9 results for chip-seq drosophila
Methods Notes - Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification
Contributors: Thomas Schwarzl, Desmond G. Higgins, Walter Kolch, David J. Duffy
ChIP-seq...ChIP-seq signal of promoter regions (5kb upstream and 500bp downstream... (F) ChIP-seq signal of promoter regions (5kb upstream and 500bp downstream ... Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.
Contributors: Frauke Goeman, Francesca De Nicola, Paolo D’Onorio De Meo, Matteo Pallocca, Berardino Elmi, Tiziana Castrignanò, Graziano Pesole, Sabrina Strano, Giovanni Blandino, Maurizio Fanciulli
promoters. ChIP-Seq and RNA-Seq data from six genomic regions showing...ChIP-Seq...ChIP-Seq and RNA-Seq data from six genomic regions showing the occupancy ... There is growing evidence that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) plays a role in breast cancer prevention and survival. It elicits a variety of antitumor activities like controlling cellular differentiation, proliferation and angiogenesis. Most of its biological effects are exerted via its nuclear receptor which acts as a transcriptional regulator. Here, we carried out a genome-wide investigation of the primary transcriptional targets of 1α,25(OH)2D3 in breast epithelial cancer cells using RNA-Seq technology. We identified early transcriptional targets of 1α,25(OH)2D3 involved in adhesion, growth regulation, angiogenesis, actin cytoskeleton regulation, hexose transport, inflammation and immunomodulation, apoptosis, endocytosis and signaling. Furthermore, we found several transcription factors to be regulated by 1α,25(OH)2D3 that subsequently amplify and diversify the transcriptional output driven by 1α,25(OH)2D3 leading finally to a growth arrest of the cells. Moreover, we could show that 1α,25(OH)2D3 elevates the trimethylation of histone H3 lysine 4 at several target gene promoters. Our present transcriptomic analysis of differential expression after 1α,25(OH)2D3 treatment provides a resource of primary 1α,25(OH)2D3 targets that might drive the antiproliferative action in breast cancer epithelial cells.
Contributors: N. Coughlan, G. Thillainadesan, J. Andrews, M. Isovic, J. Torchia
al. (top) and ChIP-Seq analysis performed by Welboren et al. (bottom)...ChIP-Seq analysis performed by Welboren et al. (bottom). (B) Venn diagram...ChIP-Seq reported by Welboren et al. 59 p/CIP/CARM1 target genes were ... identified by ChIP-Seq reported by Welboren et al. 59 p/CIP/CARM1 target ... The steroid receptor coactivator p/CIP, also known as SRC-3, is an oncogene commonly amplified in breast and ovarian cancers. p/CIP is known to associate with coactivator arginine methyltransferase 1 (CARM1) on select estrogen responsive genes. We have shown, using a ChIP-on-chip approach, that in response to stimulation with 17β-estradiol (E2), the p/CIP/CARM1 complex is recruited to 204 proximal promoters in MCF-7 cells. Many of the complex target genes have been previously implicated in signaling pathways related to oncogenesis. Jak2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the Jak2 promoter are reflective of a transcriptionally permissive gene, and modest changes in RNA and protein expression lead us to suggest that an additional factor(s) may be required for a more notable transcriptional and functional response. Bioinformatic examination of the 204 proximal promoter sequences of p/CIP/CARM1 targets supports the idea that transcription factor crosstalk is likely the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. This data may have implications towards understanding the oncogenic role of p/CIP in breast cancer and ultimately allow for the identification of new prognostic indicators and/or viable therapeutic targets.
Dynamic phosphorylation of the kinesin Costal-2 in vivo reveals requirement of fused kinase activity for all levels of hedgehog signalling
Contributors: S. Raisin, L. Ruel, N. Ranieri, L. Staccini-Lavenant, P.P. Thérond
Drosophila larvae. In the eye disc, Cos2 is phosphorylated in cells anterior...Drosophila...Drosophila cultured cells, phosphorylation of the kinesin-like Costal2 ... Hedgehog (Hh) proteins are secreted molecules that play an essential role in development and tumorigenesis. In Drosophila cultured cells, phosphorylation of the kinesin-like Costal2 (Cos2) protein at Ser572 is triggered by the kinase fused (Fu) upon Hh pathway activation. Here, we validate the first phospho-antibody for one of the Hh pathway components, Cos2, as a universal in situ readout of Hh signal transduction. For the first time, this tool allows the visualisation of a gradient of signalling activity and therefore the range of the activating Hh ligand in different tissues. We also show that, in vivo, Fu kinase is activated by and necessary to transduce all levels of intracellular Hh signalling. Our study fills a gap in the understanding of the Hh pathway by showing that the molecular cascade leading to Cos2 phosphorylation is conserved in all cells activated by Hh. Therefore, we propose that the extracellular Hh information is conveyed to an intracellular signal through graded Fu kinase activity.
The endogenous siRNA pathway in Drosophila impacts stress resistance and lifespan by regulating metabolic homeostasis
Contributors: Do-Hwan Lim, Chun-Taek Oh, Langho Lee, Jae-Sang Hong, Su-Hyun Noh, Seungwoo Hwang, Sungchan Kim, Sung-Jun Han, Young Sik Lee
Drosophila....Drosophila heads and bodies. ...proteins identified in Drosophila heads and bodies. ...Drosophila...Drosophila, Dicer-2 (Dcr-2) functions in the biogenesis of endogenous ...Drosophila impacts stress resistance and lifespan by regulating metabolic ... Small non-coding RNAs regulate gene expression in a sequence-specific manner. In Drosophila, Dicer-2 (Dcr-2) functions in the biogenesis of endogenous small interfering RNAs (endo-siRNAs). We identified 21 distinct proteins that exhibited a ⩾1.5-fold change as a consequence of loss of dcr-2 function. Most of these were metabolic genes implicated in stress resistance and aging. dcr-2 Mutants had reduced lifespan and were hypersensitive to oxidative, endoplasmic reticulum, starvation, and cold stresses. Furthermore, loss of dcr-2 function led to abnormal lipid and carbohydrate metabolism. Our results suggest roles for the endo-siRNA pathway in metabolic regulation and defense against stress and aging in Drosophila.
