.  No signs of pathology were detected in the animals used for the studies reported (n=4 per group).  Organs were removed rapidly, flash frozen, and stored in liquid nitrogen.  The weights of the mice were monitored bi-weekly.  Mouse weights are given in Table 1.
Measurement of specific mRNA levels.  Liver total RNA was isolated as described from livers of pathology-free mice in each group (n=4 per group) (13). mRNA levels were measured using Affymetrix mouse U74Av2 arrays according to standard Affymetrix protocols (Affymetrix).
Probe set expression measurement and normalization.  After hybridization and scanning, raw image files were converted to probe set data (*.CEL files) using Microarray Suite (MAS 5.0). Probe set data from all 32 arrays were simultaneously analyzed with the Robust multichip Average (RMA) method to generate normalized expression measures for each probe set (Fig. 2; Ref. 24)  The data were further filtered to exclude probe data sets that were “Absent” across all 32 arrays according to the MAS 5.0 detection algorithm (1,47).

MCF10A cells in 3-D culture after treatment with BRCA1-RNAi or CtIP-RNAi

Alzheimer’s disease: neurofibrillary tangles (Rogers-3U24NS043571-01S1)

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder affecting approximately 4 million people in the U.S. alone.  AD is characterized by the presence of senile plaques and neurofibrillary tangles in cortical regions of the brain.  These pathological markers are thought to be responsible for the massive cortical neurodegeneration and concomitant loss of memory, reasoning, and often aberrant behaviors that are seen in patients with AD.  Understanding the molecular mechanisms whereby these histopathological markers develop will greatly enhance our understanding of AD development and progression.  A clearer understanding of the mechanisms underlying neurofibrillary tangle formation specifically may help to clarify the basis for dementia of AD as well as the dementias associated with other diseases that are collectively referred to as "tauopathies."
To expression profile both neurons containing neurofibrillary tangles and normal neurons from the entorhinal cortex of 10 mid-stage AD cases.
The gene expression profile of neurons that contain neurofibrillary tangles will differ from the expression profile of histopathologically normal neurons from the same patient and from the same brain region.  Some of these differences will be informative as to the mechanisms of tangle formation.

Optimal treatment for nonalcoholic steatohepatitis (NASH) has not yet been established, particularly for individuals without diabetes.
We examined the effects of metformin, commonly used to treat patients with type 2 diabetes, on liver pathology in a non-diabetic NASH mouse model.

Laser microdissection and microarray analysis of the hippocampus of Rasgrf1 knockout mice

We used manual macrodissection or laser capture microdissection (LCM) to isolate tissue sections of the hippocampus area of Ras-GRF1 wild type and knockout mice brains, and analyzed their transcriptional patterns using commercial oligonucleotide microarrays. Comparison between the transcriptomes of macrodissected and microdissected samples showed that the LCM samples allowed detection of significantly higher numbers of differentially expressed genes, with higher statistical rates of significance. These results validate LCM as a reliable technique for in vivo genomic studies in the brain hippocampus, where contamination by surrounding areas (not expressing Ras-GRF1) increases background noise and impairs identification of differentially expressed genes. Comparison between wild type and knockout LCM hippocampus samples revealed that Ras-GRF1 elimination caused significant gene expression changes, mostly affecting signal transduction and related neural processes. The list of 36 most differentially expressed genes included loci concerned mainly with Ras/G protein signaling and cytoskeletal organization (i.e. 14-3-3γ/ζ, Kcnj6, Clasp2) or related, cross-talking pathways (i.e. jag2, decorin, strap). Consistent with the phenotypes shown by Ras-GRF1 knockout mice, many of these differentially expressed genes play functional roles in processes such as sensory development and function (i.e. Sptlc1, antiquitin, jag2) and/or neurological development/neurodegeneration processes affecting memory and learning. Indeed, potential links to neurodegenerative diseases such as Alzheimer disease (AD) or Creutzfeldt-Jacobs disease (CJD), have been reported for a number of differentially expressed genes identified in this study (Ptma, Aebp2,Clasp2, Hebp1, 14-3-3γ/ζ, Csnk1δ, etc.). These data, together with the previously described role of IRS and insulin (known Ras-GRF1 activators) in AD, warrant further investigation of a potential functional link of Ras-GRF1 to neurodegenerative processes.
Keywords: knockout analysis

Gene expression changes associated with progression and response in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematopoetic stem cell disease with distinct biological and clinical features. The biological foundation of the stereotypical progression from chronic phase through accelerated phase to blast crisis is poorly understood. We used DNA microarrays to compare gene expression in 91 cases of CML in chronic (42 cases), accelerated (17 cases), and blast phases (32 cases). Three thousand genes were found to be significantly (p<10-10) associated with the progression from chronic to blast phase. A comparison of the gene signatures of chronic, accelerated, and blast phases suggest that the progression of chronic phase CML from chronic advanced phase (accelerated and blast crisis) CML is a two-step rather than a three-step process, with new gene expression changes occurring early in accelerated phase before the accumulation of increased leukemia blast cells. The genetic signature of advanced phase CML is similar to that of normal CD34+ cells; however, progression also involved novel genes not expressed in normal CD34+ cells. Especially noteworthy is deregulation of the WNT/b-catenin pathway, the decreased expression of both JunB and Fos, and dysregulation of genes under the control of MZF1 and delta EF1 zinc finger transcription factors. Studies of CML patients who relapsed after initially successful treatment with imatinib mesylate demonstrated a gene expression pattern closely related to advanced phase disease. Take together, these data suggest that CML progression begins relative early and before clinical and pathological detection, and features distinct genetic differences compared to normal hematpoetic cells that might provide diagnostic and therapeutic targets.

Expression changes following pharmacological reversal of epigenetic silencing

A screen to identify novel tumor suppressor genes silenced by methylation in melanoma

Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival, emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.
Keywords: Expression changes following pharmacological reversal of epigenetic silencing

Since targeting of specific pathogenic pathways may be more efficient than current unspecific heart failure treatment, we obtained genomewide expression profiles of a DCM subtype characterized by cardiac inflammation (DCMi) in association with parvovirus B19. This study was entirely based on RNA isolated from endomyocardial biopsies so far only rarely used for genomic expression profiling.

We generated skeletal muscle-specific knockout mice lacking the transcription factor Yin Yang 1 (YY1) and analyzed expression patterns in the skeletal muscle these mice.
We used microarrays to detail the global programme of gene expression regulated by YY1.

Expression data from trisomy 21 and euploid induced pluripotent stem cell hematopoietic progenitors

We modeled human Trisomy 21 primitive hematopoiesis using induced pluripotent stem cells (iPSCs).  Primitive multipotent progenitor populations generated from Trisomy 21 iPSCs showed normal proliferative capacity and megakaryocyte production, enhanced erythropoiesis and reduced myeloid development compared to euploid iPSCs.

Gene expression was evaluated in 9 appendix samples removed from patients who went to the operating room with the diagnosis of acute appendicitis and 4 samples removed for non-inflammatory reasons.
A circumferential piece of tissue was obtained at the distal aspect of each specimen.  The tissue was flash frozen at -80 degrees for later processing.  Frozen specimens were homogenized into TriReagent  and RNA was isolated according to the manufacturer's instruction.  RNA was processed and hybridized to Affymetrix microarray.

PPARg and IL-4-induced gene expression data from wild-type and STAT6 knockout mouse bone marrow-derived macrophages

C57Bl/6 wild-type and STAT6 KO mice were used to study PPARg and IL-4 signaling. Bone marrow of 3 mice per group was isolated and differentiated to macrophages with M-CSF (20 ng/ml). 20 ng/ml IL-4 was used to induce alternative macrophage activation and 1 uM Rosiglitazone (RSG) was used to activate PPARg. From each mouse 4 samples were generated: 1. M-CSF, 2. M-CSF+RSG, 3. IL-4 and 4. IL-4+RSG. All compounds were added throughout the whole differentiation process, and frech media was added every other day. Control cells were treated with vehicle (DMSO:ethanol). After 10 days, RNA was isolated and gene expression profiles were analyzed using Mouse Genome 430 2.0 microarrays from Affymetrix.

Effect of fluoxetine treatment on translational profiles of S100a10 cortical pyramidal cells in p11 KOs

Molecular phenotyping of cell types and neural circuits underlying pathological neuropsychiatric conditions and their responses to therapy provides one avenue for the development of more specific and effective treatments. In this study, we identify a cell population in the cerebral cortex that shows robust and specific molecular adaptations following long-term SSRI treatment.

We employed the bacTRAP strategy, which uses BAC transgenic mice expressing EGFP-tagged ribosomal protein L10a in specific cell populations, to affinity purify polysome-bound mRNAs from S100a10-expressing corticostriatal projection neurons. We show that the S100a10 cells are a unique population of cortical cells that are strongly and specifically responsive to chronic SSRI administration and that this response requires p11 (the protein product of S100a10).

Our data demonstrate that the beneficial actions of antidepressant therapy can be mediated by a single cell type that is positioned to normalize activity between cortical and subcortical sites, and suggest that development of drugs that specifically target the activity of these cells may result in improved therapies to treat depression.

Embryos were collected at 2 cell stage.  cRNA from four biological replicates of each were generated and the expression profiles were determined using Affymetrix MOE430 v2.

Idiopathic inflammatory myopathies (polymyositis and dermatomyositis) are heterogeneous group of muscle disorders of unknown etiology.The pathogenic pathways responsible for muscle fiber damage and dysfunction in myositis are not currently well defined. Identification of such pathways may help to design novel therapeutic interventions and also help to develop diagnostic tests.
Keywords: Development or differentiation design

Transcription factor paralogs may share a common role (e.g. Hox) in staged or overlapping expression in specific tissues.   In other examples, members have distinct roles in a range of embryologic, differentiation or response pathways (e.g. Tbx, Pax). For the Interferon Regulatory Factor (IRF) family of transcription factors, mice deficient in Irf1, Irf2, Irf3, Irf4, Irf5, Irf7, Irf8 or Irf9, have defects in the immune response but display no embryologic abnormalities. Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two Mendelian orofacial clefting syndrome, and genetic variation in IRF6 confers risk for isolated cleft lip and palate.
Mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation.  This study describes a novel role for an IRF family member in epidermal development.

Olfactory bulb transcript levels for GFAP transgenic mice compared to wildtype at 3wks and 4mos
Keywords = GFAP

Genome-wide gene expression analysis in Thoc5- and CFIm68-depleted HeLa cells

We found that CFIm68, a mRNA cleavage and polyadenylation factor implicated for alternative polyadenylation site choice, was co-purified with Thoc5, a component of human THO/TREX. Microarray analysis using human HeLa cells reveals knockdown of Thoc5 affects the expression of a subset of non-heat shock genes. Notably, depletion of Thoc5 attenuated the expression of the mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68.

Laminin (merosin) deficient muscular dystrophy in dy/dy mouse diaphragm muscle, 8 weeks old
Keywords: muscle, muscular dystrophy, laminin, merosin, diaphragm, mouse

Exaggerated neuroinflammation and sickness behavior in aged mice after activation of the peripheral innate immune system

Acute cognitive impairment (i.e., delirium) is common in elderly emergency department patients and frequently results from infections that are unrelated to the central nervous system. Since
activation of the peripheral innate immune system induces brain microglia to produce inflammatory cytokines that are responsible for behavioral deficits, we investigated if aging exacerbated neuroinflammation and sickness behavior after peripheral injection of
lipopolysaccharide (LPS). Microarray analysis revealed a transcriptional profile indicating the presence of primed or activated microglia and increased inflammation in the aged brain.
Furthermore, aged mice had a unique gene expression profile in the brain after an intraperitoneal injection of LPS, and the LPS-induced elevation in the brain inflammatory cytokines and oxidative stress was both exaggerated and prolonged compared with adults. Aged mice were anorectic longer and lost more weight than adults after peripheral LPS administration. Moreover, reductions in both locomotor and social behavior remained 24 h later in aged mice, when adults had fully recovered, and the exaggerated neuroinflammatory response in aged mice was not reliably paralleled by increased circulating cytokines in the periphery. Taken together, these data
establish that activation of the peripheral innate immune system leads to exacerbated neuroinflammation in the aged as compared with adult mice. This dysregulated link between the
peripheral and central innate immune system is likely to be involved in the severe behavioral deficits that frequently occur in older adults with systemic infections.

Expression profiling of human adult postmortem brain tissue from subjects with bipolar disorder and healthy controls

Bipolar affective disorder is a severe psychiatric disorder with a strong genetic component but unknown pathophysiology. We used microarray technology (Affymetrix HG-U133A GeneChips) to determine the expression of approximately 22 000 mRNA transcripts in post-mortem brain tissue (dorsolateral prefrontal cortex and orbitofrontal cortex) from patients with bipolar disorder and matched healthy controls.

Expression profiling of human adult postmortem brain tissue from Down syndrome and healthy control subjects

Down syndrome (DS) is the result of trisomy chromosome 21 but the mechanisms by which the genotype leads to the characteristic disease phenotype are unclear. We performed a microarray study using human adult brain tissue (dorsolateral prefrontal cortex) from DS subjects and healthy controls to characterise for the first time the human adult Down syndrome brain

Expression signature of cardiac muscle as a potential diagnostic or prognostic tool for dilated cardiomyopathy

There is an emerging hypothesis that dilated cardiomyopathy (DCM) is a manifestation of end-stage heart failure (ESHF) resulting from “final common pathway” despite heterogeneous primary etiologies. We performed genome-wide expression profiling by means of high-density oligonucleotide microarrays using cardiac muscles from patients with DCM or specific cardiomyopathy as well as non-disease control hearts. Differentially expressed genes between ESHF and non-disease samples should include both genes reactive to heart failure (HF) and those responsible for ESHF. With the aid of samples with acute HF without DCM and those with DCM without HF (corrected with left ventricular assist device), we successfully distinguished ESHF genes from HF genes. Our findings implicate that transcriptional signature of cardiac muscle can be potentially applied as a diagnostic or prognostic tool for severe HF.

This dataset is a time series (1 hour [h], 4 hours, 24 hours, 48 hours, 1 week [w], and 8 weeks) intended to compare normal functioning left ventricles [lv + lv2] with infarcted [ilv] and non-infarcted left ventricles [nilv]. Ilv samples are taken from the region between the LAD artery and the apex on a mouse with myocardial infarction. Lv2 samples are from the same region in a sham operated mouse. Nilv samples are taken from the region above the infartion and the left ventricle [lv] samples mimic that region in a sham mouse. The lv and lv2 samples can be compared as both are from normal functioning hearts. For more information visit http://cardiogenomics.med.harvard.edu/groups/proj1/pages/mi_home.html

Expression data from normal adrenal gland and aldosterone-producing adenoma

The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study.

Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy
Keywords = Diabetes

Extraocular muscle, comparison of wild type and mdx mice, 14 to 112 Days (Porter lab)

Determination of gene expression changes in extraocular muscle of mdx (dystrophin-deficient) mice at postnatal ages 14, 28, 56, and 112 days.  3 independent replicates/age/strain.

Sprague-Dawley rat retina post-injury and control samples
Keywords = Gliosis, CNS injury

Effect of genetic Zfx deletion on Myc induced gene expression in bone marrrow myeloid progenitors

Particulate Matter effect on Mouse Model of Cardiac Failure: Lung and Heart Left Ventricle

Particulate Matter Triggers Carotid Body Dysfunction, Respiratory Dysynchrony and Cardiac Arrhythmias in Mice with Cardiac Failure

The mechanistic link between human exposure to airborne particulate matter (PM) pollution and the increased cardiovascular morbidity and mortality observed in people with congestive heart failure (CHF) is unknown. We now show that exposure of genetically-engineered mice with CHF (expressing a cardiac-specific CREB mutant transcription factor) to ambient PM (collected in Baltimore, mean aerodynamic diameter 1.9 um) unmasks severe autonomic morbidities manifested as significant reductions in heart rate variability, respiratory dysynchrony and increased frequency of serious ventricular arrhythmias, features not observed in PM-challenged wild type mice without CHF. PM exposure in CREB mice with CHF reflexly triggers autonomic dysfunction via heightened carotid body function as evidenced by pronounced afferent nerve responses to hypoxia and marked depression of breathing by hyperoxia challenge. Genomic analyses of lung and ventricular tissues revealed PM-induced molecular signatures of inflammation and oxidative stress. These findings in a murine model of cardiac failure provide the first direct assessment of autonomic function in response to PM challenge and are highly consistent with current epidemiologic findings on cardiovascular morbidity in susceptible PM-exposed human populations.
We utilized a murine model of dilated cardiomyopathy to address potential mechanistic links between PM exposure and the development of life-threatening cardiac dysrhythmias.

The murine pulmonary response is strain specific.  Susceptible strains such as C57BL6/J experience an inflammatory response followed by progressive lung disease.  Resistant strains such as Balb/c do not experience significant degrees of inflammation or fibrosis.  This study aims to determine the genetic basis of sensitivity differences between strains

hippocampus, rat, young or aged, control or pioglitazone-treated

Effects of Long-term Pioglitazone Treatment on Peripheral and Central Markers of Aging

Background: Thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor gamma (PPARgamma) and are used clinically to help restore peripheral insulin sensitivity in Type 2 diabetes (T2DM). Interestingly, long-term treatment of mouse models of Alzheimer’s disease (AD) with TZDs also has been shown to reduce several well-established brain biomarkers of AD including inflammation, oxidative stress and Abeta accumulation. While some of the TZD actions are becoming clear in AD models and may mediate their reported beneficial impact in AD patients, little is known about the functional consequences of TZDs in animal models of normal aging. Because aging is a common risk factor for both AD and T2DM, we investigated whether the TZD, pioglitazone could alter brain aging under non-pathological conditions.  Findings: The TZD pioglitazone (PIO) was incorporated into the diet to yield a final dose of approximately 2.3 mg/kg body weight/day. PIO reduced insulin levels irrespective of age. Interestingly, a significant reduction in the Ca2+-dependent afterhyperpolarization was seen in the aged animals with no significant change in LTP maintenance or learning and memory performance. Finally, a combination of microarray analyses on hippocampal tissue and serum-based multiplex cytokine assays revealed that age-dependent inflammatory changes in brain and periphery were not reversed by PIO.
Conclusions:  While current research efforts continue to address the underlying processes responsible for the progressive decline in cognitive function seen during aging, available medical treatments are still very limited. Our study, therefore, was aimed at elucidating potentially novel actions of TZDs in the aging brain. Using a clinically-relevant dose and delivery method, PIO had no detectable impact on several indices of brain aging and failed to alter both peripheral and central age-related increases in inflammatory signaling.

Gene expression data from MCF-7 cells treated with Lacciac Acid A

Lacciac Acid A was indentified as an inhibitor of DMNT1. MCF-7 cells were treated with Lacciac Acid A (200 uM) for 5 days. Changes in gene expression were identified by using Affymetrix Human gene ST1.0 arrays. We used microarrays to determine global changes in gene expression upon treatment with Lacciac Acid A an inhibitor of DMNT1.

LMP-420: a novel purine nucleoside analogue with potent cytotoxic effects for chronic lymphocytic leukemia cells

[original title] LMP-420: a novel purine nucleoside analogue with potent cytotoxic effects for chronic lymphocytic leukemia cells and minimal toxicity for normal hematopoietic cells.

LMP-420 induces cytotoxicity and apoptosis to CLL cells in vitro without any negative effects to normal immune cells.
This gene expression experiment compares CLL cells treated with LMP-420 versus media alone to investigate the mechanism of action of LMP-420.

Intestinal Phenotype of Variable Weight Cystic Fibrosis Knockout Mice

Cystic fibrosis transmembrane conductance regulator (Cftr) knockout mice present the clinical features of low body weight and intestinal disease permitting an assessment of the interrelatedness of these phenotypes in a controlled environment.  To identify intestinal alterations which affect body weight in CF mice the histological phenotypes of crypt-villus axis height, goblet cell hyperplasia, and mast cell infiltrate were measured, cardiac blood samples assessed, and gene expression profiling of the ileum was completed for 12 week old (C57BL/6xBALB) F2 Cftrtm1UNC and non-CF mice presenting a range of body weight.  Crypt-villus axis height decreased with increasing weight in CF, but not control, mice.  Goblet cell hyperplasia and mast cell infiltration in the submucosa and muscularis externa layers of the CF intestine, were identified to be independent of bodyweight.  Blood triglyceride levels were found to be significantly lower in CF mice than control mice (p = 3.02 x 10-5) but were not dependent on CF mouse body weight.  By expression profiling, genes of DNA replication and lipid metabolism were among those altered in CF mice relative to non-CF controls; and no differences in gene expression were measured between samples from CF mice in the 25th and 75th percentile for weight.  This study indicates that the absence of Cftr leads to altered morphology in the CF intestine the extent of which is correlated with body weight in CF mice while CF related changes in blood triglyceride levels and in the intestinal gene expression profile were not dependent on body weight in this model.

ChIP-on-chip, electrocardiography, fetal gene program, OXPHOS, PGC-1a, sodium current, Single time point (E18.5) to compare genotype differences

3 ventricles from E18.5 male mice were pooled for each array. Three arrays per genotype.

Title: ERRγ Directs and Maintains the Transition to Oxidative Metabolism in the Post-Natal Heart

Abstract: At birth the heart undergoes a critical metabolic switch to transition from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism. This remains the principle metabolic state throughout life; although pathologic conditions such as heart failure and cardiac hypertrophy reactivate components of the fetal genetic program to increase carbohydrate utilization. Disruption of the ERRγ gene, which is expressed at high levels in the fetal and postnatal mouse heart, blocks this switch resulting in lactatemia, electrocardiographic (ECG) abnormalities and death during the first week of life. Genomic ChIP-on-chip and expression analysis at E18.5 clearly identifies ERRγ as both a direct and indirect regulator of a nuclear-encoded mitochondrial genetic network that coordinates the postnatal metabolic transition. These findings reveal an unexpected and essential molecular genetic component of the oxidative metabolic gene program in the heart and highlight ERRγ in the study of cardiac hypertrophy and failure.

Homozygous disruption of Bteb2/Klf5, a homolog of Drosophila gap gene Krüppel, led to increased expression of various differentiation marker genes, such as Fgf5, Cdx2, and Brachyury in mouse ES cells without compromising their ability to differentiate into all three germ layers. Upon removal of LIF, Klf5-deficient ES cells showed faster differentiation kinetics than wild-type ES cells. In contrast, overexpression of Klf5 in ES cells suppressed the transcription of differentiation marker genes, and maintained pluripotency in the absence of LIF. In order to search downstream genes of Klf5, we surveyed genes implicated in ES cell proliferation by microarray analysis

The Insecticides Cyfluthrin & Chlorpyrifos Alter Expression of Genes with Diverse Functions in Primary Human Astrocytes

Given the widespread use of insecticides in the environment, it is important to perform studies evaluating their potential effects on humans.  Organophosphate insecticides, such as chlorpyrifos, are being phased out; however, the use of pyrethroids in household pest control is increasing.  While chlorpyrifos is relatively well studied, much less is known about the potential neurotoxicity of cyfluthrin and other pyrethroids.  To gain insights into the neurotoxicity of cyfluthrin, we compared and evaluated the toxicity profiles of chlorpyrifos and cyfluthrin in primary human fetal astrocytes.  We found that at the same concentrations, cyfluthrin exerts as great as, or greater toxic effects on the growth, survival, and proper functioning of human astrocytes.  By using microarray gene expression profiling, we systematically identified and compared the potential molecular targets of chlorpyrifos and cyfluthrin, at a genome-wide scale.  We found that chlorpyrifos and cyfluthrin targeted a similar number of transcripts.  These targets include chaperones, signal transducers, transcriptional regulators, transporters, and those involved in behavior and development.  Further computational and biochemical analyses suggest that cyfluthrin and chlorpyrifos up-regulated certain targets of the interferon-gamma and insulin signaling pathways, and that they increased the protein levels of activated ERK1/2, a key component of insulin signaling; IL-6, a key inflammatory mediator; and GFAP, a marker of inflammatory astrocyte activation.  These results suggest that inflammatory activation of astrocytes might be an important mechanism underlying neurotoxicity of both chlorpyrifos and cyfluthrin.

