Contributors: Luis Rueda, Iman Rezaeian

Date: 2014-01-01

ChIP-Seq data. However, the complexity of analyzing various patterns of...ChIP-Seq signals still needs the development of new algorithms. Most current...ChIP-Seq has emerged as an alternative to ChIP-chip methods. ChIP-Seq ...Drosophila melanogaster...Drosophila melanogaster and the H3K4ac antibody dataset....ChIP-Seq data. CMT employs a constraint-based module that can target regions ... Genome-wide profiling of DNA-binding proteins using ChIP-Seq has emerged as an alternative to ChIP-chip methods. ChIP-Seq technology offers many advantages over ChIP-chip arrays, including but not limited to less noise, higher resolution, and more coverage. Several algorithms have been developed to take advantage of these abilities and find enriched regions by analyzing ChIP-Seq data. However, the complexity of analyzing various patterns of ChIP-Seq signals still needs the development of new algorithms. Most current algorithms use various heuristics to detect regions accurately. However, despite how many formulations are available, it is still difficult to accurately determine individual peaks corresponding to each binding event. We developed Constrained Multi-level Thresholding (CMT), an algorithm used to detect enriched regions on ChIP-Seq data. CMT employs a constraint-based module that can target regions within a specific range. We show that CMT has higher accuracy in detecting enriched regions (peaks) by objectively assessing its performance relative to other previously proposed peak finders. This is shown by testing three algorithms on the well-known FoxA1 Data set, four transcription factors (with a total of six antibodies) for Drosophila melanogaster and the H3K4ac antibody dataset.

Contributors: Chen, Kai, Johnston, Jeff, Shao, Wanqing, Meier, Samuel, Staber, Cynthia, Zeitlinger, Julia

Date: 2013-08-14

ChIP-seq experiments on tightly staged Drosophila embryos and show that...ChIP-seq enrichment values at the transcription start site (TSS) and transcription...Drosophila melanogaster...Drosophila embryos and show that massive recruitment of RNA polymerase...Drosophila midblastula transition ... Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. We have performed ChIP-seq experiments on tightly staged Drosophila embryos and show that massive recruitment of RNA polymerase II (Pol II) with widespread pausing occurs de novo during the midblastula transition. However, ∼100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties.

Contributors: unknown

Drosophila that can be used to assess protein–DNA-binding rhythms at specific...Drosophila Clock...ChIP-seq. ChIP analysis has revealed the relationship between clock factor...Drosophila...ChIP-seq). ChIP has been widely used in circadian biology to assess rhythmic ... In eukaryotes, the circadian clock controls 24h rhythms in physiology, metabolism, and behavior via cell autonomous transcriptional feedback loops. These feedback loops keep circadian time and control rhythmic outputs by driving rhythms in transcription; thus, it is important to determine when clock transcription factors bind their target sequences in vivo to promote or repress transcription. Interactions between proteins and DNA can be measured in cells, tissue, or whole organisms using a technique called chromatin immunoprecipitation (ChIP). The principle underlying ChIP is that protein is cross-linked to associated chromatin to form a protein–DNA complex, the DNA is then sheared, and the protein of interest is immunoprecipitated. The cross-links are then removed from the antibody–protein–DNA complex, and the associated DNA fragments are purified. The DNA is then used to quantify specific targets by real-time quantitative PCR or to generate libraries for global analysis of protein target sites by high-throughput sequencing (ChIP-seq). ChIP has been widely used in circadian biology to assess rhythmic binding of clock components, RNA polymerase II, and rhythms in chromatin modifications such as histone acetylation and methylation. Here, we present a detailed method for ChIP analysis in Drosophila that can be used to assess protein–DNA-binding rhythms at specific genomic target sites. With minor modifications, this technique can be used to assess protein–DNA-binding rhythms at all target sites via ChIP-seq. ChIP analysis has revealed the relationship between clock factor binding, transcription, and chromatin modifications and promises to reveal circadian transcription networks that control phase and tissue specificity.

Contributors: unknown

Date: 2014-10-09

ChIP-Seq Data...ChIP-seq) is a prevailing methodology used to investigate chromatin-based...ChIP-seq experiments enables the discovery of disease-relevant changes ... Epigenomic profiling by chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) is a prevailing methodology used to investigate chromatin-based regulation in biological systems such as human disease, but the lack of an empirical methodology to enable normalization among experiments has limited the precision and usefulness of this technique. Here, we describe a method called ChIP with reference exogenous genome (ChIP-Rx) that allows one to perform genome-wide quantitative comparisons of histone modification status across cell populations using defined quantities of a reference epigenome. ChIP-Rx enables the discovery and quantification of dynamic epigenomic profiles across mammalian cells that would otherwise remain hidden using traditional normalization methods. We demonstrate the utility of this method for measuring epigenomic changes following chemical perturbations and show how reference normalization of ChIP-seq experiments enables the discovery of disease-relevant changes in histone modification occupancy.