Global methylation profiling in serous ovarian cancer is indicative for distinct aberrant DNA methylation signatures associated with tumor aggressiveness and disease progression
Contributors: Mamadou Keita, Zhi-Qiang Wang, Jean-Francois Pelletier, Magdalena Bachvarova, Marie Plante, Jean Gregoire, Marie-Claude Renaud, Anne-Marie Mes-Masson, Éric R. Paquet, Dimcho Bachvarov
ChIP-seq results in ES cells. Using also the Genomic Regions Enrichment...ChIP-seq data (see Supplemental Fig. 2 for details). Similarly, upon applying...events with H3K27me3 ChIP-seq results in ES cells. Using also the Genomic ... To characterize at high resolution the DNA methylation changes which occur in the genome of serous epithelial ovarian cancer (EOC) in association with tumor aggressiveness.
Contributors: Rony Nehmé, Olivier Joubert, Michel Bidet, Benoît Lacombe, Ange Polidori, Bernard Pucci, Isabelle Mus-Veteau
Drosophila Schneider 2 cells. Drosophila S2 cells transfected with 0.5μg...Smoothened by Hedgehog in the Drosophila Schneider 2 cells. Drosophila ... Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-β-d-maltoside (DDM). The fluorinated surfactant C8F17TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C8F17TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.
Genomic analysis of the response of mouse models to high-fat feeding shows a major role of nuclear receptors in the simultaneous regulation of lipid and inflammatory genes
Contributors: Arja J. Kreeft, Corina J.A. Moen, Gordon Porter, Soemini Kasanmoentalib, Ronit Sverdlov, Patrick J. van Gorp, Louis M. Havekes, Rune R. Frants, Marten H. Hofker
... The mechanisms of diet induced hyperlipidemia and atherosclerosis have been widely studied by delineating the role of candidate genes in transgenic and gene targeted mouse models. However, diet induced hyperlipidemia represents a complex process determined by many lipid genes that is only partly understood. This study is aimed at delineating the events induced by dietary intervention in different mouse models at the level of gene expression using microarray analysis. The focus is on the liver as the organ primarily responding to diet, and crucial in determining plasma lipid levels. Firstly, the effect of the genotype was studied. Expression profiles of liver genes were compared between APOE3Leiden (E3L), APOE knockout (E−/−) and C57BL/6JIco (B6) mice using the Incyte GEM 2.03 array carrying 9552 genes. Several hundred differentially expressed genes were identified indicating that the genotype alone effects gene expression. Secondly, the response of E3L mice to high-fat feeding was investigated using a mild and severe high-fat diet (diet W and N, respectively). Diet W caused differential regulation of 200 genes, while diet N affected the expression of 788 genes in B6 and 1010 genes in E3L mice. Annotation of these genes using the Gene Ontology (GO) database showed that two major processes were strongly affected by genotype and diet, namely lipid metabolism and inflammation, the latter as determined by “immune/defense response and detoxification” processes. Many nuclear receptor target genes were differentially regulated, with the largest effects modulated by the severe high-fat diet N, leading to the suppression of genes involved in bile acid, sterol, steroid, fatty acid, and detoxification metabolism. Strikingly, a substantial part of these nuclear receptor target genes were commonly regulated during the different experimental conditions. The common regulation of many nuclear receptor target genes underlying lipid and detoxification processes as found in this study, suggest a defense mechanism involving many nuclear receptors to protect against the accumulation of toxic endogenous lipids and bile acids. These results further strengthen the close link between hyperlipidemia and inflammatory processes.
Clinical Investigation - A common gene signature across multiple studies relate biomarkers and functional regulation in tolerance to renal allograft
Contributors: Daniel Baron, Gérard Ramstein, Mélanie Chesneau, Yann Echasseriau, Annaick Pallier, Chloé Paul, Nicolas Degauque, Maria P. Hernandez-Fuentes, Alberto Sanchez-Fueyo, Kenneth A. Newell
... Patients tolerant to a kidney graft display a specific blood cell transcriptional pattern but results from five different studies were inconsistent, raising the question of relevance for future clinical application. To resolve this, we sought to identify a common gene signature, specific functional and cellular components, and discriminating biomarkers for tolerance following kidney transplantation. A meta-analysis of studies identified a robust gene signature involving proliferation of B and CD4 T cells, and inhibition of CD14 monocyte related functions among 96 tolerant samples. This signature was further supported through a cross-validation approach, yielding 92.5% accuracy independent of the study of origin. Experimental validation, performed on new tolerant samples and using a selection of the top-20 biomarkers, returned 91.7% of good classification. Beyond the confirmation of B-cell involvement, our data also indicated participation of other cell subsets in tolerance. Thus, the use of the top 20 biomarkers, mostly centered on B cells, may provide a common and standardized tool towards personalized medicine for the monitoring of tolerant or low-risk patients among kidney allotransplant recipients. These data point to a global preservation of genes favoring the maintenance of a homeostatic and ‘healthy’ environment in tolerant patients and may contribute to a better understanding of tolerance maintenance mechanisms.