Quercetin has been described to have a wide range of beneficial effects in humans, ranging from anti-carcinogenic properties to reduced risk of cardiovascular disease. We tested whether a daily supplementation of quercetin leads to reproducible changes in gene expression profiles of human monocytes.
Keywords: individual compound treatment

PLAGL2 regulates Wnt signaling to impede differentiation in neural stem cells and glioma

A hallmark feature of glioblastoma (GBM) cells is its strong self-renewal potential and immature differentiation state -- stem cell-like properties which may contribute to the plasticity and intense therapeutic resistance of GBM.  The molecular basis of the immature differentiation profile remains an area of active investigation. Here, integrated genomic and biological analyses identified PLAGL2 as a potent proto-oncogene targeted for amplification/gain in malignant gliomas as well as in colorectal cancers. High level of PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell (GIC) differentiation while promoting their proliferation and self-renewal capacity under differentiation induction conditions. On the mechanistic level, the PLAGL2 transcriptome analysis revealed that these differentiation suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling via up-regulation of Wnt6 ligand as well as Fzd9 and Fzd2 receptor expression. Correspondingly, inhibition of Wnt signaling in PLAGL2-expressing NSC partially restores their differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics on malignant cells.

Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans

Resveratrol is a naturally occurring compound that profoundly affects energy metabolism and mitochondrial function and serves as a calorie restriction mimetic, at least in animal models of obesity. Here we treated 10 healthy, obese men with placebo and 150 mg/day resveratrol in a randomized double-blind cross-over study for 30 days. Resveratrol supplementation significantly reduced sleeping- and resting metabolic rate. In muscle, resveratrol activated AMPK, increased SIRT1 and PGC-1alpha protein levels, increased citrate synthase activity, and improved muscle mitochondrial respiration on a fatty acid-derived substrate. Furthermore, resveratrol elevated intramyocellular lipid levels, and decreased intrahepatic lipid content, circulating glucose, triglycerides, alanine-aminotransferase, and inflammation markers. Systolic blood pressure dropped and HOMA index improved after resveratrol. In the postprandial state, adipose tissue lipolysis and plasma fatty acid and glycerol decreased. In conclusion, we demonstrate that 30 days of resveratrol supplementation induces profound metabolic changes in obese subjects, mimicking the effects of calorie restriction.

Gene expression study of developing mammalian vestibular organ of wild type and Insulin-Like Growth Factor I deficient mice using Affymetrix whole transcript arrays

Insulin like growth factor 1 (IGF-1) has a central role in mammalian hearing and hearing loss. The auditory and vestibular systems form the inner ear and have a common developmental origin. During chicken early development IGF-1 modulates neurogenesis of the cochleovestibular ganglion but no further studies have been conducted to explore the potential role of IGF-1 in the vestibular system.
In this study we have compared the whole transcriptome of the vestibular organ from wild type and Igf1-/- mice at different developmental times.

Divergent dysregulation of gene expression in Fmr1 and Tsc2 knockout mouse models of autism spectrum disorders

Fragile X syndrome and tuberous sclerosis are genetic syndromes that both have a high rate of comorbidity with autism spectrum disorder (ASD). Several lines of evidence suggest that these two monogenic disorders may converge at a molecular level through the dysfunction of activity-dependent synaptic plasticity.
To explore the characteristics of transcriptomic changes in these monogenic disorders, we profiled genome-wide gene expression levels in cerebellum and blood from murine models of fragile X syndrome and tuberous sclerosis.
Differentially expressed genes and enriched pathways were distinct for the two murine models examined, with the exception of immune response-related pathways. In the cerebellum of the Fmr1 knockout (Fmr1-KO) model, the neuroactive ligand receptor interaction pathway and gene sets associated with synaptic plasticity such as long-term potentiation, gap junction, and axon guidance were the most significantly perturbed pathways. The phosphatidylinositol signaling pathway was significantly dysregulated in both cerebellum and blood of Fmr1-KO mice. In Tsc2 heterozygous (+/−) mice, immune system-related pathways, genes encoding ribosomal proteins, and glycolipid metabolism pathways were significantly changed in both tissues.
Our data suggest that distinct molecular pathways may be involved in ASD with known but different genetic causes and that blood gene expression profiles of Fmr1-KO and Tsc2+/− mice mirror some, but not all, of the perturbed molecular pathways in the brain.

comparison between Rettsyndrome  suffering  and non-suffering patients

Expression in Superior Frontal Gyrus of normal individuals or females of different ages affected by Rett syndrome

Rett syndrome (RTT, OMIM #312750) is a severe X-linked neurodevelopmental disorder linked to heterozygous de novo mutations in the MECP2 gene. MECP2 encodes methyl-CpG-binding protein 2 (MeCP2), which represses gene transcription by binding to 5-methylcytosine residues in symmetrically positioned CpG dinucleotides. The disorder is almost exclusively diagnosed in females, because males affected by the disease usually die perinatally due to severe encephalopathy.  Direct MeCP2 target genes underlying the neuropathogenesis of RTT remain largely unknown.
To identify genes involved in the neuropathogenesis of RTT we assessed the gene expression profile of postmortem brains affected by RTT and that of age-matched normal brains using Affymetrix arrays.

Reduced Expression of the Proto-oncogene Myc Increases Mouse Longevity and Enhances Healthspan

MYC is a pleiotropic transcription factor that regulates numerous pathways and whose deregulation promotes cancer. Myc+/- mice have extended lifespan relative to their wild type littermates. To better understand the effects of the Myc+/- genotype on cellular processes, microarrays were performed on young (5 month) and old (24 month) Myc+/- and WT males in liver, skeletal muscle, and adipose tissues.
Microarray data were used to compare the effects of aging with the effects of the Myc+/- genotype at each age, to identify pathways enriched for differentially expressed genes, and to compare the Myc+/- gene expression signature with that from other long-lived mice.

Hepatic gene expression in streptozotocin-induced diabetic mice fed a quercetin diet

Quercetin is a food component that may ameliorate the diabetic symptoms.  We examined hepatic gene expression of BALB/c mice with streptozotocin (STZ)-induced diabetes to elucidate the mechanism of the protective effect of dietary quercetin on diabetes-associated liver injury.
We fed STZ-induced diabetic mice with diets containing 0.1% or 0.5% quercetin for 2 weeks and compared the patterns of hepatic gene expression in these groups of mice using a DNA microarray. Diets containing 0.1% or 0.5% quercetin lowered the STZ-induced increase in blood glucose levels and improved plasma insulin levels. A cluster analysis of the hepatic gene expressions showed that 0.5% quercetin diet suppressed STZ-induced alteration of gene expression.  Gene set enrichment analysis (GSEA) and quantitative RT-PCR analysis showed that the quercetin diets had their greatest suppressive effect on the STZ-induced elevation of expression of cyclin dependent kinase inhibitor p21(WAF1/Cip1) (Cdkn1a).

Adult postmortem brain tissue (dorsolateral prefrontal cortex) from subjects with bipolar disorder and healthy controls

Bipolar affective disorder is a severe psychiatric disorder with a strong genetic component but unknown pathophysiology. We used microarray technology (Affymetrix HG-U133A GeneChips) to determine the expression of approximately 22 000 mRNA transcripts in post-mortem brain tissue (dorsolateral prefrontal cortex) from patients with bipolar disorder and matched healthy controls. A cohort of 70 subjects was investigated and the final analysis included 30 bipolar and 31 control subjects. Differences between disease and control groups were identified using a rigorous statistical analysis with correction for confounding variables and multiple testing.

To define the contribution of CDK8 versus CDK19 to gene expression control, we performed a series of microarray assays for cells where each kinase was stably knocked down.
Toward this end, we subjected HCT116 cells to three different stress stimuli: 5-fluorouracil (5FU), glucose deprivation, and hypoxia. We found that CDK8, but not CDK19, functions as a widespread coactivator of HIF1A target genes in hypoxia.

Inflammatory bowel disease;Crohn's disease;ulcerative colitis;expression screening;expression profiling;other

The series was designed to identify new genes involved in the pathophysiology of inflammatory bowel disease (Crohn's disease and ulcerative colitis).
This series represents a group of 31 samples, subdivided into 3 groups: 1) Normal controls: 11 samples; 2) patients with Crohn's diseases: 10 samples; 3) patients with ulcerative colitis: 10 samples. Each sample originated from a different patient or normal control, in total n=31 individuals were examined.

Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in 3 normal testes.

Intestinal crypt stem and transit cell proliferation and villus cell migration in mice lacking Klf9

Krüppel-like factor 9 (Klf9), a zinc-finger transcription factor, is implicated in the control of cell proliferation, cell differentiation and cell fate in brain and uterus.  Using Klf9 null mutant mice, we have investigated the involvement of Klf9 in small intestine crypt-villus cell renewal and lineage determination.  We report the predominant expression of Klf9 gene in small intestine smooth muscle (muscularis externa).  Jejunums null for Klf9 have shorter villi, reduced crypt stem/transit cell proliferation, and altered lineage determination as indicated by decreased and increased numbers of Goblet and Paneth cells, respectively.  A stimulatory role for Klf9 in villus cell migration was demonstrated by BrdU labeling.  Results suggest that Klf9 controls the elaboration, from small intestine smooth muscle, of molecular mediator(s) of crypt cell proliferation and lineage determination, and of villus cell migration.

Age-related macular degeneration has a strong epidemiological association with cardiovascular disease. One pathogenic hypothesis that applies to both diseases is the concept of an abnormal cellular response to injury resulting in a disease phenotype. It has been hypothesized that this phenotype is also present in dermal fibroblasts. This study tests this hypothesis by examination of the expression profiles of fibroblasts obtained from diseased patients and subjected to sublethal cell injury.

Expression data from wild-type and HMGN1 knockout mice injected with N-nitrosodiethylamine

HMGN1 contributes to the shortened latency of liver tumorigenesis by changing a chromatin structure and expression of relevant genes
To assess the molecular mechanisms underlying accelerated tumor development in Hmgn1-/- mice, we performed the gene expression profiling of liver cells at early stages

Gene expression profiles of dasatinib-resistant and dasatinib-sensitive pancreatic cancer cell lines

Gene profiles from three dasatinib-resistant and three dasatinib-sensitive pancreatic cancer cell lines were compared by microarray analysis.

Control of CD8+ T cell proliferation by the transcription factor ELF4

Transcription factors that regulate quiescence, proliferation, and homing of lymphocytes are critical for effective immune system function. In the present study, we demonstrated that the transcription factor ELF4 directly activates the tumor suppressor KLF4 downstream of T cell receptor (TCR) signaling to induce cell cycle arrest in naïve CD8+ T cells. Elf4- and Klf4-deficient mice accumulated CD8+CD44hi T cells during steady-state conditions and generated more memory T cells after immunization. The homeostatic expansion of CD8+CD44hi T cells in Elf4-null mice resulted in a redistribution of cells to non-lymphoid tissue due to reduced expression of the transcription factor KLF2, and the surface proteins CCR7 and CD62L. This work describes the combinatorial role of lymphocyte-intrinsic factors in the control of T cell homeostasis, activation and homing.

Differential gene expression between ERRa KO and WT mouse kidneys

Estrogen-related receptor (ERR) alpha is an orphan nuclear receptor highly expressed in the kidneys. ERRalpha is implicated in renal sodium and potassium homeostasis and blood pressure regulation. We used microarray analysis to identify differentially expressed genes in ERR alpha knockout mice kidneys versus wild-type. The results provide insight on the roles of ERRalpha in the kidney.

Extraocular and hindlimb muscle, comparison of wild type and mdx mice, 56 days (Porter lab)

Determination of gene expression changes in extraocular and hindlimb (gastrocnemius/soleus) of mdx (dystrophin-deficient) mice at postnatal day 56.  5 independent replicates/muscle group/strain.

293 cells,  infected with RCAS-beta-cateninS37A or RCAS-GFP
Keywords = 293cells

Effect of the HF diet and Akt1-mediated muscle growth on gene expression in liver tissue.

In contrast to the well-established role of oxidative muscle fibers in regulating fatty acid oxidation and whole body metabolism, little is known that about the function of fast/glycolytic muscle fibers in these processes. Here, we generated a skeletal muscle-specific, conditional transgenic mouse expressing a constitutively-active form of Akt1. Transgene activation led to muscle hypertrophy due to the growth of type IIb muscle fibers, which was accompanied by an increase in strength. These mice were then used to assess the consequence of building fast/glycolytic muscle fibers on adiposity and metabolism. Akt1 transgene induction in obese mice resulted in reductions in body weight and fat mass, a resolution of hepatic steatosis and improved metabolic parameters. These effects were achieved independent of changes in physical activity and levels of food consumption.  Akt1-mediated skeletal muscle growth opposed the effects of high fat/sucrose diet on transcript expression patterns in the liver, and increased hepatic fatty acid oxidation and ketone body production. Our findings indicate that an increase in fast/glycolytic muscle mass can result in the regression of obesity and obesity-related metabolic disorders in part through its ability to alter fatty acid metabolism in remote tissues.

Expression data from adult rat testes treated with 3 mg/kg b.w. cadmium chloride via i.p.

This is a study to explore the transcriptional changes after cadmium treatment in adult rat testes at three time points (control--0 hour,  8 hour and 4 day). Cadmium is an environmental toxicant that is known to affect the male reproductive system. It disrupts the blood-testis barrier irreversibly,  and affects the Sertoli-germ cells adhesion, causing germ cell depletion from the seminiferous epithelium.
Keywords: Cadmium effect in rat testes

TALEN gene knockouts reveal no requirement for the shelterin protein Rap1 in telomere protection and length regulation

Gene expression of human knockouts of telomere-protein Rap1 were compared to their wildtype counterparts in mutliple cell lines (available from the ATCC repository). We discovered that Rap1, desite being mostly associated with the telomere, performed a conserved role in transcriptional regulation, as evidenced by a number of genes that were differentially regulated upon deletion of Rap1.

Retroviral overexpression or siRNA mediated knockdown of FOXP1 in DLBCL cell lines and primary human B cells

To provide insight into the role of and target genes of the transcription factor FOXP1 in mature human B cells and in B cell non-Hodkgin lymhomas, we performed gene expression microarray studies, upon ectopic overexpression or silencing of FOXP1 in these cells.

We performed Affymetrix MG-U74Av2 GeneChip experiements on mRNA from the adrenal glands of the BPH hypertensive and BPL hypotensive mouse strains. All mice were aged-matched at 5 weeks. We obtained the mice from Jackson Laboratories, Bar Harbor, ME.

We measured gene expression in the adrenal glands of the Spontaneously Hypertensive Rat (SHR) and Wistar-Kyoto rat (WKY) using Affymetrix RG-U34A GeneChips. All rats were aged-matched at 4-weeks. The rats were obtained from the colonies at the Univeristy of California San Diego, La Jolla, CA.

DNA damage response (NCS treatment) in several siRNA-manipulated cellular systems

Response to treatment with the radiomimetic drug NCS in cells knocked-down for ATM, p53, and the Rel_A subunit of NFkB; and in control un-infected cells and cells infected with siRNA against LacZ
Keywords = DNA damage

Expression data from transgenic Aire expressing pancreatic islets

The aim of this study was to determine the effect of transgenic Aire expression on the transcriptional profile of a tissue that normally does not express Aire: pancreatic islets. The transcriptional profile of transgenic RIP-Aire27 islets was compared to non-transgenic littermate islets as well as to archival NOD thymic medullary epithelial cells (MEC) data. All data were from non-obese diabetic (NOD) mice

YAP1 plays importance roles in development of colorectal cancer as evidenced by their overexpression in colorectal cancer and their expression promoted cell proliferation and survival of colorectal cancer cells. In order to understand potential roles of YAP1 in colorectal cancer, we over-expressed constitutively active YAP1 mutant in NCI-H716 colorectal cancer cells and identified and analyzed genes whose expression is activated by YAP1 activation in colorectal cancer.

siRNA-mediated knockdown of MITF and PAX3 in metastatic melanoma cell lines

The transcription factors PAX3 and MITF are required for the development of the neural crest and melanocyte lineage, and both proteins play important roles in melanoma cell growth and survival.  PAX3 transcriptionally activates MITF expression during neural crest development, but the relationship between these transcription factors during melanocyte development and in melanoma cells is currently poorly understood. This study aimed to further our understanding of the interaction between transcriptional networks controlled by PAX3 and MITF by assessing the effect of siRNA-mediated knockdown of PAX3 and MITF in metastatic melanoma cell lines. The goals of this study were to determine (i) if PAX3 is required for maintaining expression of MITF in melanoma and melanocyte cell lines; (ii) whether PAX3 and MITF independently, or redundantly, influence growth and survival in melanoma cell lines; and (iii) to investigate the respective roles of PAX3 and MITF expression in melanoma cell differentiation.
Microarrays were used to measure global changes in transcript expression in response to siRNA-mediated knockdown of PAX3 or MITF compared to non-targeting controls in two metastatic melanoma cells lines.

Expression profiling of mouse ing2 -/- mice with spermatogenic arrest and infertility

Expression profiling of mouse ing2 -/- testis vs WT reveals gene expression differences consistent with spermatogenic arrest and infertility. Ing2 is indispensable for male germ cell development in mice. While mice deficient for Ing2 were born and grew without apparent abnormalities, male, but not female, were infertile, consistent with the highest expression of Ing2 in testes in wild-type mice and in humans. Histological and DNA content analyses in Ing2-/- testes revealed a spermatogenesis arrest at meiotic phase and enhanced apoptosis associated with increased p53, resulting in a  decline in mature spermatozoa, which became more severe in older age. HDAC1 accumulation and core histone deacetylation at pachytene stage were impaired in Ing2-/- testes, suggesting that the recruitment of HDAC1 by Ing2 plays a critical role in spermatogenesis. This study establishes Ing2 as a novel mammalian regulator of spermatocyte differentiation, which coordinates spermatogenesis stage-specific histone modifications. This study has implications in understanding human male infertility.

The Transcriptome of Nrf2-Deficient Mice in Cigarette Smoke-Induced Emphysematous Changes

Cigarette smoke (CS) imposes a strong oxidative burden on exposed tissues resulting in a severely disturbed oxidant/antioxidant balance, which in the context of chronic exposure is assumed to be a key contributor to CS-related diseases. Because of its emerging central role in orchestrating the general cellular antioxidant response, the pathway leading to the activation of the transcription factor Nrf2 has received mounting attention over the past decade in investigations aimed at elucidating CS-induced patho-physiological mechanisms. To comprehensively characterize the impact of Nrf2 in acute and sub-chronic smoking scenarios, Nrf2 knock-out mice and their wildtype ICR littermates were exposed to either ambient air (sham exposure) or to one of three doses of CS for up to 5 months with two post-exposure endpoints of 1 and 13 days. The lungs of the mice were monitored for transcriptomic changes on a genome-wide level.

Pompe disease is a genetic disorder resulting from a deficiency of lysosomal acid alpha-glucosidase (GAA) that manifests as a clinical spectrum with regard to symptom severity and rate of progression. In this study, we used microarrays to examine gene expression from the muscle of two cohorts of infantile-onset Pompe patients to identify transcriptional differences that may contribute to the disease phenotype. We found strong similarities among the gene expression profiles generated from biceps and quadriceps, and identified a number of signaling pathways altered in both cohorts. We also found that infantile-onset Pompe patient muscle had a gene expression pattern characteristic of immature or regenerating muscle, and exhibited many transcriptional markers of inflammation, despite having few overt signs of inflammatory infiltrate. Further, we identified genes exhibiting correlation between expression at baseline and response to therapy. This combined dataset can serve as a foundation for biological discovery and biomarker development to improve the treatment of Pompe disease.

Gene expression differences between TAF4b-KO, Het and WT ovaries at 3 weeks of age

The rapid decline of ovarian function in TAF4b-null mice begins in early postnatal life and follicle depletion is completed by sixteen weeks.
To uncover differences in gene expression that may underlie accelerated ovarian aging, we compared genome-wide expression profiles of three week old, pre-pubescent TAF4b-null and wild-type ovaries.

Psoriasis is a common chronic inflammatory and hyperproliferative immune-mediated skin disorder. Narrow-band UVB (NB-UVB) phototherapy is a convenient first-line treatment of psoriasis, though the mechanisms underlying its efficacy have not been completely elucidated. In order to improve our understanding of NB-UVB phototherapy, gene expression profiling was used to characterize gene expression in lesional epidermis from psoriasis patients undergoing NB-UVB phototherapy. Increased expression of melanogenesis pathway genes was observed to be the earliest response. At the end of treatment, genes involved in diverse biological processes were affected, such as pigmentation, cell adhesion, ectodermal development and metabolism. The relationship between gene expression and treatment outcome was further studied using Partial Least Squares Discriminant Analysis (PLS-DA). Gene ontology analysis showed that genes responding to phototherapy and highly correlated to treatment outcome were involved in oxidation reduction, growth and mitochondria organization. In particular SPATA18, a key regulator of mitochondria quality, was found to be significantly downregulated in psoriasis, and its upregulation following phototherapy was required for optimal clinical improvement. Our data suggest that oxidation reduction is a critical event for the resolution of psoriatic plaques.

TNM,Tumor-node-metastasis LCM,Laser capture microdissection OSCC,Oral squamous cell Carcinoma

Association b/w gene expression profile & tumor invasion in OSCC

There are limited studies attempting to correlate the expression changes in oral squamous cell carcinoma with clinically relevant variables. We determined the gene expression profile of 16 tumor and 4 normal tissues from 16 patients by means of Affymetrix Hu133A GeneChips. The hybridized RNA was isolated from cells obtained with laser capture microdissection, then was amplified and labeled using T7 polymerase-based in vitro transcription. The expression of 53 genes was found to differ significantly (33 upregulated, 20 downregulated) in normal versus tumor tissues under two independent statistical methods. The expression changes in four selected genes (LGALS1, MMP1, LAGY, and KRT4) were confirmed with reverse transcriptase polymerase chain reaction. Two-dimensional hierarchical clustering of the 53 genes resulted in the samples clustering according to the extent of tumor infiltration: normal epithelial tissue, tumors less than or equal to 4 cm in dimension, and tumors more than 4 cm in dimension (P=0.0014). The same pattern of clustering was also observed for the 20 downregulated genes. We did not observe any associations with lymph node metastasis (P=0.097).

Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

Background: Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals.
Methods: Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography ? mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL.
Results: Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells.
Conclusion: Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

Effect of DBC1/CCAR2 depletion on global gene expression in MDA-MB-231 cells

Deleted in breast cancer (DBC1; also known as CCAR2) is a coactivator for nuclear receptors (NRs) as well as a negative regulator of epigenetic modifiers such as deacetylases SIRT1 and HDAC3.
We performed genome-wide gene expression analysis in control (shNS) and DBC1-depleted (shDBC1) MDA-MD-231 cells to investigate global gene expression changes induced by depletion of DBC1

Imaging-guided microarray: Identifies molecular markers in the pathogenesis of Parkinson’s disease

The full complement of molecular pathways contributing to Parkinson’s disease (PD) pathogenesis remains unknown.  Here, to address this issue, we began by using a high-resolution variant of functional magnetic resonance imaging (fMRI) to pinpoint brainstem regions differentially affected by, and resistant to, the disease. Then, relying on the imaging information as a guide, we profiled gene expression levels of postmortem brain samples and used a factorial statistical model to identify a disease related decrease in the expression of the polyamine enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1).  Next, a series of studies were performed to confirm the pathogenic relevance of this finding. First, to test for a causal link between polyamines and α-synuclein toxicity, we investigated a yeast model expressing α-synuclein. Polyamines were found to enhance the toxicity of α-synuclein, and an unbiased genome-wide screen for modifiers of α-synuclein toxicity identified Tpo4, a member of a family of proteins responsible for polyamine transport.   Second, to test for a causal link between SAT1 activity and PD histopathology we investigated a mouse model expressing α-synuclein.  DENSPM (N1, N11-diethylnorspermine), a polyamine analog that increases SAT1 activity, was found to reduce PD histopathology, while Berenil (diminazene aceturate), a pharmacological agent that reduces SAT1 activity, worsened the histopathology.  Third, we genotyped PD patients and controls and isolated a rare but novel variant in the SAT1 gene, although the functional significance of this genetic variant was not identified.  Taken together, the results suggest that the polyamine pathway contributes to PD pathogenesis.