Contributors: unknown

Date: 2015-10-07

ChIP-seq and microarray gene expression data, we identified 60 genomic...ChIP-seq datasets were obtained from samples using two different GAL4 ...ChIP-seq experiments. (C) Tissue expression of fkh-GAL4 and sage-GAL4 ...ChIP-seq analysis were linked to changes in gene expression, we performed...Drosophila embryonic salivary gland through transcriptional activation...ChIP-seq analysis identifies Rib binding sites in salivary gland cells...Drosophila salivary gland via a diverse array of targets through both ...ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically...Drosophila...Drosophila salivary gland (SG). Notably, the morphogenetic changes in ...ChIP-Seq. ... Transcription factors affect spatiotemporal patterns of gene expression often regulating multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape/volume increases during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the changes in cell shape/volume in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib, we performed ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib {} embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites bound by Rib likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell growth and tissue shape in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.

Contributors: unknown

Date: 2013-04-30

ChIP-Seq...ChIP-Seq-based NF-κB p65 genes....ChIP-Seq-based NF-κB p65 target genes. Entrez Gene IDs of 918 ChIP-Seq-based...ChIP-Seq peaks in the intronic region of target genes. The genomic location ... The transcription factor nuclear factor-kappa B (NF-κB) acts as a central regulator of immune response, stress response, cell proliferation, and apoptosis. Aberrant regulation of NF-κB function triggers development of cancers, metabolic diseases, and autoimmune diseases. We attempted to characterize a global picture of the NF-κB target gene network relevant to the immunopathogenesis of multiple sclerosis (MS).

Contributors: unknown

Date: 2015-06-30

ChIP-seq data (see Materials and methods). This sequence matches the Nrf2...ChIP-seq signal and the ARE motif score in each Nrf2-bound region. ARE...ChIP-seq peak and ARE sequence at the VCP locus. Bottom: Nrf2 ChIP-seq...Drosophila to human. The ancient network consists of canonical antioxidant...Drosophila class I genes) and gene expression changes after treatment ...ChIP-seq signal from LCL cells treated with DMSO or SFN as indicated. ...Drosophila class I, II, or III genes. Orthologs were identified using ...ChIP-seq data from human LCL cells treated with SFN. Top: Nrf2 ChIP-seq...Drosophila class I genes....Drosophila. (A) Box plot representing the range of gene expression changes...Drosophila class III genes and human orthologs....Drosophila...Drosophila and humans. These data have allowed us to construct the deeply ... Nrf2, a basic leucine zipper transcription factor encoded by the gene NFE2L2, is a master regulator of the transcriptional response to oxidative stress. Nrf2 is structurally and functionally conserved from insects to humans, and it heterodimerizes with the small MAF transcription factors to bind a consensus DNA sequence (the antioxidant response element, or ARE) and regulate gene expression. We have used genome-wide chromatin immunoprecipitation and gene expression data to identify direct Nrf2 target genes in Drosophila and humans. These data have allowed us to construct the deeply conserved ancient Nrf2 regulatory network—target genes that are conserved from Drosophila to human. The ancient network consists of canonical antioxidant genes, as well as genes related to proteasomal pathways and metabolism and a number of less expected genes. We have also used enhancer reporter assays and electrophoretic mobility-shift assays to confirm Nrf2-mediated regulation of ARE activity at a number of these novel target genes. Interestingly, the ancient network also highlights a prominent negative feedback loop; this, combined with the finding that Nrf2-mediated regulatory output is tightly linked to the quality of the ARE it is targeting, suggests that precise regulation of nuclear Nrf2 concentration is necessary to achieve proper quantitative regulation of distinct gene sets. Together, these findings highlight the importance of balance in the Nrf2–ARE pathway and indicate that Nrf2-mediated regulation of xenobiotic metabolism, glucose metabolism, and proteostasis has been central to this pathway since its inception.