Gene expression analysis of H9 hESC derived neuron stem cells (NSC) harboring pathogenic LRRK2 (G2019S) mutation

Genetic mutations on leucine-rich repeat kinase 2 (LRRK2) have been associated with an increased risk of Parkinson's disease. The Gly2019Ser (G2019S) mutation on LRRK2 gene is a relatively common cause of familial Parkinson's disease in Caucasian population.
In this study, we generated H9 hESC harboring LRRK2 (G2019S) mutation by gene knockin. Wildtype and LRRK2 mutant hESC were differentiated into NSC using a chemically defined protocol.

The effect of intermittent hypoxia on gene expression in the liver of ob/ob mice

All animals were kept in a controlled environment (22-24 °C with a 12 h : 12 h light : dark cycle; lights on at 09.00) on a standard chow diet with free access to water. Five ob/ob mice (sample_ids GSM32865-GSM32869) were placed in the intermittent hypoxia (IH) chamber and subjected to IH for 12 consecutive weeks and compared to pair-fed control exposed to intermittent room (IA) air for 12 weeks in identical chambers (n = 5, sample_ids GSM32860-GSM32864). The IH and IA states were induced during the light phase alternating with 12 h of constant room air during the dark phase. The IH and IA groups were weight-matched daily during the experiment by varying food intake. Weight-matching was conducted in pairs.
The signal intensity fluorescent images produced during samples hybridizations to Affymetrix GeneChip were read using the Agilent Gene Array Scanner and converted into GeneChip Cell files (CEL) using MAS 5.0 software (Affymetrix, Santa Clara, CA). Gene expression values were generated form CEL files using Robust Microarray Analysis (RMA, Bioconductor affy package). Briefly, the rma module of this package was used for background correction, across array normalization, and extraction of the probe level data.
Ref:  Irizarry R, Gautier L, and Cope L, An R package for analyses of Affymetrix oligonucleotide arrays. The Analysis of Gene Expression Data: Methods and Software, ed. G Parmigiani,ES Garrett, RA Irizarry,and SL Zeger. 2003, New York: Springer

This series contain mouse lung samples treated with Simvastatin and LPS and corresponded controls.

Spleen samples with B lymphoma from TRAF3 knockout mice compared with littermate controls

Diabetes is a disease now affecting more than 20 million people in the United States alone.  When left untreated it has numerous debilitating effects including diabetic retinopathy, which is characterized by the death of retinal neurons as well as a breakdown of the retinal vascularization.  This leads to nearly 24,000 cases of blindness each year among diabetic patients.  Studies have shown the importance of both nitric oxide and the gene VEGF in retinopathy, however little is known about the condition's progression.  This study would determine which genes are affected early in diabetes and more generally, give insite into how neural degeneration occurs in the retina.
We aim to show the gene-level changes in the diabetic mouse retina five weeks after induction of diabetes.  Our laboratory's primary focus is on NO production through nNOS, iNOS and endothelial (eNOS).  We hope to be able to correlate some of the transcript level changes with genes known to be involved in the NO signalling pathways.
Previous data by our lab and others have shown that neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) are upregulated under diabetic conditions in the retina.  Additional work in our lab has shown that the lack of  nitric oxide (NO) produced by nNOS may affect neurotransmitter production, particularly with GABA.  We have also shown that mice with induced diabetes show neurochemical changes in the retina after only one to three weeks.  Thus, we expect to see gene level changes associated with the retina before psysological changes in newly diabetic mice.
Adult CD1 mice of random sexes are injected with the drug streptozotocin on three successive days while fasting 8 hours before the injection.  This drug selectivley destroys  the beta-islet cells of the pancrease.  Their blood sugar levels are then tested after a resting period.  Mice with glucose levels above 250mg/dL are considered diabetic from the date of the last injection.
Mice are then sacrificed after the waiting period by breif isoflourine inhalation followed by decapitation.  Retinas are immediately removed in cold HBSS and RNA is extracted using Trizol extraction.  To get adequate amounts of RNA, we will pool four retinas (2 animals) together.

Neurofibromatosis type 1 (NF1) is a multi-system disease caused by mutations in the NF1 gene encoding a Ras-GAP protein, neurofibromin, which negatively regulates Ras signalling. Besides neuroectodermal malformations and tumours, the skeletal system is often affected (e.g. scoliosis and long bone dysplasia), demonstrating the importance of neurofibromin for development and maintenance of the musculoskeletal system. Here we focus on the role of neurofibromin in skeletal muscle development. Nf1 gene inactivation in the early limb bud mesenchyme using Prx1-cre (Nf1Prx1) resulted in muscle dystrophy characterised by fibrosis, reduced number of muscle fibres, and reduced muscle force. To gain insight into the molecular changes of the observed muscle dystrophy and fibrosis and to compare these with other known muscle dystrophies, we performed transcriptional profiling of the entire triceps muscles of threemonth-old wild type (wt) and mutant animals using Affymetrix high-density microrrays.

Transcriptional targets of EphB2 and Ephrin-A4 signaling in epidermal keratinocytes

Human epidermal keratinocytes were treated with 25 ng.ml EphB2 or EFNA4, both as-Fc conjugates (Sigma).

Postnatal developmental changes in Sprague-Dawley rats in the model of neuropathic pain 'spare nerve injury'

Neuropathic pain is an apparently spontaneous experience triggered by abnormal physiology of the peripheral or central nervous system, which evolves with time. Neuropathic pain arising from peripheral nerve injury is characterized by a combination of spontaneous pain, hyperalgesia and allodynia. There is no evidence of this type of pain in human infants or rat pups; brachial plexus avulsion, which causes intense neuropathic pain in adults, is not painful when the injury is sustained at birth. Since infants are capable of nociception from before birth and display both acute and chronic inflammatory pain behaviour from an early neonatal age, it appears that the mechanisms underlying neuropathic pain are differentially regulated over a prolonged postnatal period.
We used microarrays to detail the global programme of gene expression underlying the differences in nerve injury between along the postnatal development and identified distinct classes of regulated genes during the injury

Gene expression in minor salivary gland of patients with Sjogren's syndrome (SS) and control

To study the gene expression profile of salivary glands with varying degrees of inflammation in Sjogren's and non Sjogren's patients

Functional classes of bronchial mucosa genes that are differentially expressed in asthma

Asthma pathogenesis and susceptibility involves a complex interplay between genetic and environmental factors.
We report the results of expression microarray studies using tissue obtained from bronchial biopsies of healthy controls and of subjects with allergic asthma, both before and following inhaled corticotherapy.

VAChT KDHOM mice have a 70% decrease in the vesicular acetylcholine transporter (VAChT) and this leads to a systemic decrease in ACh release and cardiac dysfunction.
We used this microarray to determine whether there were transcriptional alterations in the hearts of mutant mice as a result of this long-term decrease in cholinergic tone.

Kinase expression in each tumor was compared to expression in normal brain and expression values from normal human astrocytes were used as an additional control.

Gene expression profiling of human gliomas and human glioblastoma cell lines

To identify signaling pathways that are differentially regulated in human gliomas, a microarray analysis on 30 brain tumor samples (12 primary glioblastomas (GBM), 3 secondary glioblastomas (GBM-2), 8 astrocytomas (Astro) and 7 oligodendrogliomas (Oligo)) and on  5 glioblastoma cell lines (LN018, LN215, LN229, LN319 and BS149) was performed. Normal brain tissue (NB) and normal human astrocytes (NHA) were used as a control. Kinase expression in each tumor was compared to expression in normal brain and expression values from normal human astrocytes were used as an additional control.

Effect of genetic Zfx deletion on gene expression in c-Kit + MLL-AF9 AML cells

Acute myeloid leukemia (AML) propagates as a cellular hierarchy which is maintained by a rare subpopulation of self-renewing leukemia-initiating cells (LICs). These LICs phenotypically resemble HSCs and early myeloid progenitors, and they are functionally defined by their ability to reconstitute AML in xenografted mice.
Common transcriptional regulators are believed to drive self-renewal of LICs and normal stem cells. Examples include the histone methyltransferase, MLL and its main targets, the transcription factors HOXA9 and MEIS1; the polycomb group protein, BMI-1; and the Wnt/β-catenin signaling pathway4-9. In AML patients, LICs have been shown to share broad gene expression signatures with hematopoietic stem cells (HSCs) and, in some cases, with embryonic stem cells (ESCs). Moreover, increased expression of these stem cell signatures has been linked to tumor aggressiveness.
Elucidating the molecular determinants of stem cell properties in LICs has the potential to improve AML therapy and clarify the relationship between cancer and stem cell biology in general. Here we show that the propagation of LICs in AML depends on Zfx, a transcription factor required for the self-renewal of ESCs and HSCs. Using mouse models, we found that Zfx is required for AML propagation and LIC maintenance. We defined a Zfx-driven gene expression program in murine LICs that correlates with existing stem cell-related gene expression signatures in AML patients. Using a novel, stroma-based RNAi screening strategy, we identified functionally important Zfx target genes. Two of these genes – FAM92A1 and DOCK7 - are required for human AML cell propagation; moreover, their expression levels strongly correlate with AML patient survival across the full spectrum of AML subtypes. Together, our results characterize a novel gene expression program that orchestrates critical stem cell properties of LICs, and drives aggressiveness of AML.

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane treated human breast epithelial cells

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level.  Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.

Left ventricular gene expression profiles from 12-, 16- and 20-months old spontaneously hypertensive rats (SHRs) were compared with left ventricular profiles seen in age-matched Wistar-Kyoto (WKY) rats by screening Affymetrix U34A arrays (there are 4 samples in each timepoint except 3 samples of 20-months old WKYs).

The DNA damage response network modulates a wide array of signaling pathways, including DNA repair, cell cycle checkpoints, apoptotic pathways and numerous stress signals. The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia (A-T), is a master regulator of this network when the inducing DNA lesions are double strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combines gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation (IR) in murine lymphoid tissue. Cluster analysis revealed six major expression patterns in the data. Prominent among them was a gene cluster that contained dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of the transcription factors NF-kB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and anti-apoptotic signals were simultaneously induced, with the pro-apoptotic pathway mediated by p53, and the pro-survival pathway by NF-kB. These findings further elucidate the molecular network induced by IR and have implications for cancer management as they suggest that a combined treatment that restores the p53-mediated apoptotic arm while blocking the NF-kB-mediated pro-survival arm could be most successful in increasing the radiosensitivity of lymphoid tumors.

Allograft versus isograft mouse heterotopic tracheal transplant grafts

Exploration of genes associated with acute and chronic rejection associated with obliterative airway disease

Immune Response to Nippostrongylus brasiliensis in the mouse lung

The goal of this experiment was to examine the innate immune response to helminth infection in the lung. Hookworms (like many other helminths) use an obligate migration pathway through the lung. Their infection has been characterized in the gut in detail, but early immune responses in the lung have not been fully characterized.
Keywords: Time Course

Cytotoxic effects of Bortezomib in Myelodysplastic syndrome/Acute Myeloid Leukemia depend on autophagy-mediated lysosomal degradation of TRAF6 and repression of PSMA1

Bortezomib (Velcade©) is widely used for the treatment of various human cancers, however, its mechanisms of action are not fully understood, particularly in myeloid malignancies. Bortezomib is a selective and reversible inhibitor of the proteasome. Paradoxically, we find that Bortezomib induces proteasome-independent degradation of TRAF6 protein, but not mRNA, in Myelodysplastic syndrome (MDS) and Acute Myeloid Leukemia (AML) cell lines and primary cells. The reduction in TRAF6 protein coincides with Bortezomib-induced autophagy, and subsequently with apoptosis in MDS/AML cells. RNAi-mediated knockdown of TRAF6 sensitized Bortezomib-sensitive and -resistant cell lines, underscoring the importance of TRAF6 in Bortezomib-induced cytotoxicity. Bortezomib-resistant cells expressing an shRNA targeting TRAF6 were resensitized to the cytotoxic effects of Bortezomib due to down-regulation of the proteasomal subunit alpha-1 (PSMA1). To uncover the molecular consequences following loss of TRAF6 in MDS/AML cells, we applied gene expression profiling and identified an apoptosis gene signature. Knockdown of TRAF6 in MDS/AML cell lines or patient samples resulted in rapid apoptosis and impaired malignant hematopoietic stem/progenitor function. In summary, we describe novel mechanisms by which TRAF6 is regulated through Bortezomib/autophagy-mediated degradation and by which it alters MDS/AML sensitivity to Bortezomib by controlling PSMA1 expression.

We investigated the ability of transferrin receptor1 (TfRc) knockout cells to populate different domains of the developing kidney by using a chimeric approach. The TfRc cells developed into all segments of the developing nephron, but there was a relative exclusion from the ureteric bud and a positive bias towards the stromal compartment. Here we conducted a microarray analysis of differential gene expression between TfRc deficient and wild type (wt) cells in chimeric embryonic kidneys derived from embryos created by blastocyst injection of wt blastocysts with TfRc-/- green fluorescent protein-expressing (GFP+) embryonic stem cells.
Keywords: cell type comparison, genetic modification, iron signaling

Autism and increased paternal age related changes in global levels of gene expression regulation

A causal role of mutations in genes encoding for multiple general transcription factors in neurodevelopmental disorders including autism suggested that alterations at the global level of gene expression regulation might also relate to disease risk in sporadic cases of autism. This premise can be tested by evaluating for global changes in the overall distribution of gene expression levels. For instance, in mice, we recently showed that variability in hippocampal-dependent behaviors was associated with variability in the pattern of the overall distribution of gene expression levels, as assessed by variance in the distribution of gene expression levels in the hippocampus. We hypothesized that a similar change in the variance in gene expression levels might be found in children with autism. Gene expression microarrays covering greater than 47,000 unique RNA transcripts were done on purified RNA from peripheral blood lymphocytes of children with autism (n=82) and controls (n=64). The variance in the distribution of gene expression levels from each microarray was compared between groups of children. Also tested was whether a risk factor for autism, increased paternal age, was associated with variance in the overall distribution of gene expression levels. A decrease in the variance in the distribution of gene expression levels in peripheral blood lymphocytes (PBL) was associated with the diagnosis of autism and a risk factor for autism, increased paternal age. Traditional approaches to microarray analysis of gene expression suggested a possible mechanism for decreased variance in gene expression. Gene expression pathways involved in transcriptional regulation were down-regulated in the blood of children with autism and children of older fathers. Thus, results from global and gene specific approaches to studying microarray data were complimentary and supported the hypothesis that alterations at the global level of gene expression regulation are related to autism and increased paternal age. Regulation of transcription, thus, represents a possible point of convergence for multiple etiologies of autism and other neurodevelopmental disorders.

Ovarian Cancer gene expression profiling identifies the surface of the ovary as a stem cell niche

In contrast to epithelial derived carcinomas that arise in most human organs, ovarian surface epithelial cells become more rather than less differentiated as the malignancy progresses. To test the hypothesis that ovarian surface epithelial cells retain properties of relatively uncommitted pluripotent cells until undergoing neoplastic transformation, we conducted gene expression profiling analysis (Affymetrix, U133 Plus 2.0) of 12 ovarian surface epithelial cells and 12 laser capture microdissected serous papillary ovarian cances. We find that over 2000 genes are significantly differentially expressed between the surface epithelial and cancer samples. Network analysis implicates key signaling pathways and pathway interactions in ovarian cancer development. Genes previously associated with adult stem cell maintenance are expressed in ovarian surface epithelial cells and significantly down-regulated in ovarian cancer cells. Our results indicate that the surface of the ovary is an adult stem cell niche and that deregulation of genes involved in maintaining the quiescence of ovarian surface epithelial cells is instrumental in the initiation and development of ovarian cancer.

“Memory-like T cells” are a subset of thymic cells that acquire effector function through the maturation process rather than interaction with specific antigen. Disruption of genes encoding T cell signaling proteins or transcription factors have provided insights into the differentiation of such cells. We show here that in BALB/c but not C57BL/6 mice, a large portion of thymic CD4-CD8+ T cells exhibit a memory-like phenotype. In BALB/c mice, IL-4 secreted by invariant natural killer T (iNKT) cells is both essential and sufficient for the generation of memory-like T cells. In C57BL/6 mice, iNKT cells are less abundant, producing IL-4 that is insufficient to induce thymic memory-like CD8+ T cells. BALB/c mice deficient in the transcription factor Kruppel-like factor (KLF) 13 have comparable numbers of iNKT cells to C57BL/6 mice and extremely low levels of thymic memory-like CD8+ T cells.  This work documents the dramatic impact of a small number of KLF13-dependent iNKT cells.

Expression data from severe asthmatics, mild asthmatics and healthy controls

Background: Around 5% of children with asthma suffer from chronic symptoms and/or severe exacerbations despite extensive treatment. The causes of severe therapy-resistant childhood asthma are poorly understood. Objectives: To define global patterns of gene expression in severe therapy-resistant vs. controlled asthma and healthy controls. Methods: Children with severe, therapy-resistant (SA, n=20) and controlled asthma (CA, n=20) were identified from a Swedish nation-wide study including extensive clinical and immunological characterisation. In addition, healthy controls were recruited (Ctrl, n=19). White blood cells were isolated and the global transcriptome profile was characterised using the Affymetrix Human Gene ST 1.0 chip. Results: 1378 genes were differentially expressed in one or several of the CA vs. Ctrl, SA vs. CA or SA vs. Ctrl contrasts. A large number could uniquely differentiate the SA group from the CA (n=351 genes) and Ctrl (n=315) groups, whereas fewer genes differentiated the CA from the Ctrl group (n=149). Several non-coding RNAs were found up-regulated in SA compared to CA or Ctrl. Three significantly enriched KEGG pathways were represented; bitter taste transduction, TAS2Rs (up-regulated mostly in SA), natural killer cell mediated cytotoxicity (up-regulated in CA) and N-Glycan biosynthesis (down-regulated in SA). An unsupervised hierarchical clustering of the 1378 genes could crudely separate the SA, CA and Ctrl individuals. Conclusion: Our data indicate a separation in gene expression patterns between children with severe, therapy-resistant asthma and controlled persistent asthma, and suggest novel pathways characterizing the severe therapy-resistant asthma phenotype.

The effect of ablation of Tak1 gene in the liver was characterized.

Omega-6 fatty acids, arachidonic acid (AA) activates PI3K signaling and induces gene expression in prostate cancer

Essential fatty acids (FA) are not only energy-rich molecules; they are also an important component of the membrane bilayer and recently have been implicated in induction of fatty acid synthase (FAS) and other genes.  Using gene chip analysis, we have found that arachidonic acid (AA), an omega-6 fatty acid, induced 11 genes that are regulated by NFkappaB.  We verified gene induction by omega-6 fatty acids including COX2, IKBA, NFKB, GMCSF, IL1B, CXCL1, TNFA, IL6, LTA, IL8, PPARG, and ICAM1 using qRTPCR.  PGE2 synthesis was increased within 5min of addition of AA.  Analysis of upstream signal transduction showed that within 5min of FA addition, phophatidylinositol 3-kinase (PI3K) was significantly activated followed by activation of Akt at 30min.  ERK1 and 2, p38, and SAPK/JNK were not phosphorylated after omega-6 FA addition.  Thirty minutes after FA addition, we found a significant 3-fold increase in translocation of NFkappaB transcription factor to the nucleus.  Addition of non-steroidal anti-inflammatory drug (NSAID) caused a decrease in cox-2 protein synthesis, PGE2 synthesis as well as inhibition of PI3K activation.  We have previously shown that AA induced proliferation is also blocked (P<0.001) by PI3K inhibitor LY294002.  LY294002 also significantly inhibited the AA induced gene expression of COX2, IL1B, GMCSF, and ICAM1.  Taken together, the data suggests that AA via conversion to PGE2 plays an important role in stimulation of growth related genes and proliferation via PI3K signaling and NFkappaB translocation to the nucleus.
Keywords: Time course of Prostate Cancer Activation by Arachidonic Acid

Torcetrapib induces aldosterone and cortisol production in an intracellular calcium-dependent mechanism

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib’s effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

Gene expression analysis in Panc-1 cells in response to treatment with Gli3T, a dominant-negative repressor of Gli

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most aggressive human malignancies. In our studies, we find that the Gli transcription factors are required for Kras initiation of pancreatic tumorigenesis. In order to identify the downstream transcriptional targets of Gli in PDAC, we conducted gene expression analysis using Gli3T, a transcriptional repressor of Gli.
In this data set we include the expression data from 6 samples with three of them expressing a control vector and another three expressing Gli3T

To identify differences in gene expression between peptidylprolyl isomerase F (cyclophilin D; Ppif)-null hearts and WT control hearts.

Gene Expression Profile in kidneys from Cldn16 +/+, +/-, -/- female mice

Claudin-16 deficiency leads to renal salt wasting in humans and mice. Analysis of renal gene expression in Claudin-16 deficient mice, compared to heterozygous and wild type littermates, was performed to gain insights into molecular mechanisms compensating salt loss. Our results indicate the upregulation of known and putative genes for renal transcellular transporters. Furthermore, we could identify a transcript so far not associated with renal salt metabolism, which will provide a first link to a human electrolyte disorder disease.

Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells (mRNA)

Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.

Gene expressing profiling of PC12 cells expressing the FGFR3 mutation responsible for thanatophoric dysplasia type II

To investigate signaling pathways activated by FGFR3 mutations through an analysis of altered expression of downstream transcriptional targets, we performed gene expression profiling of stably transfected inducible PC12 cells expressing a chimeric receptor (PFR3) consisting of the extracellular domain of human platelet derived growth factor receptor (PDGFR) and the transmembrane and intracellular domains of human FGFR3 under the control of the zinc inducible metalliothionein promoter.  Cells expressing the wildtype receptor (i.e. zinc induced) were compared to cells expressing the mutant receptor PFR3K650E, which contains the thanatophoric dysplasia type II mutation, K650E.  See PubMed ID for further description of samples.

THREE INDEPENDENT REPLICATES AND ARE THE CONTROL NON-INFECTED CELLS:
GSM49939, GSM49940, GSM49941

THREE INFECTED INDEPEDENDENT REPLICATES:
GSM49942, GSM49943, GSM49944

We have used primary MEFs derived from wild type and E2F4 null mice growing asynchrounously in serum to generate a signature for E2F4 pathway activation.  10 wild type and 10 E2F4 null samples were each assayed using the Affymetrics Mouse Genome 430A 2.0 array.
Keywords: Primary MEFs from wild type and E2F4 null mice

We overexpressed the spliced form of transcription factor XBP1 in mature F442A adipocytes by adenoviral infection.  Control virus expressed GFP alone.

Polycomb protein EED is required for selective silencing of pluripotency genes upon ESC differentiation

Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation.

Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell (NPC) to a clonal PC and from an indolent clonal PC to a malignant PC, we performed gene expression profiling in 20 patients with MGUS, 33 with high-risk SMM and 41 with MM. The analysis showed that 126 genes were differentially expressed in MGUS, SMM and MM as compared to NPC. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules (snoRNA) and zinc finger proteins. GADD45A was the most significant up-regulated gene in clonal PC compared to NPC. Several proapoptotic genes (AKT1 and AKT2) were downregulated and antiapoptotic genes (APAF1 and BCL2L1) were upregulated in MM, both symptomatic and asymptomatic, compared to MGUS. Myc mediated apoptosis signaling is one of the top canonical pathways differentiating the asymptomatic and symptomatic myeloma. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation in the transition from MGUS to SMM and to MM. In conclusion, our data show that although MGUS, SMM and MM are not clearly distinguishable groups according to their GEP, several signaling pathways and genes were significant deregulated in the different steps of transformation process.

Expression data for the different phases of ulcerative colitis-associated carcinogenesis in a mouse model

Nonresolving inflammation is correlated to carcinogenesis. Ulcerative colitis-associated colorectal cancer (UC-CRC), one of the typical carcinoma generated by inflammation that cannot be resolved properly, has been widely believed to involve a multistep process contains “inflammation-dysplasia-cancer” sequence. The exact molecular mechanisms underlying the step-wise development of UC-CRC is still not fully understood. Detecting the changes in gene expression profiles may help to reveal why and how does the prolonged inflammatory response lead to carcinogenesis, and to characterize potential diagnostic/prognostic markers or additional therapeutic targets for UC-CRC. There for, we performed temporal genome expression profiling analysis using the Affymetrix genome wide microarray system to identify broad scale changes in gene expression associated with the development of colitis-associated cancer, based on an AOM/DSS induced mouse model of UC-CRC.

Gene expression data from Hexose-6-phosphate dehydrogenase knockout mouse muscle at 4 weeks

Hexose-6-phosphate dehydrogenase (H6PD)is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver, H6PD is required for the 11-oxoreductase activity of 11ss-hydroxysteroid dehydrogenase type 1 (11ss-HSD1), which converts inactive 11-oxo glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in Type II (fast) fibers which have increased glycogen content. They also display a progressive vacuolar myopathy evident after 4 weeks of age.
We carried out microarray analysis on TA and soleus muscles from 4 week old WT and KO mice to determine an expression profile predicting myopathy.

Genome-wide analysis of gene expression patterns in the liver tumors of mir-122 knockout mice

To invesigate the physiological roles of mir-122 in hepatocarcinogenesis, we performed expression profiling of the liver tumors of mir-122 knockout mice and the liver tissues of the control B6/129 mice.

mouse embryonic fibroblasts, gene expression profiling, microarray, Affymetrix Mouse Genome 430 2.0, cell type comparison

The roles of histone demethylase RBP2 in gene expression were assessed using gene expression profiling experiments with wild type and RBP2-/- primary MEFs. Several cytokine genes including SDF1 and Kit ligand were upregulated upon inactivation of RBP2.
Keywords: mouse embryonic fibroblasts, gene expression profiling, microarray, Affymetrix Mouse Genome 430 2.0, cell type comparison

Cardiac muscle, comparisons of wild type and mdx mice, 8 week and 10.5 month (Porter lab)

Determination of gene expression changes in cardiac muscle of mdx (dystrophin-deficient) mice at postnatal ages 8 weeks and 10.5 months.  3 independent replicates/age/strain.

Endometriosis is a common disease seen by gynecologists. Clinical features involve pelvic pain and unexplained infertility. Although endometriosis is pathologically characterized by endometrial tissue outside the normal uterine location, endometriosis is otherwise not easily explained. Endometriomas, endometriotic cysts of the ovary, typically cause pain and distortion of pelvic anatomy. To begin to understand the pathogenesis of endometriomas, we carried out transcriptome:microRNAome analysis of endometriomas and eutopic endometrium, using gene expression arrays and next generation small RNA sequencing technology.

Vitamin C induces Tet-dependent DNA demethylation in ES cells to promote a blastocyst-like methylome [Affymetrix]

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germline and may be mediated by Tet (ten-eleven-translocation) enzymes, which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Tet enzymes have been extensively studied in mouse embryonic stem (ES) cells, which are generally cultured in the absence of Vitamin C, a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of Vitamin C to ES cells promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by Vitamin C treatment. Importantly, Vitamin C, but not other antioxidants, enhances the activity of recombinant human Tet1 in a biochemical assay and the Vitamin C-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ES cells. Vitamin C has the strongest effects on regions that gain methylation in cultured ES cells compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) elements, which are resistant to demethylation in the early embryo, are resistant to Vitamin C-induced DNA demethylation. Collectively, this study establishes that Vitamin C is a direct regulator of Tet activity and DNA methylation fidelity in ES cells.

Novel Foxo1-dependent Transcriptional Programs Control Treg Cell Function [Affymetrix gene expression data]

Regulatory T (Treg) cells characterized by expression of the transcription factor forkhead box P3 (Foxp3) maintain immune homeostasis by suppressing self-destructive immune responses1-4.  Foxp3 operates as a late acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg cell lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6-10. However, whether Foxo proteins act beyond the Treg cell commitment stage to control Treg cell homeostasis and function remains largely unexplored.  Here we show that Foxo1 is a pivotal regulator of Treg cell function.  Treg cells express high amounts of Foxo1, and display reduced T-cell receptor-induced Akt activation, Foxo1 phosphorylation, and Foxo1 nuclear exclusion.  Mice with Treg cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to Foxp3-deficient mice, but without the loss of Treg cells.  Genome-wide analysis of Foxo1 binding sites reveals ~300 Foxo1-bound target genes, including the proinflammatory cytokine Ifng, that do not appear to be directly regulated by Foxp3.  These findings demonstrate that the evolutionarily ancient Akt-Foxo1 signaling module controls a novel genetic program indispensable for Treg cell function.

Expression data from control and Phospholipid dependent kinase 1 (PDK1) null cytotoxic T-lymphocytes (CTL) and from control and Akt inhibitor treated CTL

In cytotoxic T cells (CTL), Protein Kinase B /Akt is activated by the T cell antigen receptor (TCR) and the cytokine Interleukin 2 (IL2), in part by phosophorylation of Akt by Phospholipid dependent kinase 1 (PDK1).
The role of PDK1 and Akt in CTL has however not been fully defined.
In order to explore the relative roles of these kinases in CTL we used microarrays to profile the gene expression of control and PDK1 null CTL.
In separate experiments we compared the gene expression profiles of control and Akt inhibitor treated CTL.

Gene expression profiling of CD34+ cells from MDS patients and normal controls

In order to gain insight into the poorly understood pathophysiology of the myelodysplastic syndromes (MDS), we have determined the gene expression profiles of the CD34+ cells of 55 MDS patients using the Affymetrix GeneChip U133 Plus2.0 platform

Expression data from mitochondrial protease ClpP knock-out mice.

The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism.

Mouse pancreas from wild type and MistKO animals were induced either with caerulein or saline as control and processed for RNA.  Targets from three biological replicates of each were generated and the expression profiles were determined using Affymetrix Mouse Expression chips 430.  Comparisons between the sample groups allow the identification of genes with differential expression patterns of genes which might contribute to pancreatitis.

We studied the molecular mechanisms of hepatocellular carcinoma (HCC) initiation and promotion using the Mdr2-knockout (Mdr2-KO) mice at pre-cancerous stages of liver disease. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by the development of HCC between the ages of 12 and 15 months. Liver tissue samples of Mdr2-KO and control Mdr2-heterozygotes mice aged 3 and 12 months, were subjected to histological, biochemical and gene expression profiling analysis using Affymetrix Mouse Genome Array.
Keywords: HCC, murine model, precancerous stages, chronic liver desease

Effect of AT1 receptor anatagonist on brain microvessels from hypertensive and normotensive rats

In hypertension, abnormal regulation of microcirculation and endothelial dysfunction enhances vulnerability to hypertensive brain damage. In addition to lowering blood pressure, blockade of Angiotensin II AT1 receptors protects against stroke and stress in different animal models and this treatment may be of therapeutic advantage. We studied gene expression using Affymetrix Rat Genome U34A arrays from brain microvessels of spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto controls (WKY) rats treated with an AT1 antagonist (candesartan, 0.3 mg/kg/day) or vehicle via osmotic minipumps for 4 weeks.

Expression data in H1299 after retinoblastoma binding protein-2 (RBP2) knockdown

To identify genes whose expression was affected by RBP2, we performed gene expression microarray analysis in control and RBP2 depletion human lung cancer cell line. Two individual shRNA (RBP2 KD1 and RBP2 KD2) were used to limit potential off-target effects.

Gene expression changes in cultured LNCaP cells after DHT-stimulation and various treatment conditions

Suppression of androgen receptor mediated gene expression by a sequence-specific DNA binding polyamide

Androgen Receptor (AR) is essential for the growth and progression of prostate cancer in both hormone-sensitive and hormone-refractory disease.  We have designed a sequence-specific DNA binding polyamide (1) that targets the consensus androgen response element (ARE).  This polyamide binds the PSA promoter ARE, inhibits androgen-induced expression of PSA and several other AR-regulated genes in cultured prostate cancer cells, and reduces AR occupancy at the PSA promoter and enhancer.  Down-regulation of PSA by this polyamide was comparable to that produced by the synthetic anti-androgen bicalutamide (Casodex) at the same concentration.  Genome-wide expression analysis reveals that a similar number of transcripts are affected by treatment with the polyamide and with bicalutamide.  Direct inhibition of AR-DNA binding by sequence-specific DNA binding small molecules could offer an alternative approach to antagonizing AR activity.  A polyamide (2) that targets a different DNA sequence is included as a control.
Keywords: Gene expression changes in cultured LNCaP cells after DHT-stimulation and various treatment conditions

Although activated CD4+ T cell-driven overproduction of cytokines, especially TNF and IL1, is generally regarded as the major factor in the development of rheumatoid arthritis (RA), little is known about the precise role of CD4+ T cells in the initiation and progression of this disease. In this study, this issue was addressed using a time-course microarray analysis in a mouse model of RA, Il1rn deficient mice.No obvious cytokine gene expression changes reflecting T cell activation was observed in CD4+ T cells in the Il1rn deficient mice during the course of spontaneous arthritis. On the contrast, majority of dysregulated genes were those predominantly expressed in myeloid lineage cells, suggesting T cell reprogramming involvement in arthritis development. Distinct gene expression patterns were identified for different stages of disease, including downregulated expression of immunoglobulin heavy chain constant region genes and increased expression of inflammatory genes in the early phase, and downregulation of MHC class II genes in the late phase. The common changes occurred in both early and late phases included upregulation of Arl2bp and Mfap1, which are involved in the regulation of cytoskeletal dynamics.

Macrophages from RA synovial fluids were compared to primary human blood-derived macrophages.

Gata5 is a zinc finger transcription factor that is expressed in embryonic pulmonary mesenchyme and becomes upregulated in the lungs, gut, and bladder during postnatal development. We used microarray to comapre gene expression profiles of mouse lung between Gata5 knockout and wild type mice. We hope to identify the differentially expressed genes that affected by Gata5 gene deletion and their functional clusters or pathways.
The goal of this study is  to identify the differentially expressed genes that affected by Gata5 gene deletion and their functional clusters or pathways.

Effect of 4-Hydroxynonenal on gene expression. Comparison of HSF1-siRNA silenced vs control cells

4-Hydroxynonenal (HNE), a cytotoxic and diffusible electrophile generated by the spontaneous decomposition of oxidized lipids, has a suspected role in neurodegenerative and inflammatory disease processes. In addition to promoting cell death, elevated levels of HNE lead to the engagement of cytoprotective signaling pathways, including the heat shock, antioxidant, DNA damage, and ER stress responses. Activation of the heat shock response, mediated by the transcription factor heat shock factor 1 (HSF1), is critical for maintaining cellular viability in the presence of HNE. Accordingly, silencing HSF1 expression using siRNA enhances the toxicity of HNE. Microarray analysis of samples from control and HSF1-silenced cells was performed to investigate which associated changes in gene could be responsible for the decrease in cellular viability.

Impaired neutrophil recruitment in CXCL5 knockout mice during Mtb infection

The effect of trichloroethylene on gene expression in mouse liver

Trichloroethylene (TCE) is a widely used industrial chemical, and a common environmental contaminant. It is a well-known carcinogen in rodents and a probable carcinogen in humans.   Studies utilizing panels of mouse inbred strains afford a unique opportunity to understand both metabolic and genetic basis for differences in responses to TCE. We tested the hypothesis that individual and liver-specific toxic effects of TCE are genetically controlled and that the mechanisms of toxicity and susceptibility can be uncovered by exploring responses to TCE using a diverse panel of inbred mouse strains. TCE (2100 mg/kg) or corn oil vehicle were administered by gavage to 6-8 wk old male mice of 15 mouse strains. Serum and liver were collected at 2, 8, and 24 hr post dosing and were analyzed for TCE metabolites, hepatocellular injury and gene expression of liver. TCE metabolism, as evident from the levels of individual oxidative and conjugative metabolites, varied considerably between strains. TCE treatment-specific effect on the liver transcriptome was strongly dependent on the individual’s genetic background. PPAR-mediated molecular networks, consisting of the metabolism genes known to be induced by TCE, represent some of the most pronounced molecular effects of TCE treatment in mouse liver that are dependent on the individual’s genetic background. Conversely, cell death, liver necrosis, and immune mediated response pathways which are affected by TCE treatment in liver are largely genetic background-independent. These studies provide better understanding of the mechanisms of TCE-induced toxicity anchored on metabolism and genotype-phenotype correlations that may define susceptibility or resistance.

A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke.

White and grey matter in the distribution on the middle cerebral artery 6 hours after administration of the saline/catalytic antioxidant AEOL 10150 and ischemia/reperfusion of the MCAO; pooled mRNA from 6 brains.

The influence of the deletion of Serum Response Factor in B cells

Serum response factor (SRF), a MADS-box transcription factor, is essential for murine embryonic development and for the function of muscle cells and neurons. SRF and its transcriptional co-factors are broadly expressed. To determine the in vivo role of SRF in developing lymphocytes we specifically inactivated the murine Srf gene during T or B cell development using lymphocyte-specific Cre transgenic mouse lines. T cell-specific Srf deletion led to a severe block in thymocyte development at the transition from double to single positive stage. The few residual T cells detectable in the periphery retained at least one functional Srf allele, thereby demonstrating the importance of SRF in T cell development. In contrast, deletion of Srf in developing B cells did not interfere with the growth and survival of B cells in general, yet led to a complete loss of marginal zone B cells and a marked reduction of the CD5+ B cell subset. Our study also revealed a contribution of SRF to the expression of the surface molecules IgM, CD19, and the chemokine receptor 4 in B lymphocytes.

5-aza-2'-deoxycytidine; methylation; dermal fibroblast; chondrocyte; osteoblast

Global analysis of transcriptional changes following genomic demethylation in chondrocytes, osteoblasts, and fibroblasts

DNA methylation can contribute to the stable transcriptional silencing of mammalian genes. Oftentimes, these genes are important developmental regulators, and their silencing in cell types where they are not supposed to be active is important for the phenotypic stability of the cells. To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2’-deoxycytidine, and changes in gene expression monitored by microarray analysis.
Keywords: 5-aza-2'-deoxycytidine; methylation; dermal fibroblast; chondrocyte; osteoblast

Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant.  Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant.  This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner.  This activity depends upon the zinc ion chelating properties of the compound as well as redox changes.  These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development.
We utilized gene expression microarrays to examine the transcriptional activity of p53 targets in TOV112D cells after treatment with NSC319726.

mRNA expression analysis of undifferentiated Dicer +/- (D4) and Dicer -/- (27H10) embryonic cell lines

We have analyzed the transcript expression levels in Dicer heterozygous and Dicer knock-out embryonic stem (ES) cells  in order to identify which transcripts are regulated by RNAi pathway in mouse ES cells.
Keywords: Cell type comparison of cell line with or without knock-out

Gene expression data from IL13-induced allergic airway inflammation of mice lungs

Asthma is a common chronic inflammatory airway condition with a strong genetic and inheritability component, as siblings and first-degree relatives of those with the disease are often affected.
For our studies, we used a well-characterized transgenic mouse model of allergic airway inflammation induced by IL13. In this model, IL13 is conditionally overexpressed in the mouse lung when treated with doxycycline. Upon IL13 induction, these mice showed inflammatory cell infiltration, pronounced emphysema, increased pulmonary compliance, lung volume enlargement, mucus metaplasia, and increased expression of matrix metalloproteinases and cathepsins in the lung. We performed gene expression microarray to examine the changes in gene expression during IL13-induced allergic airway inflammation.

IL-13 involvement in eosinophilic esophagitis: transcriptome analysis

3 eosinophilic esophagitis biopsies, cultured and stimulated with IL-13 : each of them was either left unstimulated or stimulated (100ng for 48h)
We used microarray to uncover the IL-13-induced genes in esophageal epithelial cells of the esophagus

Fresh Atypical ductal hyperplasia (ADH) tissue collected from breast of a women who either (1) had no prior history of breast cancer and had not developed breast cancer in five years after diagnosis, (2) had cancer before ADH, or had cancer at the time as ADH or developed cancer after ADH diagnosis

Parkinson’s disease (PD) progresses relentlessly and affects five million people worldwide. Laboratory tests for PD are critically needed for developing treatments designed to slow or prevent progression of the disease. We performed a transcriptome-wide scan in 105 individuals to interrogate the molecular processes perturbed in cellular blood of patients with early-stage PD. The molecular marker here identified is strongly associated with risk of PD in 66 samples of the training set (third tertile cross-validated odds ratio of 5.7 {P for trend 0.005}). It is further validated in 39 independent test samples (third tertile odds ratio of 5.1 {P for trend 0.04}). The genes differentially expressed in patients with PD, or Alzheimer’s or progressive supranuclear palsy offer unique insights into disease-linked processes detectable in peripheral blood. Combining gene expression scans in blood and linked clinical data will facilitate the rapid characterization of candidate biomarkers as demonstrated here with respect to PD.

The goal of  this experiment was to investigate the early mechanisms of human fulminant hepatitis through ConA-induced hepatitis model.Early diagnosis and interventions are important for patients with fulminant hepatitis and gene expression may be pivotal in the early diagnosis.

Keyword :ConA-induced hepatitis model

Expression changes in mouse brains following nicotine-induced seizures; the modulation of transcription factor networks

Nicotine, acting through the neuronal nicotinic acetylcholine receptors (nAChR), can induce seizures in mice. We aimed to study brain transcriptional response to seizure and to identify genes whose expression is altered after nicotine-induced seizures. Whole brains of untreated mice were compared to brains one hour after seizure activity, using Affymetrix U74Av2 microaarays. Experimental groups included wild-type mice and both nicotine-induced seizures sensitive and resistant nAChR mutant mice. Each genotype group received different nicotine doses to generate seizures. This approach allowed the identification of significantly changed genes whose expression was dependent on seizure activity, nicotine administration or both, but not on the type of nAChR subunit mutation or the amount of nicotine injected. Significant expression changes were detected in 62 genes (p < 0.05, FDR correction). Among them, GO functional annotation analysis determined that the most significantly over-represented categories were of genes encoding MAP kinase phosphatases, regulators of transcription and nucleosome assembly proteins. In-silico bioinformatic analysis of the promoter regions of the 62 changed genes detected the significant enrichments of 16 transcription regulatory elements (TREs), creating a network of transcriptional regulatory responses to seizures. The TREs for ATF and SRF were most significantly enriched, supporting their association with seizure activity. Our data suggest that nicotine-induced seizure in mice is a useful model to study seizure activity and its global brain transcriptional response. The differentially expressed genes detected here can help understand the molecular mechanisms underlying seizures in animal models, and may also serve as candidate genes to study epilepsy in humans.
Keywords: treatment, seizure activity, genotypes

Genetic modification to analyze transgenic overexpression of ABCA1 in a knock-out LDLr genetic backgrounds.

Gene expression series from murine liver samples comparing ABCA1 over-expression in LDL receptor -/- genetic background

Changes in the transcript profile due to ABCA1 expression in murine liver samples was evaluated in LDL receptor -/- genetic backgrounds.
Keywords: Genetic modification to analyze transgenic overexpression of ABCA1 in a knock-out LDLr genetic backgrounds.

Pyruvate induces mitochondrial biogenesis by a PGC-1alpha independent mechanism

The present study examines the impact of altering energy provision on mitochondrial biogenesis in muscle cells.  C2C12 myoblasts were chronically treated with supraphysiological levels of sodium pyruvate for 72 hr.  Treated cells exhibited increased mitochondrial protein expression, basal respiratory rate and maximal oxidative capacity.  The increase in mitochondrial biogenesis was independent of increases in PGC-1alpha and PGC-1alpha mRNA expression.  To further assess whether PGC-1alpha expression was necessary for pyruvate action, cells were infected with adenovirus containing shRNA for PGC-1alpha prior to treatment with pyruvate.  Despite a 70% reduction in PGC-1alpha mRNA the effect of pyruvate was preserved.  Furthermore, pyruvate induced mitochondrial biogenesis in primary myoblasts from PGC-1alpha null mice.  These data suggest that regulation of mitochondrial biogenesis by pyruvate in myoblasts is independent of PGC-1alpha, suggesting the existence of a novel energy-sensing pathway regulating oxidative capacity.

Expression data from wild type and Gata6-deficient tissue resident peritoneal macrophages

Tissue resident macrophages are notoriously heterogeneous, exhibiting discrete phenotypes as a consequence of tissue- and micro-anatomical niche-specific functions, but the molecular basis for this is not understood. We resolved a restricted transcriptional profile for the self-renewing population of peritoneal resident macrophages, which is expressed during homeostasis and inflammation and distinct from other MØ. Prominent within this profile was the expression of Gata6. This study represents a characterisation of the role of Gata6 in peritoneal resident macrophage phenotype. We used microarrays to determine the patterns of gene expression in peritoneal resident MØ in the absence of GATA-6 against wild type.

Expression data from MCF-7 cells stimulated by Estrogen or IGF-I

Although estrogen receptor (ER) and insulin-like growth factor (IGF) signaling are important for normal mammary development and breast cancer, cross-talk between these pathways, particularly at the level of gene transcription, remains poorly understood.  We performed microarray analysis on MCF-7 breast cancer cells treated with estradiol (E2) or IGF-I for 3hr or 24hr.  IGF-I regulated mRNA of 5-10-fold more genes than estradiol, and many genes were co-regulated by both ligands.  Importantly, expression of these co-regulated genes correlated with poor prognosis of human breast cancer.  Closer examination revealed enrichment of repressed transcripts.  Interestingly, a number of potential tumor suppressors were down-regulated by IGF-I and estradiol.  In fact, BLNK, one of the top repressed genes, is a potential growth suppressor in breast cancer cells.  Analysis of three down-regulated genes showed that E2-mediated repression occurred independently of IGF-IR, and IGF-I-mediated repression occurred independently of ER.  However, repression by IGF-I or estradiol required common downstream kinases.  In conclusion, E2 and IGF-I co-regulate a set of genes that affect breast cancer outcome.  There is enrichment of repressed transcripts, and the down-regulation is independent at the receptor level.  This may be important clinically, as tumors with active ER and IGF-IR signaling may require co-targeting of both pathways.
KEYWORDS: multiple group comparison

Protective Role of IL-10 in Ozone-induced Pulmonary Inflammation

Background: The mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear.  Interleukin (IL)-10 is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators.
Objectives: The current study investigated the molecular mechanisms underlying IL-10-mediated attenuation of O3-induced pulmonary inflammation in mice.
Methods: Il10-deficient (Il10-/-) and wild type (Il10+/+) mice were exposed to 0.3-ppm O3 or filtered air for 24, 48 or 72 hr.  Immediately following exposure, differential cell counts, and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF).  mRNA and protein levels of cellular mediators were determined from lung homogenates.  We also utilized global mRNA expression analyses of lung tissue with Ingenuity Pathway Analyses (IPA) to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation.
Results: Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10-/- mice than in Il10+/+ mice after exposure to O3 at all time points tested.  O3-enhanced nuclear NF-kB translocation was elevated in the lungs of Il10-/- compared to Il10+/+ mice.  Gene expression analyses revealed several key IL-10 and O3-dependent mediators, including IL-6, MIP-2, IL-1 and CD86.
Conclusions: Results indicated that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation.  Moreover, gene expression analyses identified three response pathways and several novel genetic targets (e.g. Ccr1, Socs3, Il33, Hat1, and Gale) through which IL10 may modulate the innate and adaptive immune response.  These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals.