Contributors: unknown

Date: 2013-03-04

ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging...ChIP-Seq sequencing tracks on the selected targets of Egr2. Egr2 binding...ChIP-Seq analysis. (A) Distribution of the types of Egr2 binding sites...ChIP-Seq analyses. (B) Consensus sequence of Egr2 binding sites derived...ChIP-Seq and gene expression profiling analyses. CAR Tg×Egr2flox/flox ...ChIP-Seq analyses were overlapped. The dark gray and light gray-colored ... T cell anergy is one of the mechanisms contributing to peripheral tolerance, particularly in the context of progressively growing tumors and in tolerogenic treatments promoting allograft acceptance. We recently reported that early growth response gene 2 (Egr2) is a critical transcription factor for the induction of anergy in vitro and in vivo, which was identified based on its ability to regulate the expression of inhibitory signaling molecules diacylglycerol kinase (DGK)-α and -ζ. We reasoned that other transcriptional targets of Egr2 might encode additional factors important for T cell anergy and immune regulation. Thus, we conducted two sets of genome-wide screens: gene expression profiling of wild type versus Egr2-deleted T cells treated under anergizing conditions, and a ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging of these data sets revealed 49 targets that are directly regulated by Egr2. Among these are inhibitory signaling molecules previously reported to contribute to T cell anergy, but unexpectedly, also cell surface molecules and secreted factors, including lymphocyte-activation gene 3 (Lag3), Class-I-MHC-restricted T cell associated molecule (Crtam), Semaphorin 7A (Sema7A), and chemokine CCL1. These observations suggest that anergic T cells might not simply be functionally inert, and may have additional functional properties oriented towards other cellular components of the immune system.

Contributors: Joshi, Anagha, Beck, Yvonne, Michoel, Tom

Date: 2014-07-24

Drosophila species at the 10% recall level (in brackets for each TF the...Drosophila species....ChIP-seq gold standard interactions conserved in one to three species ...ChIP-seq network measured in its own species. This overall good performance...Drosophila species and compared predicted networks to gold standard networks...Drosophila species at the same developmental time point are not greater...Drosophila species reconstructed from the inferred interactions at 10%...ChIP-seq interactions for developmental transcription factors in five ...Drosophila species measured at 9–13 time points during early embryonic...ChIP-seq networks for D. melanogaster (b, 4 TFs), D. ananassae (c, 1 TF...Drosophila species using a gold standard network of ChIP-chip interactions...Drosophila species (D. melanogaster (“amel”), D. ananassae (“ana”), D....Drosophila species. The gold standard networks were the ChIP-chip network...Drosophila species. We created gene lists at each branch point containing...Drosophila ... Gene regulatory network inference uses genome-wide transcriptome measurements in response to genetic, environmental or dynamic perturbations to predict causal regulatory influences between genes. We hypothesized that evolution also acts as a suitable network perturbation and that integration of data from multiple closely related species can lead to improved reconstruction of gene regulatory networks. To test this hypothesis, we predicted networks from temporal gene expression data for 3,610 genes measured during early embryonic development in six Drosophila species and compared predicted networks to gold standard networks of ChIP-chip and ChIP-seq interactions for developmental transcription factors in five species. We found that (i) the performance of single-species networks was independent of the species where the gold standard was measured; (ii) differences between predicted networks reflected the known phylogeny and differences in biology between the species; (iii) an integrative consensus network which minimized the total number of edge gains and losses with respect to all single-species networks performed better than any individual network. Our results show that in an evolutionarily conserved system, integration of data from comparable experiments in multiple species improves the inference of gene regulatory networks. They provide a basis for future studies on the numerous multi-species gene expression datasets for other biological processes available in the literature.

Contributors: Zabet, Nicolae Radu, Adryan, Boris

Date: 2014-04-22

Drosophila TFs during early embryonic development: Bicoid, Caudal, Giant...ChIP-seq profiles of Hunchback and Kruppel display some sharp peaks, which...ChIP-seq profile of Hunchback A C and Kruppel B D . The analytical model...ChIP-seq profiles. We plotted heatmaps for the correlation ( A ) and (...ChIP-seq peaks is that these two TFs bind cooperatively to the genome....ChIP-seq profiles when using the binding motif from JASPAR database; see...ChIP-seq data set and the model predicted profiles....ChIP-seq data set we selected the regions where the mean ChIP-seq signal...ChIP-seq profiles, we backwards inferred copy number and specificity for...ChIP-seq data sets is similar to the one found in ....ChIP-seq data is not available. While the ChIP-seq profiles were generated...ChIP-seq signal higher than the thresholds are listed in Table ... The binding of transcription factors (TFs) is essential for gene expression. One important characteristic is the actual occupancy of a putative binding site in the genome. In this study, we propose an analytical model to predict genomic occupancy that incorporates the preferred target sequence of a TF in the form of a position weight matrix (PWM), DNA accessibility data (in case of eukaryotes), the number of TF molecules expected to be bound specifically to the DNA and a parameter that modulates the specificity of the TF. Given actual occupancy data in form of ChIP-seq profiles, we backwards inferred copy number and specificity for five Drosophila TFs during early embryonic development: Bicoid, Caudal, Giant, Hunchback and Kruppel. Our results suggest that these TFs display thousands of molecules that are specifically bound to the DNA and that, while Bicoid and Caudal display a higher specificity, the other three transcription factors (Giant, Hunchback and Kruppel) display lower specificity in their binding (despite having PWMs with higher information content). This study gives further weight to earlier investigations into TF copy numbers that suggest a significant proportion of molecules are not bound specifically to the DNA.