Progressive loss of PGC-1alpha expression in aging muscle potentiates glucose intolerance and systemic inflammation

Decreased mitochondrial mass and function in muscle of diabetic patients is associated with low PGC-1alpha, a transcriptional coactivator of the mitochondrial gene program. To investigate whether reduced PGC-1alpha and oxidative capacity in muscle directly contributes to age-related glucose intolerance, we compared the genetic signatures and metabolic profiles of aging mice lacking muscle PGC-1alpha. Microarray analysis revealed that a significant proportion of PGC-1alpha-dependent changes in gene expression overlapped with age-associated effects, and aging muscle and muscle lacking PGC-1alpha shared gene signatures of impaired electron transport chain activity and TGFbeta signalling.

Expression data from naïve and MPTP-exposed cholinergic transgenic mice

PD  is the second most common neurodegenerative disease worldwide with growing prevalence. MPTP is a neurotoxin which causes the appearance of Parkinson's disease (PD) pathology.  The involvement of the cholinergic system in PD has been identified decades ago and anti-cholinergic drugs were upon the first drugs used for symptomatic treatment of PD. Of note, MPTP intoxication is a model of choice for symptomatic neuroprotective therapies since it have been quite predictive. Mice were exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), with or without the protective acetylcholinesterase (AChE-R) variant. Transgenic AChE-S (the synaptic variant), AChE-R (the shorter, protective variant) and FVB/N control mice were included in this study. Two brain regions were examined: the pre-frontal cortex (PFC) and the striatal caudate-putamen (CPu). Each condition (i.e brain region and transgenic variant) was examined on both naive and MPTP-exposed mice.

This study was performed to identify genes that are regulated following iFGFR1 activation in mammary glands from MMTV-iFGFR1 transgenic mice.

Expression data from wild-type and Zmpste24-/- mouse embryonic fibroblasts (MEFs) at an early passage (passage 3, P3)

Zmpste24 is a metalloproteinase processing prelamin A into mature lamin A, a nuclear structure protein. Zmpste24-/- mice which accumulate prelamin A in cells recapitulate accelerated aging phenotypes observed in human premature aging disorder, Hutchinson Gilford progeria sydrome (HGPS). Zmpste24-/- mouse embryonic fibroblasts (MEFs) exhibited genomic instabiliy and accelerated aging at cellular level, which is premature senescence.
We performed microarray analysis on Zmpste24-/- MEFs, compared to wild-type littermates' MEFs, at an early passage (P3), which is a pre-symptom stage before cellular senescence occurs in the mutant MEFs, in order to examine gene expression profile and figure out the underneath mechanism triggering the premature aging process.

Expression analysis of Reh cells after transfection with shRNA targeting CBFA2T3 and/or constitutively active IKKβ(EE)

Genome-wide gene expression analysis of Reh cells following transfection with shRNA targeting CBFA2T3, constitutively active IKKβ(EE), or both in combination.

Gene expression profile of squamous lung cancer cells are used to identify genes that are differentially regulated.
Keywords: Cell type comparison

In this study we used the d337T TRb transgenic mouse that has been created to reproduce the human genetic disease known as resistance to thyroid hormone (RTH) as a model to determine if the d337T TRb mutation would have an effect on PPARa activation.  A single amino acid deletion (d337T)  abrogates thyroid hormone (T3) binding and transforms the thyroid hormone receptor (TRb) into a constitutive repressor.  The principle goal was to determine if T3 regulates myocardial energy metabolism through its nuclear receptors.  We introduced a known PPARa activator (WY14, 643) into control and d337T TRb transgenic mice then examined cardiac gene expression using Affymetrix 430_2  expression arrays and RT-PCR.  We compared the gene expression of  PPARa, RXRb and TRa,b and three PPARa target genes among four studies groups [control, control with WY14, 643, d337T TRb, and d337T TRb with WY14, 643] consisting of seven mice per group.  Microarray analysis revealed that these genes responded to the WY14, 643 treatments of control and d337T TRb mice.  Analysis of the array and RT-PCR data indicates that mRNA expression levels of PPARa and mRXRb decrease after a six hour drug treatment in both control and d337T TRb mice (P<0.01) as did the array mRNA expression levels for TRa & b (P<0.025).  Three target genes (AMPD3, PDK4 and UCP3) of PPARa were up regulated in control and down regulated in the d337T TRb transgenic mouse, indicating a direct action on these metabolic genes when the TRb becomes a repressor.   In conclusion, PPARa activation by WY14, 643 has a positive effect on control mice and a negative effect on the TRb transgenic mice which supports our hypothesis that T3 regulates myocardial energy metabolism through its nuclear receptors.
Keywords: treatment and deletion effects

Investigation of expression profile in XDP brains.
Keywords: an XDP patient and neurologically normal control

Gene expression profiling of two diffuse large B-cell lymphoma (DLBCL) cell lines, DHL4 and DHL6, treated with the SYK inhibitor, R406

The two DLBCL cell lines were treated with R406 to assess the signature of SYK inhibition. In previous studies, R406 decreased the proliferation and induced apoptosis of these surface Ig+ cell lines. In the previous studies, R406 inhibited the autophosphorylation of SYK 525/526 and SYK-dependent phosphorylation of BCR signaling components such as BLNK.

Changes in gene expression induced by CDK9 inhibition alone and in combination with fludarabine

Using a transcriptomics approach we explored the mechanism(s) of synergy observed between CDKI-73 and fludarabine in primary CLL cells. The cytotoxic effects of CDKI-73 were associated with transcriptional inhibition of cdk9 target genes including MCL1 and XIAP. In contrast, fludarabine induced the transcription of these genes, an effect that was reversed by the combination of CDKI-73 and fludarabine.
We used microarrays to explore the cytoxic synergy observed in primary CLL cells when we combined a novel CDK9 inhibitor with the purine nucleoside analogue fludarabine

Analysis of gene expression changes with human SNRK-GFP overexpression.  The purpose of the study was to determine the expression of the genes that are altered by SNRK in order to obtain evidence about what cellular functions SNRK regulates.

Expression Data from PtenF341V and Null Mouse Embryonic Fibroblasts

PTEN imparts tumor suppression in mice by cell autonomous and non-autonomous mechanisms. Whether these two tumor suppressor mechanisms are mediated through similar or distinct signaling pathways is not known. Here we generated and analyzed knockin mice that express a series of human cancer-derived mutant alleles of PTEN that differentially alter the Akt axis in either stromal or tumor cell compartments of mammary glands. We find that cell non-autonomous tumor suppression by Pten in stromal fibroblasts strictly requires activation of P-Akt signaling, whereas cell autonomous tumor suppression in epithelial tumor cells is independent of overt canonical pathway activation. These findings expose distinct Akt-dependent and independent tumor suppressor functions of PTEN in stromal fibroblasts and tumor cells, respectively, that can be used to guide clinical care of breast cancer patients

Gene expression profile of VIN lesions in comparison to controls

In order to understand the molecular mechanism behind Vulvar Intraepithelial Neoplasia (VIN), we have analyzed the gene expression profile of VIN lesions in comparison to controls.

Gene expression profile on ischemic kidneys from wildtype C57BL/10 and TLR4 null C57BL/10ScNJ mice

TLR4 deficiency attenuates kidney injury after ischemic reperfusion as measured by both renal function and morphology. To better understand the role of TLR4 during the acute kidney injury, we used DNA microarray to identify genes that were differentially expressed on kidneys in wildtype B10 mice and TLR4 null mice during the early stage of injury.

C57/BL6 to C57/BL6
Balb/c to C57/BL6
Balb/c to C57/BL6 with anti CD80/86 mAb

Identificaton of gene expression profile in tolerizing murine cardiac allograft by co-stimulatory blockade.

The induction of specific tolerance would be the ultimate achievement in transplant immunology, but the precise mechanisms of immunological tolerance remain largely unknown. Here, we investigated global gene expression analysis in tolerizing murine cardiac allografts by means of oligonucleotide microarrays. Tolerance induction was achieved in cardiac allografts from BALB/c to C57BL/6 mice by daily intraperitoneal injection of anti-CD80 and CD86 mAbs. Comparative analysis revealed 64 genes to be induced more extensively in the tolerizing than in the syngeneic isografts, and 16 genes than in the rejecting allografts. Two genes were specifically upregulated in the tolerizing allografts. In the tolerizing allografts there were induced marked expressions of a number of genes for proinflammatory factors, including interferon-gamma inducible cytokines and chemokines, as well as apoptosis-related genes which were also upregulated in the rejecting allografts. Moreover, these gene expression patterns continued to be upregulated more than 70 days post transplant. These results provide evidence that immunological tolerance can be induced and maintained in the presence of prominent proinflammatory gene expression in vivo.

Adult postmortem brain tissue (orbitofrontal cortex) from subjects with bipolar disorder and healthy controls

Bipolar affective disorder is a severe psychiatric disorder with a strong genetic component but unknown pathophysiology. We used microarray technology (Affymetrix HG-U133A GeneChips) to determine the expression of approximately 22 000 mRNA transcripts in post-mortem brain tissue (orbitofrontal cortex) from patients with bipolar disorder and matched healthy controls. Orbitofrontal cortex tissue from a cohort of 30 subjects was investigated and the final analysis included 10 bipolar and 11 control subjects. Differences between disease and control groups were identified using a rigorous statistical analysis with correction for confounding variables and multiple testing.

Helicobacter pylori (H. pylori) infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation. Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients. Thus, the impact of H. pylori on the expression of a broad set of genes has not yet been analyzed. cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H. pylori infected patients. Two main gene expression profiles were identified based on cluster analysis. The data obtained suggest a strong involvement of selected Toll-like receptors (TLRs)3, adhesion molecules, chemokines, and interleukins in the mucosal response. This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H. pylori. The genotype of the bacteria (presence of genes encoding CagA, VacA and BabA) was analyzed by PCR and shown to be associated with different expression profiles. The presence of a
Keywords = H. pylori

To begin to identify genes involved in the transdifferentiation process we analyzed Barrett’s esophagus (with no dysplasia), normal esophagus and small intestine biopsy samples by Affymetrix microarray.

High cholesterol diet and phenobarbital affect cholesterol homeostasis and drug metabolism

High cholesterol diet and xenobiotic treatment induce changes in cholesterol homeostasis and drug metabolism. Mice were either 7 days on high cholesterol diet or were treated with phenobarbital. Liver samples were anayzed using Affymetrix GeneChip MOE430A.
Keywords: treatment and diet effects

RNA samples prepared from lymphoblastic cells or cell lines from normal and FCHL (familial combined hyperlipemia) patients

Exposure of cystic fibrosis bronchial epithelial cells (CFBE 41 o-) to Pseudomonas aeruginosa (PA01) biofilms

In the clinical setting, mutations in the CFTR gene enhance the inflammatory response to P. aeruginosa (PA01) infection, but measurements of the inflammatory response to pathogen stimulation by isolated airway epithelia can yield variable results. In this series, we exposed CFBE41o- cells over-expressing ∆F508/∆F508 CFTR and CFBE41o- cells rescued with wt-CFTR to P. aeruginosa  biofilms. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL2,  CXCL3, CXCR4 and TNF-α) in CFBE-wt-CFTR cells compared to CFBE-∆F508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o- ∆F508/∆F508-CFTR cells.

124 gastric biopsies comprising of Normals, Intestinal Metaplasia, Chronic Gastritis and Carcinomas are profiled.

The ratio metric is given by (Biological sample / Universal Reference). The Biological Samples are labeled with Cy5 (Green) and the Pooled Normal Biopsies  RNA is always labeled with Cy3 (Red).
The arrays were scanned using a ScanArray 5000 scanner (Perkin-Elmer) and data extraction was done using Quantarray (GSI Lumonics). Subsequently, the data was imported into GeneSpring (Silicon Genetics) and intensity dependent LOWESS normalization was performed. The 7383 genes were filtered using an intensity in the reference channel (Cy3) of 300. This corresponded to 2 standard deviations above the mean for the background signal. Background singal was calculated using the empty wells/spots on the array and the average of 4 different positions on the slide were used.

Anesthetic gases elicit organ protection in patients undergoing coronary artery bypass graft (CABG) surgery.  This study aimed at identifying myocardial transcriptional phenotypes and anesthetic-induced changes in gene expression to predict cardiovascular biomarkers and cardiac function after off-pump CABG.
Keywords: cardiac surgery, anesthetics

TCF-1 is an HMG family transcription factor which is known to be activated by the canonical Wnt signaling pathway and modulated by other signals such as those derived from T cell receptor. We found that during CD8 T cell responses, TCF-1 deficiency impaired long-term maintenance of antigen-specific memory CD8 T cells.
We used microarrays to detect gene expression changes in memory CD8 T cells caused by TCF-1 deficiency.

Gene array analysis of anaplastic thyroid carcinoma tissue versus matched and unmatched normal thyroid tissue

Currently there is a lack of effective therapies which result in long-term durable response for patients presenting with anaplastic thyroid carcinoma (ATC), a very rare and lethal variant of thyroid cancer. ATC is resistant to chemotherapy, radiation, and targeted therapies currently available. In an effort to identify novel tumor-specific therapeutic targets, we performed high throughput gene array analysis screening numerous patient ATC tumor tissues, and compared their gene expression levels to matched and unmatched normal thyroid tissue samples.

DOCK8 is critical for the survival and function of NKT cells [DOCK8_CPM_NKT]

Analysis of DOCK8 deficient animals revealed a key role for this protein the survival and maintenance of natural killer T cells. This work lead to the identification of genes regulated by the guanine exchange factor, DOCK8.

Limitless reproductive potential is one of the hallmarks of cancer cells1. This ability is accomplished by maintaining telomeres, which erosion otherwise causes cellular senescence or death. Human cancer cells often maintain shorter telomeres than do cells in surrounding normal tissues2-5. While most cancer cells activate telomerase, which can elongate telomeres6, it remains elusive why cancer cells keep telomeres short. Here we show that forced elongation of telomeres in cancer cells promotes their differentiation in a tumor microenvironment in vivo. We elongated telomeres of human prostate cancer PC-3 cells, which possess short telomeres7, by enhancing their telomerase activity. The resulting cells with long telomeres retain an ability to form tumors in a mouse xenograft model. Strikingly, these tumors exhibit many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These phenotypic changes are caused by telomere elongation per se but not enhanced telomerase activity. Gene expression profiling revealed that telomere elongation correlates with inhibition of cell-cycle processes. Together, our results suggest a functional contribution of short telomeres to tumor malignancy by regulating cancer cell differentiation.

Thus, mouse macrophage cultures were established from PBMCs isolated from wild-type control mice and were inoculated with SeV (Sendai virus) or UV-SeV (UV-inactivated SeV). These microarrays were performed in concert with assays of CCL5 and CCR5 expression, viral replication, and cellular apoptosis. Initial experiments indicated that wild-type mouse macrophages inoculated with SeV exhibit induction of CCL5 mRNA to the highest level of any known mouse gene product, while mRNA levels for CCL5 receptors (CCR5 as well as CCR3 and CCR1) or alternative ligands for these receptors (CCL3 and CCL4) were relatively unchanged by viral infection.
Keywords: Treatment Comparison

Activation of mTOR controls the loss of TCRζ in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation

CD3-positive T cells were negatively isolated from 10 SLE patients and 9 healthy controls without SLE.  All of the SLE samples and control samples were compared with one another to identify baseline differences in expression due to the disease.  Next, T cell preparations from 4 of the control subjects were stimulated with either Nitric Oxide (NOC-18) 600 uM for 24hr or stimulated through CD3/CD28 for 24hr to determine which genes were responsive to these signaling mechanisms. Here, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in SLE T cells. Activation of mTOR was inducible by NO, a key trigger of MHP which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in SLE T cells and, in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 over-expression was also inversely correlated with diminished TCRζ protein levels. Combined with follow up studies, these results suggest that activation of mTOR causes the loss of TCRζ in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

Accelerated leukemogenesis by truncated CBFb-SMMHC defective in high-affinity binding with RUNX1

Dominant RUNX1 inhibition has been proposed as a common pathway for CBF-leukemia.  CBFb-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). We generated knock-in mice expressing CBFb-SMMHC with a HABD deletion, CBFb-SMMHCd179-221. These mice developed leukemia highly efficiently, even though hematopoietic defects associated with Runx1-inhibition were partially rescued.
To identify changes in gene expression with the deletion of the HABD, we compared the gene expression profile in leukemia samples from mice expressing CBFb-SMMHCd179-221 with those from mice expressing full length CBFb-SMMHC.

Pax6 is required for normal cell cycle exit and the differentiation kinetics of retinal  progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 was implicated in both proliferation and differentiation of multiple regions within the CNS, its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6- retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6- precursors. These alterations were accompanied by co-expression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27kip1 and P27kip2) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks which function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.

Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis [HEL cell lines]

We used expression profiling, SNP arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status.  In addition, the activated JAK2 signature was present in patients with somatic CALR mutations.  Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK mutational status are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.

Title: Transcriptome analysis of human endometrial tissues from healthy post-menoupausal women reflecting the endometrial response to 3-weeks treatment with tibolone, E2 and E2+MPA.
In an observational, open, non-randomized, controlled study uterine tissues were collected in order to generate endometrial gene expression profiles. healthy postmenopausal women were enrolled into the following treatment groups: Control-group; Tibolone-group, 2,5 mg of tibolone (administered orally) every day, starting 21 days prior to surgery; Estradiol-group, 2 mg of estradiol (administered orally) every day, starting 21 days prior to surgery; Estradiol+progestagen-group, 2 mg of estradiol (administered orally) and 5 mg of MPA (Medroxy Progesteroneacetate, administered orally) every day, starting 21 days prior to surgery. Pure (100%) endometrium was isolated from the snap-frozen uterine tissues and used for RNA isolation. RNA was labeled and hybridized to whole genome Affymetrix U133plus2 GeneChips containing 54,614 probe sets, representing approximately 47,000 transcripts.
Relative to the control group, 940 genes are regulated in the endometrium of E2 treated patients, whereas only 198 genes are significantly regulated in endometria from tibolone or E2+MPA treated patients. Furthermore, only 9% of E2 regulated genes are also regulated by tibolone (85 out of 940), only 5% are also regulated by E2+MPA treatment and the overlap between tibolone and E2+MPA treatment is about 10%. This indicated that tibolone-treatment results in a weak endometrial profile similarity to E2 treatment and no profile similarity to E2+MPA treatment. A more detailed analysis showed that down stream processes, such as regulation of the cell cycle, angiogenesis and cell proliferation are almost not affected by tibolone treatment but, in contrast, are significantly affected by E2. For example, upon staining with Ki67, a marker for mitotic activity, significantly increased stromal as well as glandular cell proliferation was observed in the endometria from the E2-only treated group, while tibolone treatment resulted only in a slight increase in stromal cell proliferation (and no increase in glandular cell proliferation). These results indicate that in contrast to long-term tibolone use, short-term (21-days) use results in some estrogenic stimulation of the endometrium, which is clearly far less and rather different from what is observed during E2 treatment.
Klaassens et al., 2006
Hanifi-Moghaddam et al., 2007
Verheul et al., 2007

Expression data from melanoma cell lines 224 and BL transfected with siRNA-NRASQ61R

Transcriptional profiling for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRASQ61R

Genomic microarray analysis of adrenergic-deficient (Dbh-/-) vs. wild-type control (Dbh+/+) mouse heart expression at embryonic day 10.5 (E10.5).

Gene expression analysis of human iPSC generated from pathogenic LRRK2 (G2019S) mutation bearing patients

Genetic mutations on leucine-rich repeat kinase 2 (LRRK2) have been associated with an increased risk of Parkinson's disease. The Gly2019Ser (G2019S) mutation on LRRK2 gene is a relatively common cause of familial Parkinson's disease in Caucasian population. In this study, we generated human induced pluripotent stem cell (iPSC) lines from LRRK2 (G2019S) bearing patient fibroblasts by cell reprogramming.
We performed global gene expression profiling of LRRK2 (G2019S) heterozygous and homozygous patient iPSC lines, and the corresponding fibroblast lines they originated from. An age-matched wildtype human fibroblast line and H1 human embryonic stem cell (ESC) line were used as controls.

Tpl2 promotes chemokine/chemokine receptor expression and macrophage migration during acute inflammation

In autoimmune diseases, accumulation of activated leukocytes correlates with inflammation and disease progression, and therefore, disruption of leukocyte trafficking is an active area of research.  The protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity.  Herein, we addressed the contribution of Tpl2 to the regulation of macrophage chemokine and chemokine receptor expression and subsequent migration in vivo using a mouse model of Tpl2 ablation.  We found that gene expression of the chemokine ligands CCL2, CCL7, CXCL2, and CXCL3 as well as the chemokine receptors CCR1 and CCR5 were reduced in macrophages from the bone marrow and peritoneal cavities of tpl2-/- mice following stimulation with LPS.  LPS stimulation repressed chemokine receptor expression of CCR1, CCR2 and CCR5.  Notably, LPS-induced repression of CCR1 and CCR5 was significantly enhanced in Tpl2-deficient macrophages and was observed to be dependent upon Erk activation and independent of PI3K and mTOR signaling.  Consistent with alterations in chemokine and chemokine receptor expression, tpl2-/- macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation.  Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of both chemokine receptors and their ligands and provides further insight into how Tpl2 inhibition may disrupt inflammatory networks in vivo.

Determination of mRNA transcripts in HeLa cells that are regulated by RENT1

HeLa cells were treated with siRNA directed against Luciferase or RENT1 in duplicate (as described in Mendell et al., Science, 2002; PubMed ID:12228722).  Transcripts that are differentially expressed between the two experimental conditions are putatively regulated by RENT1.

Comparative gene expression analysis of repro9/repro9 and wild type testes from 14 and 17 day mice

Repro9 in an allele of Mybl1 (A-Myb) transcription factor obtained in ENU screen to identify alleles causing mouse infertility. Repro9/repro9 mutant males are infertile due to meiotic arrest at pachytene stage. Mutants show wide range of abnormalities including inefficient chromosome synapsis, sex body formation and progression through meiotic cycle. Females are unaffected. To determine genes transcriptionally regulated by MYBL1 we analyzed gene expression profiles of wild type and repro9/repro9 mutant testis at 14 and 17 days postpartum. Analysis revealed many misregulated genes, in majority downregulated, at day 14 pp and even more at day 17 pp, probably due to secondary effects of meiotic arrest. Significantly misregulated genes were characterized by Gene Ontology. Comparative gene expression analysis uncovered potential targets of MYBL1 regulation that play roles in regulation of transcription, cell cycle, apoptosis, protein phosphorylation and ubiquitination, chromosome organization and others.
Abstract:The transcriptional regulation of mammalian meiosis is poorly characterized, due to few genetic and ex vivo models.  From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several critical meiotic processes.  Spermatocytes bearing a novel separation-of-function allele (Mybl1repro9) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double strand break (DSB) repair on synapsed pachytene chromosomes, and a lack of crossing-over.  MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically.  These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation.  Meiosis in mutant females appears unaffected. These results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, including chromosome synapsis, recombination, and cell cycle progression through pachynema.

SPARC is a matricellular glycoprotein involved in regulation of the extracellular matrix, growth factors, adhesion, and migration.  SPARC-null mice have altered basement membranes and develop posterior sub-capsular cataracts with cell swelling and equatorial vacuoles.  Exchange of fluid, nutrients, and waste products in the avascular lens is driven by a unique circulating ion current.  Here we demonstrate that SPARC-null mouse lenses exhibit abnormal circulation of fluid, ion, and small molecules which leads to altered fluorescein distribution in vivo, loss of resting membrane polarization, and altered distribution of small molecules.  Microarray analysis of SPARC-null lenses showed changes in gene expression of ion channels and receptors, matrix and adhesion genes, cytoskeleton, immune response genes, and cell signaling molecules.  Our results demonstrate that the regulation of SPARC on cell-capsular matrix interactions can influence the circulation of fluid and ions in the lens, and the phenotype in the SPARC-null mouse lens is the result of multiple intersecting pathways.

creatine, life span, neurobehavioural performance, microarray, oxidative stress, aging

Here we show that oral creatine (Cr) supplementation leads to increased life span in mice. Treated mice showed improved neurobehavioral performance, decreased accumulation of the aging pigment lipofuscin and upregulation of “anti-aging” genes in brain. As Cr is virtually free of adverse effects, it may be a promising food supplement for healthy aging in man.
Keywords: creatine, life span, neurobehavioural performance, microarray, oxidative stress, aging

Studied gene regulation in bronchial smooth muscle cells following vitamin D stimulation.
Keywords: comparison

Smad4 and Trim33 dependend nodal/activin responsive genes in differentiated mouse ESCs

In differentiated mouse ESCs, most of the nodal/activin responsive genes are dependent on both Smad4 and Trim33, some are solely dependent on Smad4, and some are dependent on Trim33.

Gene expression profiling of Tgfbr2 mutant mouse models of cleft palate

The overall goal of this project is to investigate the role of TGF-beta signaling in palate development in order to discover candidate therapeutics for preventing and treating congenital birth defects. Here, we conducted gene expression profiling of embryonic palatal tissue from wild type mice as well as those with a neural crest specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of cleft palate formation.

Gene expresion changes following knockdown of KDM4C in primary fibroblasts

Epigenetic and genetic regulations are sometimes considered as separate mechanisms that influence gene expression and phenotypes.  However, there are DNA sequence variants in epigenetic regulators that could affect gene regulation.  The histone demethylase, KDM4C, promotes transcriptional activation by removing the repressive histone mark, tri-methylation of lysine 9 of histone H3 (H3K9me3), from its target genes.  In this study, we uncovered cis-acting DNA sequence variants in KDM4C that contribute to individual differences in its expression.  Utilizing this natural variation, we performed genetic analyses in B-cells in order to identify target genes that are regulated by KDM4C.
We used microarrays to investigate gene expression changes in the target genes from our genetic analyses following knockdown of KDM4C in primary fibroblasts.

Stearoyl-CoA desaturase 1-deficient (SCD1-/-) mice have impaired monounsaturated fatty acid (MUFA) synthesis.  When maintained on a very low-fat, high-carbohydrate (VLF-HC) diet, SCD1-/- mice develop severe hypercholesterolemia characterized by an increase in apolipoprotein B-containing lipoproteins and the appearance of lipoprotein-X.  Additionally, high-density lipoprotein cholesterol is dramatically reduced in VLF-HC SCD1-/- mice.  The concomitant presence of elevated plasma bile acids, bilirubin and aminotransferases in the VLF-HC SCD1-/- mouse are indicative of hepatic dysfunction.  Supplementation of the VLF-HC diet with unsaturated fat (canola oil), but not saturated fat (coconut oil), prevents these plasma phenotypes.  However, dietary oleate was not as effective as canola oil in reducing low-density lipoprotein cholesterol, signifying an additional role for dietary polyunsaturated fatty acid deficiency in the development of this phenotype.  These results indicate that lack of SCD1 results in an increased requirement for dietary unsaturated fat to compensate for impaired MUFA synthesis and to prevent hypercholesterolemia and hepatic dysfunction.
Keywords: repeat (genotype and diet)

The zinc finger protein ZNF658 regulates the transcription of genes involved in zinc homeostasis and affects ribosome biogenesis through the zinc transcriptional regulatory element (ZTRE)

We previously identified the ZTRE in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE.  ZNF658 was identified by MALDI-TOF mass spectrometry of a band excised after EMSA using a ZTRE probe.  The protein contains a KRAB domain and 21 zinc fingers.  It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658.  siRNA targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5), SLC30A10 (ZnT10) and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered large changes in rRNA.  We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins.  ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell.  ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability.

Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia

Isolated methylmalonic acidemia (MMA) is a pleiotropic enzymatic defect of branched-chain amino acid oxidation most commonly caused by deficiency of methylmalonyl-CoA mutase (MUT). End stage renal disease (ESRD) is emerging as an inevitable disease-related complication, recalcitrant to conventional therapies and liver transplantation. To establish a viable model of MMA-associated renal disease, methylmalonyl-CoA mutase (Mut) was expressed in the liver of Mut -/- mice as a stable transgene under the control of an albumin (INS-Alb-Mut) promoter. Mut -/- ;TgINS-Alb-Mut  mice were rescued from the neonatal lethality displayed by Mut -/- mice and manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstital nephritis (CTIN) and prominent ultrastructural changes in the proximal tubular mitochondria, replicating precisely the renal manifestations seen in a large MMA patient cohort.

TNFR1 controls apoptosis and chronic liver disease in hepatocyte-specific IKKγ (Nemo) mice.

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral hepatitis, alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury and cancer. Deletion of NF-ĸB essential modulator in hepatocytes (NemoΔhepa) causes the spontaneous development of hepatocellular carcinoma preceded by steatohepatitis in mice and thus serves as an excellent model for the progression from chronic hepatitis to liver cancer. In the present study we aimed to dissect the death-receptor mediated pathways that contribute to liver injury in NemoΔhepa mice. Therefore, we generated NemoΔhepa/TRAIL-/- and NemoΔhepa/TNFR1-/- animals and analyzed the progression of liver injury. NemoΔhepa/TRAIL-/- displayed a similar phenotype to NemoΔhepa mice characteristic of high apoptosis, infiltration of immune cells, hepatocyte proliferation and steatohepatitis. These pathophysiological features were significantly ameliorated in NemoΔhepa/TNFR1-/- livers. Hepatocyte apoptosis was increased in NemoΔhepa and NemoΔhepa/TRAIL-/- mice while NemoΔhepa/TNFR1-/- animals showed reduced cell death concomitant with a strong reduction in pJNK levels. Cell cycle parameters were significantly less activated in NemoΔhepa/TNFR1-/- livers. Additionally, markers of liver fibrosis and indicators of tumour progression were significantly decreased in these animals. The present data demonstrate that the death receptor TNFR1 but not TRAIL is important in determining progression of liver injury in hepatocyte-specific Nemo knockout mice.

Expression data of wild type and C-DGCR8 KO cones at different timepoints [array]

We have analyzed gene expression in cone photoreceptors isolated from wild type and C-DGCR8 (DiGeorge Syndrome Critical Region Gene 8) KO mice at five different time points to get a mechanistic inside into the altered molecular pathways after microRNAs depletion.

Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways

Keloids are benign tumors of the dermis that form during a protracted wound healing process.  Susceptibility to keloid formation occurs predominantly in people of African and Asian descent.  The key alteration(s) responsible for keloid formation has not been identified and there is no satisfactory treatment for this disorder. The altered regulatory mechanism is limited to dermal wound healing, although several diseases characterized by an exaggerated response to injury are prevalent in individuals of African ancestry.  We have observed a complex pattern of phenotypic differences in keloid fibroblasts grown in standard culture medium or induced by hydrocortisone.   In this study Affymetrix-based microarray was performed on RNA obtained from fibroblasts cultured from normal scars and keloids grown in the absence and presence of hydrocortisone.  We observed differential regulation of approximately 500 genes of the 38,000 represented on the Affymetrix chip.  Of particular interest was increased expression of several IGF-binding and IGF-binding related proteins and decreased expression of a subset of Wnt pathway inhibitors and multiple IL-1-inducible genes.  Increased expression of CTGF and IGFBP-3 was observed in keloid fibroblasts only in the presence of hydrocortisone. These findings support a role for multiple fibrosis-related pathways in the pathogenesis of keloids
Keywords: cell-type comparison, drug treatment comparison

Genome-wide analysis of human pluripotent cells after kd/oe of SNF5

Analysis of global gene expression by SNF5 level change in human pluripotent and differentiated cells. The core subunit of BAF complex SNF5 is at the nexus of the link between chromatin remodeling and tumor suppression. We demonstrated a role for the remodeler SNF5 as a key executor in regulating pluripotency gene networks during differentiation by using loss and gain of function experiments followed by gene-expression arrays.

Gene expression data from severe asthmatic children: PBMC profiles during acute exacerbation versus convalescence

Severe asthma exacerbations in children requiring hospitalisation are typically associated with viral infection, and occur almost exclusively amongst atopics, but the significance of these comorbidities is unknown. We hypothesised that underlying interactions between immunoinflammatory pathways related to responses to aeroallergen and virus are involved, and that evidence of these interactions is detectable in circulating cells during exacerbations.
To address this hypothesis we used a genomics-based approach involving profiling of PBMC subpopulations collected during acute exacerbation versus convalescence by microarray and flow cytometry.

Gene expression profiling of the tongue in Tgfbr2 mutant mouse models

The overall goal of this project is to investigate the role of TGF-beta signaling in tongue development in order to study the contribution of cranial neural crest (CNC) cells towards the patterning of cranial mesoderm for proper tongue formation. Here, we conducted gene expression profiling of embryonic tongue tissue from wild type mice as well as those with a neural crest specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of microglossia, a common congenital birth defect which is frequently observed with several syndromic conditions.

Naturally occurring FoxP3+CD4+CD25+high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing auto-reactive CD4+CD25- T cell activity. Although Tregs from T1D subjects are functionally deficient, there is little knowledge of the molecular mechanisms that orchestrate this loss of Treg function. We observed increased apoptosis (by a novel YOPRO-1/7AAD dual staining protocol) and decreased suppression in polyclonal Tregs in the periphery from high at-risk and T1D subjects. We hypothesize that prior to and during the onset of disease, Tregs lack pro-survival signals and are caught up in a relatively deficient cytokine milieu whose effects may be detectable in the periphery.
Microarray analysis was performed on un-stimulated Tregs from 12 subjects with newly diagnosed T1D and 15 healthy controls.

Expression data from laminin alpha 2 chain deficient mice vs wild type

Mutations in the gene encoding laminin a2 chain cause congenital muscular dystrophy, MDC1A. In skeletal muscle, laminin a2 chain binds at least two receptor complexes; the dystrophin-glycoprotein complex and integrin a7b1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin a2 chain deficient mouse limb muscle. One of the down-regulated genes encodes a protein called calcium and integrin binding protein 2 (Cib2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin aIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin a7b1 binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle Cib2 colocalizes with integrin a7B subunit at the sarcolemma and at the neuromuscular- and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium binding protein that interacts with integrin a7Bb1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin a7Bb1D signaling in skeletal muscle

Foxa2 programs Th2-cell mediated innate immunity in the developing lung.

Deletion of the gene encoding Foxa2, a winged helix transcription factor selectively expressed in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia.  Loss of Foxa2 induced the recruitment and activation of myeloid dendritic cells (mDCs) and Th2 cells in the lung, and was associated with the increased production of T helper 2 (Th2) cytokines and chemokines.  mRNA microarray analysis demonstrated that deletion of Foxa2 induced the expression of a number of mRNAs regulating pulmonary dendritic cell activation, Th2 mediated inflammation, and goblet cell differentiation.  The spontaneous pulmonary inflammation and goblet cell metaplasia caused by loss of Foxa2 was inhibited by treatment of newborn Foxa2∆/∆ mice with monoclonal IL-4Ralpha antibody.  Expression of Foxa2 in non-ciliated secretory cells (Clara cells) in vivo inhibited goblet cell differentiation induced by pulmonary allergen exposure.  The respiratory epithelium plays a central role in the regulation of Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2.

Expression data from wild-type, Dgcr8 knockout and Dicer knockout ES cells

Dgcr8 and Dicer are both important components of the microRNA biogenesis pathway while Dicer is also implicated in biogenesis of other types of small RNAs such as siRNAs and mirtrons. Here we performed microarray analysis of WT, Dgcr8 and Dicer knockout ES cells to identify mRNAs differentially regulated upon loss of Dgcr8 and Dicer.

Gene expression microarray analysis on hippocampus of Schnurri-2 knockout and wild-type control mice

Schnurri-2 (Shn-2), an NF-kappa B site-binding protein, tightly binds to the enhancers of major histocompatibility complex (MHC) class I genes and inflammatory cytokines, which have been shown to harbor common variant single nucleotide polymorphisms associated with schizophrenia. Shn-2 knockout mice show behavioral abnormalities that strongly resemble those of schizophrenics. We performed gene expression microarray analysis of hippocampi from Shn-2 knockout and wild-type control mice.

Early expression data of H69 xenograft tumors following PDT treatment.

The response of tumors to PDT treatment is expected to provide information on effects of oxygen depletion, induced apoptosis, induction of an inflammatory response and induction of an ani-tumor immune response.
Keywords: comparison of treatment vs control

Cardiac samples from OTT1 null/null and OTT1 null/wt embryonic mice

The infant leukemia-associated gene, Ott1(Rbm15), has broad regulatory effects within the murine hematopoiesis. However, germline Ott1 deletion  results in fetal demise prior to E10.5, indicating additional developmental requirements for Ott1. The spen gene family, to which Ott1 belongs, has a transcriptional activation/repression domain and RNA recognition motifs, and in Drosophila has a significant role in the development of the head and thorax. Early Ott1-deficient embryos show growth retardation and incomplete closure of the notochord. Further analysis demonstrated placental defects in the spongiotrophoblast and syncytiotrophoblast layers, resulting in an arrest of vascular branching morphogenesis. Rescue of the placental defect using a conditional allele with a trophoblast-sparing cre transgene allowed embryos to form a normal placenta and survive gestation. This result shows that the process of vascular branching morphogenesis in Ott1-deficient animals is regulated by the trophoblast compartment rather than the fetal vasculature. Mice surviving to term manifested hyposplenia and abnormal cardiac development. Analysis of global gene expression of Ott1-deficient embryonic hearts shows enrichment of hypoxia-related genes and significant alteration of several candidate genes critical for cardiac development. Thus, Ott1-dependent pathways in addition to being implicated in leukemogenesis, may also be important in the pathogenesis of placental insufficiency and cardiac malformations.

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer.  NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer.  However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy.  A better understanding of the mechanisms of NIS regulation in breast cancer may lead to strategies for increasing cell surface NIS and radioactive iodide uptake (RAIU) in breast cancer.  The MCF-7 cell line is the only human breast cancer cell line with inducible endogenous NIS expression. Kogai et al. [2000] first reported that trans-retinoic acid (tRA) induces NIS mRNA expression in MCF-7 cells and it was later reported that a combination treatment of tRA and hydrocortisone (tRA/H) further increases tRA-induced NIS expression/function in MCF-7 cells (Kogai et al., 2005; Dohan et al., 2006).  In this study, we used gene expression profiling to identify genes that correlate with NIS expression in MCF-7 cells such that mechanisms underlying NIS modulation may be elucidated.

Tetrahydrobiopterin compounds prolong allograft survival independent of their effect on nitric oxide synthase activity

BMDM from BAL/c and C57BL/6 BMDM were stimlated with IFN-g overnight and infected with different Yersinia strains for 3 hrs.

Targetting CD24 for treatment of colorectal and pancreatic cancer by monoclonal antibodies or siRNA

CD24 is a potential oncogene reported to be overexpressed in a large variety of human malignancies. We have shown that CD24 is overexpressed in 90% of colorectal tumors at a fairly early stage in the multistep process of carcinogenesis. Anti-CD24 monoclonal antibodies (mAb) induce a significant growth inhibition in colorectal and pancreatic cancer cell lines that express the protein. This study is designed to investigate further the effects of CD24 down-regulation using mAb or small interfering RNA in vitro and in vivo. Western blot analysis showed that anti-CD24 mAb induced CD24 protein down-regulation through lysosomal degradation. mAb augmented growth inhibition in combination with five classic chemotherapies. Xenograft models in vivo showed that tumor growth was significantly reduced in mAb-treated mice. Similarly, stable growth inhibition of cancer cell lines was achieved by down-regulation of CD24 expression using short hairpin RNA (shRNA). The produced clones proliferated more slowly, reached lower saturation densities, and showed impaired motility. Most importantly, down-regulation of CD24 retarded tumorigenicity of human cancer cell lines in nude mice. Microarray analysis revealed a similar pattern of gene expression alterations when cells were subjected to anti-CD24 mAb or shRNA. Genes in the Ras pathway, mitogenactivated protein kinase, or BCL-2 family and others of oncogenic association were frequently down-regulated. As a putative new oncogene that is overexpressed in gastrointestinal malignancies early in the carcinogenesis process, CD24 is a potential target for early intervention in the prevention and treatment of cancer.

Expression analysis of control and low protein fed mouse offspring subsequently treated with multiple low doses of STZ

Low protein (LP) during gestation leads to low birth weight and poor fetal growth, with altered islet development and glucose intolerance in adulthood. Additionally, LP offspring fail to regenerate their β-cells following depletion with streptozotocin (STZ), in contrast to control-fed offspring that are capable of β-cell regeneration.
Our objective was to identify genes and signalling pathways that may be critically altered in LP offspring rendering them susceptible to develop long term glucose intolerance and decreased β-cell plasticity.

CD3+ cells were studied in duplicate from healthy controls, patients with active ITP and ITP in remission.

Gene Expression Profiling of Cellular Dose-Response to Hypochlorous Acid (HOCl)

Hypochlorous acid (HOCl) is a potent oxidant that is produced endogenously in mammalian tissue by phagocytes. Exogenous exposure to HOCl also can occur following inhalation of chlorine gas. HOCl has been implicated as a source of oxidative stress associated atherosclerosis and other diseases.
The purpose of this study was to identify dose-dependent transitions in cellular response to hypochlorous acid (HOCl), with a  focus on understanding how various cellular defense and stress dose-responses overlap.

Array-based characterisation of SCY1-like 1 (NM_020680.3) esiRNA knockdown

Expression profiles from HEK293 cells by overexpression of TRPM7 channel

The functional activity of TRPM7 is essential for cell viability and growth, and its expression is up-regulated in certain pathological conditions. In order to assess the effects of TRPM7 activity on cellular gene expression, inducible HEK293 cell-lines harboring the wild-type mouse TRPM7 and a mutant lacking the kinase domain were established.
By using microarray analysis, we identified genetic profiles altered in transcription significantly and specifically by the expression of the functional TRPM7 channel.

Cancer genomics identifies regulatory gene networks associated with the transition from dysplasia to adenocarcinomas

Lung cancer is a leading cause of deaths in the world. There is a need to improve an understanding of mechanisms of malignant transformation and to develop genetic markers of disease for better and targeted therapies.
Here, we report findings with a transgenic disease model where targeted expression of c-raf to respiratory epithelium induced adenocarcinomas. Specifically, by use of laser microdissection we harvested either tumor or transgenic and non-transgenic but otherwise morphologically unaltered cells. We then searched genome wide for regulated genes and validated results by quantitative real-time PCR. Overall, 473 and 541 genes were significantly regulated when cancer versus transgenic and cancer versus non-transgenic cells were compared. Principal component analysis and hierarchical clustering of the data clearly separated the cancer cells from transgenic and non-transgenics and we observed predominately repression of gene expression at advanced stages of tumor growth. Nonetheless genes up-regulated in dysplasia were also up-regulated in solid tumors. We observed groups of genes acting in concert either linked to development with an unexpected high number of genes coding either for the epithelial mesenchymal or mesenchymal endothelial transition. Additionally, genes coding for cell adhesion including the integrins and the tight and gap junction proteins were repressed (integrin alpha 1, integrin alpha 8, claudin 2, claudin 5, gap junction membrane channel protein alpha 5 and cadherin 5) but ligands for the membrane bound epidermal growth factor tyrosine kinase i.e. epi- and amphiregulin were up-regulated. Moreover, molecules in the signalling of vascular endothelial growth factor receptor- 2 and VEGFD, Notch and WNT were regulated as were glycosylases that facilitate cellular recognition. Other regulated signalling molecules included exchange factor such as RAP guanine nucleotide exchange factor 3, RHO guanine nucleotide exchange factor 10, RAS guanine releasing protein 2 and 3, and RAS guanine nucleotide-releasing factor 1 that play a role in an activation of the MAP kinases.
Notably, we found the tumor suppressors MCC (mutated in colorectal cancers), HEY1 (hairy/enhancer-of-split related with YRPW motif 1), FAT3 (FAT tumor suppressor homolog 3), ARMCX1 (armadillo repeat containing, X-linked 1) and RECK (reversion-inducing-cysteine-rich protein with kazal motifs) to be significantly repressed.
Taken collectively, our study provides valuable information for new candidate genes in lung adenocarcinoma induced by exaggerted c-raf kinase activity.

Relief of feedback inhibition of HER3 transcription by RAF and MEK inhibitors attenuates their antitumor effects in BRAF mutant thyroid carcinomas.

Analysis of transcriptome kinetics of SW1736 thyroid cancer cell line vs SK-MEL-28 melanoma cell line at various times after addition of 2 µM vemurafenib. The hypothesis tested was that SW1736 cells (vemurafenib-refractory) differentially express genes compared to SK-MEL-28 cells (vemurafenib sensitive) that confer resistance to the RAF inhibitor.

The Brwd1 mutation, though having dramatic effects on the spermatocyte transcriptome, had little effect on the oocyte transcriptome.

Postmeiotic gene expression is essential for development and maturation of sperm and eggs. In B6/C3H females, Brwd1 ablation causes severe chromosome condensation and structural defects associated with abnormal telomere structure. In contrast to males, transcriptome analysis at the germinal vesicle stage revealed only minor changes in gene expression including a  >2 fold overexpression of the histone methyltransferase MLL5 and LINE-1 elements transposons. Thus, loss of BRWD1 function interferes with the completion of oogenesis and spermatogenesis through sexually dimorphic mechanisms. Our results demonstrate that BRWD1 is essential for the epigenetic control of chromosome stability during female meiosis while playing a critical role in haploid gene transcription during the post-meiotic differentiation events of spermiogenesis.

The dual bromodomain protein Brd2 is closely related to the basal transcription factor TAFII250, which is essential for cyclin A transactivation and mammalian cell cycle progression. In transgenic mice, constitutive lymphoid expression of Brd2 causes a malignancy most similar to human diffuse large B cell lymphoma. We compare the genome-wide transcriptional expression profiles of these lymphomas with those of proliferating and resting normal B cells. Transgenic tumors reproducibly show differential expression of a large number of genes important for cell cycle control and lymphocyte biology; expression patterns are either tumor-specific or proliferation-specific. Several of their human orthologs have been implicated in human lymphomagenesis. Others correlate with human disease survival time. BRD2 is underexpressed in some subtypes of human lymphoma and these subtypes display a number of similarities to the BRD2-mediated murine tumors. We illustrate with a high degree of detail that cancer is more than rampant cellular proliferation, but involves the additional transcriptional mobilization of many genes, some of them poorly characterized, which show a tumor-specific pattern of gene expression.

Gene expression analysis of crypt base epithelial cells from WT and Nod2-/- mice

Nod2 has been extensively characterized as a bacterial sensor that induces an antimicrobial and inflammatory gene expression program. Therefore, it is unclear why Nod2 mutations that disrupt bacterial recognition are paradoxically among the highest risk factors for Crohn’s disease, which involves an exaggerated immune response directed at intestinal bacteria. Previous studies from our lab have shown that mice deficient in Atg16L1, another Crohns disease susceptibility gene, develop abnormalities in Paneth cells, specialized epithelial cells in the small intestine involved in antimicrobial responses.
The goal of our study is to determine whether Nod2 deficiency leads to differences in the transcriptional profile of Paneth cells, ultimately leading to small intestinal inflammation.

DNA microarrays time course of SKO-infected MEFs and MaF pre-iPSCs treated with Vc

Inhibition of p300 impairs Foxp3+ T-regulatory cell function and promotes anti-tumor immunity

Foxp3+ T-regulatory (Treg) cells maintain immune homeostasis and limit autoimmunity, but can also curtail host responses to cancers. Tregs are therefore promising targets to enhance anti-tumor immunity. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and non-histone proteins. We found that conditional deletion or pharmacologic inhibition of one specific HAT, p300, in Foxp3+ Tregs, increased TCR-induced apoptosis in Tregs, impaired Treg suppressive function and iTreg peripheral conversion, and limited tumor growth in immunocompetent, but not in immunodeficient, hosts. Our data demonstrate that p300 is important for Foxp3+ Treg function and homeostasis in vivo and in vitro, and identify a novel mechanism to diminish Treg function without overtly impairing effector Tcell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.

Txnip deficient mice with hepatocellular carcinoma

Regulation of gene expression program by Fsp27 in white and brown adipose tissue

Background: Brown and white adipose tissues (BAT and WAT) play critical roles in controlling energy homeostasis and in the development of obesity and diabetes. Fsp27 is expressed in both BAT and WAT and promotes lipid storage and the development of obesity and diabetes. In addition, Fsp27-deficient white adipocytes acquired certain BAT-like properties including reduced lipid droplet size and increased mitochondrial activity. Using microarray and semi-quantitative real-time PCR analyses, we systematically analyzed the gene expression profile in Fsp27-deficient WAT and BAT.
Results: We observed that BAT-selective genes were significantly up-regulated, whereas WAT-selective genes were down-regulated in the WAT of Fsp27-/- mice. Expression levels of BAT-selective genes were also dramatically up-regulated in the WAT of leptin and Fsp27 double deficient mice. Furthermore, we observed that expression levels of genes in multiple metabolic pathways including oxidative phosphorylation, the TCA cycle and fatty acid synthesis and oxidation were increased in the Fsp27-/- WAT. In contrast, expression levels for extracellular matrix remodeling, the classic complement pathway and TGF-β signaling"were down-regulated in the WAT of Fsp27-/- mice. Most importantly, regulatory factors that determine BAT identity such as CEBPα/β, PRDM16 and major components in the cAMP pathway were markedly up-regulated in the WAT of Fsp27-/- mice. Interestingly, we observed distinct gene expression profiles in the BAT of Fsp27-/- mice.
Conclusion: Our data suggest that Fsp27 acts at upstream to control gene expression of diverse pathways, in particular the expression of regulatory factors that determine the identity of BAT and WAT. Therefore, Fsp27 is an important molecular determinant for the identity of WAT, and loss of Fsp27 leads to the conversion of WAT to a BAT-like tissue.

Co-expression network analysis from genes involved with neural-differentiation shows specific pattern in patients with schizophrenia

Schizophrenia is a severe and debilitating neuropsychiatric disorder with the involvement of genetic and environmental factors contributing to its pathogenesis. The specific role of the brain cell types in the disease remains uncovered due to the difficulties in accessing diseased tissue. This study analysed molecular alterations in human induced pluripotent stem cells (hiPSCs) derived from fibroblasts from control subject and individual with schizophrenia and further differentiated to neuron to identify genes relevant for the development of schizophrenia. We identified 228 genes that are involved with metabolic processes, signal transduction, nervous system development, regulation of neurogenesis and neuronal differentiation. These genes were analysed in expression microarrays of post-mortem brain tissue (frontal cortex) of additional patients with schizophrenia and normal individuals revealing only six common genes: CACNA1E, CEBPB, DUX4, EDNRA, SPHK1 and SPRY4 (GSE62191). Furthermore, a co-expression network analysis of the 228 genes revealed a non-conserved module enriched for genes associated with oxidative stress and negative regulation of cell differentiation as involved with schizophrenia. This study supports the relevance of using cellular approaches to dissect molecular aspects of neurogenesis with impact in the schizophrenic brain.

Identification of the JNK Signaling Pathway as a Functional Target of the Tumor Suppressor PTEN

Although most of the oncogenic phenotypes of PTEN loss have been attributed to AKT activation, AKT alone is not sufficient to induce all of the biological activities associated with PTEN inactivation.  We searched for additional PTEN-regulated pathways through gene set enrichment analysis (GSEA) and found that PTEN inactivation causes an enrichment of genes associated with JNK activation.  Biochemically, PTEN-null cells exhibit higher JNK activity, and genetic studies demonstrate that JNK functions parallel to and independently of AKT.  Furthermore, PTEN deficiency sensitizes cells to JNK inhibition.  We also found that negative feedback regulation of PI3K was impaired in PTEN-null cells.  These findings implicate JNK in PI3K-driven cancers and demonstrate the utility of GSEA to identify functional pathways using genetically defined systems.
Keywords: Genetic modification:  PTEN RNAi

We show that numerous miRNAs are transcriptionally up-regulated in papillary thyroid carcinoma (PTC) tumors compared with unaffected thyroid tissue.  Among the predicted target genes of the three most upregulated miRNAs (miRs 221, 222 and 146b), only less than 15% showed significant downexpression in transcript level between tumor and unaffected tissue.  The KIT gene which is known to be downregulated by miRNAs 221 and 222 displayed dramatic loss of transcript and protein in those tumors that had abundant mir-221, mir-222, and mir-146b transcript.

An RNAi Screen of Chromatin Proteins Identifies Tip60-p400 as a Regulator of Embryonic Stem Cell Identity, Experiment A

Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.
Keywords: knockdown-gene expression profiling

Genome-wide analysis of REST knockdown responsive gene expression in mouse ES cells

Analysis of gene expression profiling upon REST shRNA knockdown in mouse ES cells for 72 hours,

Gene expression analyis of primary MCL including IGHV mutated and unmutated cases
Gene set analysis was perfomed in  MCL samples, comparing IGHV mutated cases vs. IGHV unmutated cases

Gene expression in b1-integrin wild-type and knockout mouse heart

Growth and expansion of ventricular chambers is essential during cardiogenesis and is achieved by proliferation of cardiac progenitors that are not fully differentiated. Disruption of this process can lead to prenatal lethality. In contrast, adult cardiomyocytes achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Moreover, the function of embryonic cardiac fibroblasts, derived from epicardium, and their secreted factors are largely unknown. Using a novel co-culture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. b1 integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of b1 integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

rosiglitazone, PPARgamma, microarray, time course, gene expression, stem cells

Rosiglitazone (Rosi), a member of the thiazolidinedione class of drugs used to treat type 2 diabetes, activates the adipocyte-specific transcription factor peroxisome proliferator-activated receptor gamma (PPARg). This activation causes bone loss in animals and humans, at least in part due to suppression of osteoblast differentiation from marrow mesenchymal stem cells (MSC). In order to identify mechanisms by which PPARg2 suppresses osteoblastogenesis and promotes adipogenesis in MSC, we have analyzed the PPARg2 transcriptome in response to Rosi. A total of 4,252 transcriptional changes resulted when Rosi (1 uM) was applied to the U-33 marrow stromal cell line, stably transfected with PPARg2 (U-33/g2), as compared to non-induced U-33/g2 cells. Differences between U-33/g2 and U-33 cells stably transfected with empty vector (U-33/c) comprised 7,928 transcriptional changes, independent of Rosi. Cell type-, time- and treatment-specific gene clustering uncovered distinct patterns of PPARg2 transcriptional control of MSC lineage commitment. The earliest changes accompanying Rosi activation of PPARg2 included adjustments in morphogenesis, Wnt signaling, and immune responses, as well as sustained induction of lipid metabolism. Expression signatures influenced by longer exposure to Rosi provided evidence for distinct mechanisms governing the repression of osteogenesis and stimulation of adipogenesis. Our results suggest interactions that could lead to the identification of a “master” regulatory scheme controlling osteoblast differentiation.
Keywords: rosiglitazone, PPARgamma, microarray, time course, gene expression, stem cells

YAP is an oncogene and an inducer of Epithelial-to-Mesenchymal Transition (EMT).
We used microarrays to detail the global program of gene expression to identify YAP target genes.

PUBLICATION ABSTRACT:
The Hippo pathway defines a novel signaling cascade regulating cell proliferation and survival in Drosophila, which involves the negative regulation of the transcriptional coactivator Yorkie by the kinases Hippo and Warts. We have recently shown that the human ortholog of Yorkie, YAP, maps to a minimal amplification locus in mouse and human cancers, and that it mediates dramatic transforming activity in MCF10A primary mammary epithelial cells. Here we show that LATS proteins (mammalian orthologs of Warts) interact directly with YAP in mammalian cells and that ectopic expression of LATS1, but not LATS2, effectively suppresses the YAP phenotypes. Furthermore, shRNA-mediated knockdown of LATS1 phenocopies YAP overexpression. Since this effect can be suppressed by simultaneous YAP knockdown, it suggests that YAP is the primary target of LATS1 in mammalian cells. Expression profiling of genes induced by ectopic expression of YAP or by knockdown of LATS1 reveals a subset of potential Hippo pathway targets implicated in epithelial-to-mesenchymal transition (EMT), suggesting that this is a key feature of YAP signaling in mammalian cells.

The deubiquitinase BAP1 is a candidate tumor suppressor regulating cell proliferation in human and is required for development in Drosophila. BAP1 is assembled into high molecular weight transcriptional multi-protein complexes.
In order to identify potential BAP1 target genes, global mRNA expression profiling using microarrays was conducted.

Polycomb function during oogenesis is required for mouse early embryonic development (germinal vesicle oocytes)

In mammals, totipotent pre-implantation embryos are formed by fusion of highly differentiated oocytes and spermatozoa. Acquisition of totipotency concurs with remodeling of chromatin states of parental genomes (“epigenetic reprogramming”), changes in maternally contributed transcriptome and proteome, and zygotic genome activation. Genomes of mature germ cells are more proficient in supporting embryonic development than those of somatic cells. It is currently unknown whether transgenerational inheritance of chromatin states present in mature gametes underlies the efficacy of early embryonic development after natural conception. Here, we show that Ring1 and Rnf2, two core components of the Polycomb Repressive Complex 1 (PRC1), serve redundant gene regulatory functions during oogenesis that are required to support embryonic development beyond the two-cell stage. Numerous developmental regulatory genes that are established Polycomb targets in various somatic cell types are de-repressed in Ring1/Rnf2 double mutant (dm) fully grown germinal vesicle (GV) oocytes. Translation of tested aberrant maternal transcripts is, however, delayed until after fertilization. Exchange of maternal pro-nuclei between control and Ring1/Rnf2 maternally dm early zygotes demonstrates an essential role for Ring1 and Rnf2 during oogenesis in defining cytoplasmic and nuclear maternal contributions that are both essential for proper initiation of embryonic development. A large number of genes up-regulated in Ring1/Rnf2 dm GV oocytes harbor PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) in spermatozoa and in embryonic stem cells (ESCs), and are repressed during normal oogenesis and early embryogenesis. These data strongly support the model that Polycomb acts in the female and male germline to silence differentiation inducing genes and to program chromatin states, thereby sustaining developmental potential across generations.
Expression profiling of fully grown mouse GV oocytes was performed with the following genotypes: Ring1+/+Rnf2F/F (control), Ring1-/-Rnf2F/F (Ring1 mutant), Ring1+/+Rnf2F/FZp3-cre (Rnf2 mutant) and Ring1-/-Rnf2F/FZp3-cre (Ring1/Rnf2 double mutant).

The combination of peginterferon and ribavirin is the standard treatment for chronic hepatitis C.  Our recent clinical study suggests that ribavirin augments the induction of interferon stimulated genes (ISGs) in patients treated for HCV infection [1].  In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was also studied. Similar to interferon-alpha, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative PCR assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IRF7 and IRF9 are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with interferon, induction of specific ISGs is synergistic when compared to either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with interferon signaling and intracellular double stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and NF-KappaB pathways in the action of ribavirin. In conclusion, our study suggests that ribavirin, acting via a novel innate mechanism, potentiates the anti-HCV effect of interferon. Understanding the mechanism of action of ribavirin would be valuable in identifying novel antivirals.

Gene expression in DN4 thymocytes from mice with an inducible deletion of Ikaros

The aim of the experiment was to visualize genes under the control of Ikaros during early T cell development. We used mice carrying floxed Ikaros alleles that were crossed with the Rosa26-CreERT2 mice, and which were injected with tamoxifen for 4 days. DN4 thymocytes were sorted from 4 mice (5-6 week-old) which are positive or not for the Rosa26-CreERT2 transgene.

Emerging data suggest that many immunoreceptors, all of which use a conserved ITAM-module for their signaling, can couple with members of additional classes of membrane receptors to deliver unique signal(s) to the cell. Using functional and gene expression analysis we describe the role for the tonic signaling through Fcg-chain, the major ITAM-module of the innate immune system, in modifying cytokine responses in MF.

Expression data from adult lungs isolated from TMEM100-deficient mice

Tmem100 was identified as a downstream gene of BMP9-ALK1 signaling. The signaling has been implicated in hereditary hemorrhagic telangiectasia. This study was designed to identify affected genes in TMEM100-deficiect mice. Tmem100 was inactivated in adult Tmem1002loxP/2loxP;ROSA26+/CreER mice by tamoxifen tereatment. Microarray data was obtained from the lungs isolated from tamoxifen treated control and mutant mice.

HUVEC were left untreated or stimulated for 5h with 2 ng/ml TNF. Comparsion of the gene profiles revealed TNF-mediated gene expression changes in HUVEC.

HMEC cultures were left untreated or stimulated for 5h with 2 ng/ml TNF. Comparison of the gene expression profiles revealed the TNF-mediated gene expression changes.

Expression data from postnatal mouse apical and basal organ of Corti from Dicer1 conditional knockout and littermate control cochleae.

Cre recombinase-mediated conditional knockout of floxed Dicer1 alleles causes depletion of small RNAs including microRNAs, which function to repress target mRNA expression by inhibiting translation and/or stimulating mRNA degradation.
We used microarrays to examine gene expression in apical versus basal organ of Corti from the cochleae of control and mutant mice in which Dicer1 was deleted and microRNAs were depleted specifically in sensory hair cells by Atoh1 promoter-driven Cre recombinase expression.

DRG from CCI and Seltzer models of neuropathic pain and sham operated animals

L5 DRG samples from CCI, Seltzer and sham models collected at 14, 21 and 50 days after surgery from ipsilateral hind limb.  Note that all replicates are techincal, i.e., there were no biological replicates in this study as samples were pooled for each group

MHCaCre induced knockout of Fog2flox.
Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator FOG2 is known to be essential for heart morphogenesis and coronary development, its tissue specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we use spatiotemporally regulated inactivation of Fog2 to delineate its function both in embryo and adult heart. Early cardiomyocyte-restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8-14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels. This was associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in adult heart resulted in similar phenotype, as did ablation of FOG2 interaction with the transcription factor GATA4. Loss of FOG2 or FOG2-GATA4 interaction altered expression of a panel of angiogenesis-related genes. Collectively, our data indicated that FOG2 regulates adult heart function and coronary angiogenesis.

Mndal, a new interferon-inducible family member, suppresses cell growth and may modify plasmacytoma susceptibility

Mndal, a new interferon-inducible family member, is highly polymorphic, suppresses cell growth and may modify plasmacytoma susceptibility.
The human HIN-200 gene cluster and its mouse counterpart, the Interferon inducible-200 (Ifi200) family, both on Chr 1, are associated with several diseases, including solid tumors and lupus.  Our study was initiated to identify the modifier gene(s) encoded by the Pctm locus, in which mouse B cell plasmacytomas induced by pristane are associated with heterozygosity of Chr 1 genes near the Ifi200 cluster.
A screen for differentially expressed genes in granulomatous tissues induced by pristane in resistant and susceptible strains identified a new Ifi200 member whose expression was 1000-fold higher in the strain carrying the resistant allele of Pctm, and was the most highly expressed Ifi200 gene.  The gene, designated Mndal (for MNDA-like, myeloid nuclear differentiation antigen like), was absent in the susceptible genome, as were genomic sequences upstream of Ifi203, the gene adjacent to Mndal.  Ectopic expression of MNDAL suppressed cell growth, which, together with the disease susceptibility of heterozygotes at the Pctm locus, suggests that Mndal, perhaps with Ifi203, acts as a tumor suppressor and display(s) haplo-insufficiency.  Mndal is highly polymorphic among inbred mouse strains, as it is absent in 10/24 strains.  This polymorphism may have implications for other disease modifiers mapping to the same region.

The effect on gene expression of Smchd1 deletion in pre-B cells from E17.5 Eµ-Myc embryos

Smchd1 appears to act as a tumour suppressor in the Eµ-Myc mouse B cell lymphoma model. We find a small number of gene expression differences at E17.5 in the pre-B cells, before phenotypic differences are observed.

Gene Expression Profiles of Scd1 knockout mice vs wild type mice on chow diet: Liver.

Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity.

Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski CM, Yandell BS, Song Y, Cohen P, Friedman JM, Attie AD.

Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA. ntambi@biochem.wisc.edu

Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (C18:1) and palmitoleate (C16:1), which are components of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Several SCD isoforms (SCD1-3) exist in the mouse. Here we show that mice with a targeted disruption of the SCD1 isoform have reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced weight gain. The protection from obesity involves increased energy expenditure and increased oxygen consumption. Compared with the wild-type mice the SCD1-/- mice have increased levels of plasma ketone bodies but reduced levels of plasma insulin and leptin. In the SCD1-/- mice, the expression of several genes of lipid oxidation are up-regulated, whereas lipid synthesis genes are down-regulated. These observations suggest that a consequence of SCD1 deficiency is an activation of lipid oxidation in addition to reduced triglyceride synthesis and storage.

Functional analysis of the cytoprotective transcription factor Nrf2 in skin morphogenesis and disease: Identification of Nrf2 target genes

The Nrf2 transcription factor is a key player in the cellular stress response, which regulates the expression of important antioxidant enzymes and other cytoprotective proteins. We recently generated a novel transgenic mouse model to determine the function of Nrf2 in the skin. These mice revealed interesting phenotypic abnormalities, including hyperkeratosis and acanthosis. To gain insight into the underlying molecular mechanisms, we wanted to identify genes, which are differentially expressed in the skin of wild-type and mutant mice before the onset of phenotypic abnormalities.

Expression profiling of PBMC from patients with hepatocellular carcinoma

Aberrant gene expression analysis between peripheral blood mononuclear cell (PBMC) samples from healthy individuals and patients with pancreatic carcinoma, gastric carcinoma and hepatocellular carcinoma (HCC) were identified using Affymetrix gene arrays.

Comprehensive proteomic and transcriptomic characterization of hepatic expression signatures affected in p14 liver conditional knockout mice

Scaffold proteins regulate intracellular MAP kinase signaling by providing critical spatial and temporal specificity. We have shown previously that the scaffold protein MEK1 partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in livers of mice we analysed protein and transcript signatures in tissue samples. Further biological network analysis predicted that the differentially expressed transcripts and proteins are involved in cell cycle progression and regulation of cellular proliferation. Although some of the here identified signatures were previously linked to phospho-ERK activity, most of them were novel targets of late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation defect was confirmed in a chemically induced liver regeneration model in p14 liver knock-out mice.
Conditional liver p14 KO mice were generated by mating p14 f/f mice with mice expressing Cre recombinase under control of Alb promoter and alpha-fetoprotein enhancer. The obtained p14 floxed/floxed;AlfpCre(p14 liver KO) mice were used in experiments as KO mice. To prevent the influence of AlfpCre transgene we used p14 +/+;AlfpCre mice as a control. All mice were derived in a C57BL/6 background and were six weeks old.

Prolonged Drug Selection of Breast Cancer Cells and Enrichment of Cancer Stem Cell Characteristics.

Background: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells.
Methods: Cancer stem cells were defined as CD44+/CD24– cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24– phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided.
Results: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24– phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24– and CD44+/CD24+ cells), and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P<.001). No enrichment in the CD44+/CD24– or CD133+ population was detected in MCF-7/MDR.
Conclusion: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.

JAK2 Naive and Persitent Murine BaF3 cells infected with MPLW515L

Transcriptional profiling of Murine BaF3 cells infected with MPLW515L grown under either normal conditions (Naive) or in 0.8 uM INCB18424 for 4-6 weeks (Persistent). Naive and Persistent cells were then treated with either DMSO (Control) or 0.8 uM INCB18424 for 4 hours. Goal was to determine transcriptional changes conditioned upon sensitivity/resistance of BaF3 MPLW515L mutants to JAK1/2 specific inhibitor.

Background & Aims: HNF4α is an important transcriptional regulator of hepatocyte and pancreatic function. Hnf4α deletion is embryonically lethal with severe defects in visceral endoderm formation, liver maturation and colon development.  However, the precise role of this transcription factor in maintaining homeostasis of the adult intestine remains unclear.  Herein, we aimed to elucidate the adult intestinal functions of Hnf4α.  Methods: A conditional intestinal epithelial Hnf4α knockout mouse was generated.  Histological abnormality of the colonic mucosa was assessed by immunodetection and Western.  Changes in global gene expression and biological network were analyzed.  Results: Hnf4α intestine null mice developed normally until reaching young adulthood. Crypt distortion became apparent in the Hnf4α null colon at 3 months of age followed by focal areas of crypt dropout, increased immune cell infiltrates, crypt hyperplasia and early signs of polyposis later in life.  A gene profiling analysis identified cell death and cell cycle related to cancer as the most significant sets of genes altered in the Hnf4α colon null mice.  Expression levels of the tight junction proteins claudin 4, 8 and 15 were altered early in the colon epithelium of Hnf4α mutants and correlated with increased barrier permeability to a molecular tracer that does not normally penetrate normal mucosa.  Conclusion: These observations support a functional role for Hnf4α in protecting the colonic mucosa against the initiation of the changes resembling inflammatory bowel diseases and polyp formation.

TGF-beta regulated gene transcription and protein expression in human GFAP-negative lamina cribrosa cells

Primary open angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. TGF-beta is an important pro-fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non-glaucomatous controls.  We treated human lamina cribrosa (LC) cells with TGF-beta1 (10ng/ml) for 24 hours in order to examine differential gene expression patterns in repsonse to this cytokine.  In particular we focused on ECM and fibrotic genes.  We found that TGF-beta1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro-fibrotic cell type and an attractive target for novel therapeutic strategies.

Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets.  In order to better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profile of differentiating murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3.  An ESC line was created that allows for the induction of Ngn3 by adding doxycycline (Dox) to the culture medium.  Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of both undifferentiated and differentiated conditions.  Characterization of pancreatic developmental markers during embryoid body (EB) formation revealed an optimum context for Ngn3 induction. Neuroendocrine genes including neurogenic differentiation 1 (NeuroD1) and single minded 1 (Sim1) were found to be significantly upregulated.  Genes regulated by Ngn3 independent of the context were analyzed using systematic gene ontology tools and revealed Notch signaling as the most significantly regulated signaling pathway (p=0.009).  This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and conversely, more sensitive to Notch signaling inhibition.  Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p<0.0001).  Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin transcription.
Keywords: Ngn3 induction, embryonic stem cell differentiation

PPARg agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein

Brown adipose tissue dissipates energy through heat and functions as a defense against cold and obesity. PPARγ ligands have been shown to induce the browning of white adipocytes; however, the underlying mechanisms remain unclear. Here we show that PPARγ ligands require full agonism to induce a brown fat gene program preferentially in subcutaneous white adipose. These effects require expression of PRDM16, a factor that controls the development of classical brown fat. Depletion of PRDM16 blunts the effects of the PPARγ agonist rosiglitazone on the induced brown fat gene program. Conversely, PRDM16 and rosiglitazone synergistically activate the brown fat gene program in vivo. This synergy is tightly associated with an increased accumulation of PRDM16 protein, due in large measure to an increase in the half-life of the protein in agonist treated cells. Identifying compounds that stabilize PRDM16 protein may represent a novel therapeutic pathway for the treatment of obesity and diabetes.

Congenital development disorders with variable severity occur in trisomy 21. However, how these phenotypic abnormalities develop with variations remains elusive. We hypothesize that the difference in euploidy gene expression variation among trisomy 21 tissues are perturbed by the presence of an extra copy of chromosome 21 and this may contribute to the phenotypic variations in Down syndrome.

Discovery of genes regulated by the metastasis suppressor gene, RhoGDI2

A number of studies find that metastasis suppressor proteins, including RhoGDI2, may function in part though controlling expression of genes regulating metastasis (reviewed in Smith and Theodorescu, Nature Reviews Cancer, 2009, PMID: 19242414). To uncover systematically gene expression patterns dependent on RhoGDI2 expression, we profiled gene expression in stably transfected control (GFP empty vector) UM-UC-3 bladder carcinoma cells (which have lost endogenous expression of RhoGDI2, as occurs commonly in the progression of bladder cancer PMID: 15173088), as well as stably transfected GFP-tagged RhoGDI2 expressing UM-UC-3 cells.

Moissoglu K, McRoberts KS, Meier JA, Theodorescu D et al. Rho GDP dissociation inhibitor 2 suppresses metastasis via unconventional regulation of RhoGTPases. Cancer Res 2009 Apr 1;69(7):2838-44. PMID: 19276387

Wu Y, Moissoglu K, Wang H, Wang X et al. Src phosphorylation of RhoGDI2 regulates its metastasis suppressor function. Proc Natl Acad Sci U S A 2009 Apr 7;106(14):5807-12. PMID: 19321744

Smith SC, Theodorescu D. Learning therapeutic lessons from metastasis suppressor proteins. Nat Rev Cancer 2009 Apr;9(4):253-64. PMID: 19242414

Theodorescu D, Sapinoso LM, Conaway MR, Oxford G et al. Reduced expression of metastasis suppressor RhoGDI2 is associated with decreased survival for patients with bladder cancer. Clin Cancer Res 2004 Jun 1;10(11):3800-6. PMID: 15173088

Gene expression data from non-malignant fallopian tube epithelium and high grade serous carcinoma.

The purpose of this study was to identify molecular alterations potentially involved in predisposition to adnexal serous carcinoma (SerCa) in the non-malignant fallopian tube epithelium   (FTE) of BRCA1/2-mutation carriers, given recent evidence implicating the distal FTE as a common source for SerCa.

Differential gene expression analysis of parental and resistant sub-lines of melanoma cell lines treated or untreated with PLX4032
Using microarray we sought to obtain a genome-wide profile of differentially expressed genes in parental melanoma cell lines and resistant sub-lines in response to PLX4032 vs DMSO control treatment.

The neural transcription factor SOX11 is overexpressed in aggressive lymphoid neoplasms mainly in mantle cell lymphoma (MCL), but its functional role in malignant B-cells is unknown. To identify target genes transcriptionally regulated by SOX11 in malignant lymphoid cells, we have used Gene Expression Profiling (GEP) after SOX11 silencing in MCL cell lines.

Expression and functional pathway analysis of nuclear receptor NR2F2 in Ovarian cancer

Identification of transcript dysregulation in patients with Autism Spectrum Disorders

In order to unveil the molecular mechanisms at play during the development of autistic brains, we studied cells that are representative of the very early stages of ontogenesis, namely stem cells. We used nasal olfactory stem cells that are readily accessible and can be biopsied safely. We recruited a relatively homogeneous cohort of nine adults with severe autism and low to very low developmental disabilities, and included two more adults with either Asperger syndrome or high-functioning autism, to enlarge the spectrum. The cohort was then paired with 11 age- and gender-matched control individuals. Stem cells were purified, banked and used for a transcriptomic study.

In order to investigate the function of Bach2 in pre-B ALL, we isolated bone marrow cells from wildtype and Bach2 knockout mice of C57Bl6 background and transformed them with BCR-ABL1.

CBP is required for environmental enrichment-induced neurogenesis and cognitive enhancement.

The epigenetic changes of the chromatin represent an attractive molecular substrate for adaptation to the environment. We examined here the role of CBP, a histone acetyltransferase involved in mental retardation, in the genesis and maintenance of long-lasting systemic and behavioral adaptations to environmental enrichment (EE). Morphological and behavioral analyses demonstrated that EE ameliorates deficits associated to CBP-deficiency. However, CBP-deficient mice also showed a strong defect in environment-induced neurogenesis and impaired EE-enhanced spatial navigation and patter separation ability. These defects correlated with an attenuation of the transcriptional program induced in response to EE and with deficits in histone acetylation at the promoters of EE-regulated, neurogenesis-related genes. Additional experiments in CBP restricted and inducible knockout mice indicated that environment-induced adult neurogenesis is extrinsically regulated by CBP function in mature granule cells. Overall, our experiments demonstrate that the environment alters gene expression by impinging on activities involved in modifying the epigenome and identify CBP-dependent transcriptional neuroadaptation as an important mediator of EE-induced benefits, a finding with important implications for mental retardation therapeutics.
To identify those genes whose expression was altered in the hippocampus of cbp heterozygous mice both under basal conditions and after 2 weeks of exposure to environmental enrichment, we performed a gene profiling analysis of hippocampal tissue using high-density oligonucleotide microarrays.

Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis

The regeneration of diseased hyaline cartilage remains a great challenge, mainly because degeneration activities after major injury or due to age-related processes overwhelm the self-renewal capacity of the tissue. We show that repair tissue from human articular cartilage of late stages of osteoarthritis harbor a unique progenitor cell population, termed chondrogenic progenitor cells exhibiting stem cell characteristics, such as multipotency, lack of immune system activation and, in particular, migratory activity. The isolated CPC exhibit a high chondrogenic potential and were able to populate diseased tissue in vivo. Moreover, down-regulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9 and consequently the matrix synthesis potential of chondrogenic progenitor cells. Our results, while offering new insight into the biology of progenitor cells from diseased cartilage tissue, might assist future strategies to treat late stages of osteoarthritis.

The aim of this study was to investigate the molecular mechanisms implicated in this mouse model of nemaline myopathy, and to further compare the molecular disease response in different skeletal muscles. For this purpose, snap frozen skeletla muscle specimens from wild type and transgenic for alpha tropomyosin slow mice were studied. Five different muscle types were used (diaphragm, plantaris, extensor digitorum longus, tibialis anterior, gastrocnemus). Mice were sacrificed between 7 and 10 months. RNA pools from 3-5 animals were created and each pool was hybridized to a U74Av2 Affymetrix GeneChip. Datasets from 36 GeneChips were included in this study.
Keywords: disease mouse model analysis

Dengue virus (DENV) infects hundreds of millions of people annually, yet there is only a limited knowledge of the host immune response to dengue. Here, we used a systems biological approach to perform a detailed analysis of the innate immune response to DENV infection in the whole blood samples of acutely infected humans in Bangkok, Thailand. Transcriptomic analysis revealed that genes encoding pro-inflammatory mediators and type I IFN related proteins, were associated with high levels of virus during the first few days of infection. Individuals with low or negative viremia at the late stage of fever were enriched with genes associated with pathways involved in cell cycle, proliferation, cell metabolism and translational control. Meta-analysis showed significant enrichment in genes specific for innate cells (monocytes, macrophages and DCs) in the specimens with high VL and enrichment in genes specific for NK cells, CD4+ and CD8+ T cells as well as B cells in specimens with low VL. Furthermore, flow cytometric analysis revealed an expansion in the numbers of CD14+CD16+ monocytes and depletion of CD14dimCD16++ cells and BDCA-1+ myeloid DC in blood. Consistent with this, in a non-human primate model, infection with DENV boosted the numbers of CD14+CD16+ monocytes in the blood and in secondary lymphoid organs. In vitro, freshly isolated blood monocytes infected with DENV up regulated CD16 and mediated robust differentiation of resting B cells to CD27++CD38++ plasmablasts and IgG and IgM secretion. Taken together, these data provide a detailed picture of the innate response to dengue infection in humans, and highlight an unappreciated role for CD14+CD16+ monocytes in promoting the differentiation of plasmablasts and mediating antibody response to DENV.

FoxA1 is a critical determinant of Estrogen Receptor function and endocrine response (part II)

Estrogen Receptor-a (ER) is the key feature in the majority of breast cancers and ER binding to the genome correlates with the Forkhead protein FOXA1 (HNF3a), but mechanistic insight is lacking. We now show that FOXA1 is the defining factor that governs differential ER-chromatin interactions. We show that almost all ER-chromatin interactions and gene expression changes are dependent on the presence of FOXA1 and that FOXA1 dictates genome-wide chromatin accessibility. Furthermore, we show that CTCF is an upstream negative regulator of FOXA1-chromatin interactions. In ER responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity is absolute and in tamoxifen resistant cells, ER binding occurs independently of ligand, but in a FOXA1 dependent manner. Importantly, expression of FOXA1 in non-breast cancer cells is sufficient to alter ER binding and response to endocrine treatment. As such, FOXA1 is the primary determinant that regulates estrogen-ER activity and endocrine response in breast cancer cells and is sufficient to program ER functionality in non-breast cancer contexts.

Transplantation of ALDHhiSSClo Neural Stem Cells in nmd Mice, an Animal Model of SMARD1

SMARD1 is an infantile autosomal recessive motor neuron (MN) disease, caused by mutations in the Immunoglobulin mu binding protein 2 (IGHMBP2).
We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase activity (ALDH) to modify disease progression of nmd mice, an animal model of SMARD1.
ALDHhiSSClo stem cells are self-renewing and multipotent and when intratechally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns.
Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased life-span.
To further investigate the molecular events responsible for these differences, microarray and Real time RT-PCR analysis of wild-type, mutated and transplanted nmd spinal cord were undertaken.
We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared to wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd transplanted mice expressed high transcript levels for genes related to neurogenesis such as Doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogenesis may contribute to the observed nmd phenotype amelioration.

We used high density oligonucleotide arrays to measure the relative expression levels of >39,000 genes and ESTs in the NOD mouse (a murine model of T1D and other autoimmune conditions), four NOD-derived diabetes resistant congenic strains and two nondiabetic control strains. Owing to the importance of T cells in the development of T1D we decided to target two organs, the spleen and thymus.  We profiled gene expression in thymi from four week old female mice and spleens from three month old female mice.  For each of the 14 strain-tissue combinations (7 strains x 2 tissues) we performed two independent replicate experiments, making the total number of hybridizations performed 28.  In each case, an RNA population from a pool of two or three organs was generated to minimize the chances of within strain variation masking genuine variation between the strains.  To minimize any variation introduced during target preparation, all samples within a given replicate group were processed in parallel.  Three samples from group two (spleen: B10.H2g7_S2 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gsm594 (GSM594); thymus: B10.H2g7_T2 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gsm608 (GSM608), B10.H2g7 Idd3_T2 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gsm609 (GSM609)) and one from group one (spleen B10.H2g7_S1 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gsm587 (GSM587) failed a preliminary quality control check.  Consequently, the steps involved in preparing labelled cRNA from the initial starting material had to be repeated for these four samples at a later date.  A subsequent comparison of the variation seen between samples within the same replicate group (samples processed in parallel) versus samples in distinct replicate groups (samples processed at different times) revealed that the variation between the two groups (i.e. owing to independent sample preparation) was higher than the small amount of variation between the closely matched strains.  Ignoring the variability introduced during sample preparation was therefore likely to result in spurious changes being detected and genuine strain-specific differences in gene expression being masked.  Consequently, we decided to exclude the four samples that had had to be prepared independently owing to failing initial quality control.
Keywords = NOD, diabetes, congenic

This experiment investigated the role of mechanical ventilation (MV) in modulating lung's transcriptional response to LPS.   Twenty four  C57/B6 male mice were randomized to four groups: 1. Control, 2. MV, 3. LPS and 4. MV+LPS.  Expression profiling of whole lungs revealed a significant augmentation of the transcriptional response in the combined MV+LPS group relative to the other 3 conditions.

Abstract

Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), a PKA-selective cAMP analog, alters the expression of ~4500 of ~13,600 unique genes. By contrast, gene expression was unaltered in Kin– S49 cells (that lack PKA) incubated with CPT-cAMP. Changes in mRNA and protein expression of several cell-cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2 h, CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements (CRE) within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of CRE-containing genes and secondary networks that include the circadian clock.

Transcript level in F9 teratocarcinoma WT and RARalpha knockout in presence and absence of all-trans retinoic acid

Retinoic acid receptors (RARs) α, β and γ are key regulators of embryonic development. Hematopoietic differentiation is regulated by RARα, and several types of leukemia show aberrant RARα activity. We demonstrate that RARα plays an important role in cellular memory and imprinting by regulating the CpG methylation status of specific promoter regions.
We used microarrays to identify genes, which display differential expression in F9 RARalpha knockout (RARaKO) cells relative to F9 wt cells.

Gene expression profiling of 18 hepatocellular carcinoma and adjacent non-tumorous liver tissue

These paired HCC and non-tumorous liver tissues were used to determine highly dfferentially expressed genes in HCC and non-tumorous liver tissue.
Hepatocellular carcinoma (HCC) is a malignancy with poor survival outcome. Genes showing extreme differential expression between paired human HCC and adjacent non-tumorous liver tissue were investigated. PLVAP was identified as a gene specifically expressed in vascular endothelial cells of HCC but not in non-tumorous liver tissues. This finding was confirmed by RT-PCR analysis of micro-dissected cells and immunohistochemical staining of tissue sections. A recombinant monoclonal anti-PLVAP Fab fragment co-expressing extracellular domain of human tissue factor (TF) was developed. The potential therapeutic effect and toxicity to treat HCC were studied using a Hep3B HCC xenograft model in SCID mice. Infusion of recombinant monoclonal anti-PLVAP Fab-TF into the tumor feeding artery induced tumor vascular thrombosis and extensive tumor necrosis at doses between 2.5 µg and 12 µg. Tumor growth was suppressed for 40 days after a single treatment. Systemic administration did not induce tumor necrosis. Little systemic toxicity was noted for this therapeutic agent. The results of this study suggest that anti-PLVAP Fab-TF may be used to treat HCC cases for which transcatheter arterial chemoembolization (TACE) is currently used, but without major drawbacks of TACE. Anti-PLVAP Fab-TF may improve therapeutic outcome and be a viable therapeutic agent in patients with more advanced disease and compromised liver function.

Comparison of gene expression data between wild-type and DM1-affected Mesodermal Precursors Cells (MPC)

Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 Mesodermal Precursors Cells.

Embryonic stem (ES) cell lines provide, theoretically, unlimited access to any needed amount of any specific cell phenotype of an organism, due to their unique capacities at indefinite self-renewal and pluripotency (Smith 2001; Trounson 2006). These properties allow using the progeny of ES cell lines to model human pathologies (Martinat, Shendelman et al. 2004; Lerou and Daley 2005; Ben-Nun and Benvenisty 2006). In particular, human ES cell lines derived from embryos characterized as gene-carriers following pre-implantation genetic diagnosis (PGD) for any one of major monogenic diseases (Pickering, Minger et al. 2005; Mateizel, De Temmerman et al. 2006) may be considered as perfect cellular replicas of those diseases, as they exhibit the exact genotypes associated to them. Here, we confirm this hypothesis by demonstrating that the cell progeny of an ES cell line derived from an embryo with myotonic dystrophy type 1 (DM1) displayed the morphological stigma associated to the expression of the mutant gene –so-called intranuclear foci- as well as abnormal alternate splicing of the insulin receptor, a characteristic feature of DM1. Further differential transcriptomic analysis of the DM1 gene-carrying cells with phenotypically similar populations from native ES cell lines revealed abnormal expression of 89 genes, among which 48 were down-regulated and 39 over-expressed. This study demonstrates that DM1, though a disease with relatively late clinical onset, is associated with expression of genetic defects early on during development. It underlines the value of PGD-derived ES cell lines as a tool to decipher molecular mechanisms of genetic diseases.
Ben-Nun, I. F. and N. Benvenisty (2006). Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252(1-2): 154-9.
Lerou, P. H. and G. Q. Daley (2005). Therapeutic potential of embryonic stem cells. Blood Rev 19(6): 321-31.
Martinat, C., S. Shendelman, et al. (2004). Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism. PLoS Biol 2(11): e327.
Mateizel, I., N. De Temmerman, et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21(2): 503-11.
Pickering, S. J., S. L. Minger, et al. (2005). Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod Biomed Online 10(3): 390-7.
Smith, A. G. (2001). Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17: 435-62.
Trounson, A. (2006). The production and directed differentiation of human embryonic stem cells. Endocr Rev 27(2): 208-19.

Comparison of gene expression data between wild-type and DM1-affected undifferentiated hES cells.

Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 undifferentiated hES cells

Comparison of gene expression data between wild-type and DM1-affected Neural Precursors Cells (NPC)

Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 Neural Precursor cells

The mouse cytosine-5 RNA methyltransferase NSun2 is a component of the chromatoid body and required for testis differentiation

Post-transcriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and often organized by the chromatoid body. Chromatoid bodies are large cytoplasmic ribonucleoprotein granules, whose precise function and composition remain unclear. Here, we identify NSun2 as a novel component of the chromatoid body, and further show that this RNA methylase is essential for germ cell differentiation in the mouse testis. Lack of NSun2 leads to down-regulation of genes controlling RNA processing and post-transcriptional repression pathways, including Ddx4, Mili, Piwil1 (Miwi) and Tudor domain containing (Tdrd) proteins. Germ cell differentiation was blocked specifically at the pachytene stage by lack of NSun2, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 with Dnmt2, the only other characterized cytosine-5 RNA methyltransferase to date, indicating that Dnmt2 was not functionally redundant for NSun2 in spermatogonial stem cells or Sertoli cells. Thus, our data indicate that RNA methylation pathways play an essential role in male germ cell differentiation.

Expression data from isolated E17.5 mouse skin with or without Ovol2 overexpression

During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake alpha-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-a-catenin sequential repression and highlight novel functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity.

Expression data from calcium-induced isolated keratinocytes from mouse skin with or without Ovol1 and Ovol2 expression

To formally address the tumor suppressor activity of Sh2b3 in vivo, we tested the interaction between oncogenic NOTCH1 and Sh2b3 loss in a retroviral- transduction bone marrow transplantation model of NOTCH-induced T-ALL
Forced expression of activated NOTCH1 in this model typically results in full leukemia transformation 5-10 weeks later.

To study the effect of PIAS1 on transcriptional regulation, we establishedstable PIAS1 shRNA knockdown cells in breast cancer cell line MDA-MB231.
By comparing the expression profiles of control vs PIAS1 knockdown cells, we can identify potential PIAS1 target genes involved in breast tumorigenesis.

Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts

The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signaling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the non-selective S1P receptor agonist FTY720 causes increased bone formation in wildtype, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo, and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.

Posttranslational modifications on histone tails control gene expresssion levels in an either positive or negative way. Therefore enzymes which modifiy the modifications on the histon tails, like histone demethylases, are able to strictly regulate the transcriptom.
To analyse the regulatory role of histone demethylase Fbxl10 we generated a stable cell line permanently overexpressing HA-tagged Fbxl10 and identified the transcriptome changes compared to untransfected control cell line by microarray.

Expression data of A375 melanoma cells after DMSO or MLN4924 treatment from 1 hour to 24 hour

Microarrays were used to determine the change in gene expression of genes involved in the CDT1/NAE pathway

Medullary Thymic Epitherlial Cell (mTEC) Gene Expression in MBD1 WT and KO Mice

To explore global gene expression using microarray analysis in mTEC's isolated from MBD1 WT and MBD1 KO mice

DECREASED FIBRONECTIN PRODUCTION SIGNIFICANTLY CONTRIBUTES TO DYSREGULATED REPAIR OF ASTHMATIC EPITHELIUM

Rationale: Damage to airway epithelium is followed by deposition of extracellular matrix (ECM) and migration of adjacent epithelial cells. We have shown that epithelial cells from asthmatic children fail to heal a wound in vitro. Objectives: To determine whether dysregulated ECM production by the epithelium plays a role in aberrant repair in asthma. Methods: Airway epithelial cells (AEC) from children with asthma (n=36), healthy atopic (n=23) and healthy non-atopic controls (n=53) were investigated by microarray, gene expression and silencing, transcript regulation analysis and ability to close mechanical wounds. Results: Wound repair of AEC from healthy and atopic children were not significantly different and were both faster than AEC from asthmatics. Microarray analysis revealed differential expression of multiple gene sets associated with repair and remodeling in asthmatic AEC. Fibronectin (FN) was the only ECM component whose expression was significantly lower in asthmatic AEC. Expression differences were verified by qPCR and ELISA, and reduced FN expression persisted in asthmatic cells over passage. Silencing of FN expression in non-asthmatic AEC inhibited wound repair, while addition of FN to asthmatic AEC restored reparative capacity. Asthmatic AEC failed to synthesize FN in response to wounding or cytokine/growth factor stimulation. Exposure to 5’, 2’deoxyazacytidine had no effect on FN expression and subsequent analysis of the FN promoter did not show evidence of DNA methylation. Conclusions: These data show that the reduced capacity of asthmatic epithelial cells to secrete FN is an important contributor to the dysregulated AEC repair observed in these cells.

Effects of RNA interference-mediated c-MYC depletion on gene expression profiles in human cancer cell lines

Affymetrix Hu133 GeneCHIP Microarray data for Control and c-MYC knock-down (KD) human cancer cell lines.

Data related to article 'Novel c-MYC target genes mediate differential effects on cell proliferation and migration', from D. CAPPELLEN et al., EMBO Reports

Note: Samples GSM136093 and GSM136094 (Hela cell line, expt 1) were hybridized and normalized separately from the other 18 Samples.

Identification of rapamycin as a glucocorticoid resistance reversal agent

Drug resistance remains a major obstacle to successful cancer treatment. Here we use a novel approach to identify rapamycin as a glucocorticoid resistance reversal agent. A database of drug-associated gene expression profiles was screened for molecules whose profile overlapped with a gene expression signature of glucocorticoid (GC) sensitivity/resistance in Acute Lymphoblastic Leukemia (ALL) cells. The screen indicated the mTOR inhibitor rapamycin profile matched the signature of GC-sensitivity. We thus tested the hypothesis that rapamycin would induce GC sensitivity in lymphoid malignancy cells, and found that it sensitized cells to glucocorticoid induced apoptosis via modulation of antiapoptotic MCL1. These data indicate that MCL1 is an important regulator of GC-induced apoptosis, and that the combination of rapamycin and glucocorticoids has potential utility in ALL. Furthermore this approach represents a novel strategy for identification of promising combination therapies for cancer.

gene expression data from wild-type and Bcl6-/- regulatory T cells
in order to find genes regulated by Bcl6 in Treg cells

Molecular signature of CD34+ hematopoietic stem and progenitor cells in chronic phase CML

Chronic myelogenous leukaemia (CML) is a malignant disorder of the hematopoietic stem cell, which is characterized by the reciprocal translocation between chromosomes 9 and 22 (t(9;22)(q34;q11)) The translocation results in the formation of the BCR-ABL fusion oncogene encoding a protein with constitutive activated tyrosine kinase activity which plays a central role in the pathogenesis of the disease. There are still several open questions with respect to BCR-ABL-induced malignant transformation. A large limitation of the existing data about BCR-ABL effects is that they are derived to a great proportion from human hematopoietic cell lines, BCR-ABL-transformed murine cell lines or fibroblasts and mouse models, which might not be representative for chronic phase CML. A suitable cell population for studies on CML biology are primary hematopoietic stem and progenitor cells from patients with CML. Therefore, we provide in this study a genome-wide expression signature of highly enriched CD34+ cells from bone marrow (BM) of untreated patients with CML in chronic phase. Gene expression profiles of immunomagnetically enriched BM CML CD34+ cells (n=9) were compared with those of normal BM CD34+ cells (n=8) using microarrays covering 8.746 genes. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 918 genes were significantly differentially expressed (q value <0.1%; fold change > 1.3). Several of the BCR-ABL-induced effects described in cell lines and BCR-ABL-transduced cells could also be found in primary CML progenitor cells as for example the transcriptional activation of the classical MAPK pathway and the PI3 kinase/AKT pathway and the down-regulation of the pro-apoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include an up-regulation of components of the TGFb signalling pathway and the non-canonical Wnt/Ca2+ pathway, a transcriptional activation of fetal haemoglobin genes and genes associated with early hematopoietic stem cells (HSC) such as HoxA9 and MEIS1 and up-regulation of genes involved in fatty acid metabolism, of the thrombin receptor PAR1 and the neuroepithelial cell transforming gene 1. Differential expression of differentiation-associated genes suggested an alteration of the composition of the CD34+ cell population in CML. This was confirmed by immunophenotypical subset analyses of chronic phase CML CD34+ cells showing an increase of erythroid progenitors and a decrease of granulocyte-macrophage progenitor cells while the proportion of HSC was similar in normal and CML CD34+ cells. In conclusion, our results give novel insights into the biology of CML hematopoietic stem and progenitor cells and could be the basis for identification of new targets for therapy.

In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis.
Keywords: Gene Expression, C2C12 Myotubes, Oxidative Stress, Adaptation