Contributors: unknown

Date: 2009-02-27

ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide...ChIP-seq), or unmappable regions (ChIP-seq)....ChIP-seq data allow for high-resolution localization of protein–DNA binding...Drosophila S2 cells and binding was detected with NimbleGen HD2 Drosophila...Drosophila S2 cells were crosslinked, sonicated, and total Pol II (Rpb3...Drosophila Whole Genome 2-ChIP sets. Pol II-bound genomic regions were...ChIP-seq experiments. Following experimental manipulation (yellow boxes...Drosophila Whole Genome 2-ChIP sets with average probe spacing of 250bp...ChIP-seq...Drosophila whole genome arrays) or ChIP-seq (Illumina Genome Analyzer ...ChIP-seq. Drosophila S2 cells were crosslinked, sonicated, and total Pol...Drosophila Release 3 Genomic sequence as previously described [4,5,48]...ChIP-seq with genetic, chemical, and experimental manipulation to obtain...ChIP-seq provides information about all mappable sequences located at ... Transcription is a sophisticated multi-step process in which RNA polymerase II (Pol II) transcribes a DNA template into RNA in concert with a broad array of transcription initiation, elongation, capping, termination, and histone modifying factors. Recent global analyses of Pol II distribution have indicated that many genes are regulated during the elongation phase, shedding light on a previously underappreciated mechanism for controlling gene expression. Understanding how various factors regulate transcription elongation in living cells has been greatly aided by chromatin immunoprecipitation (ChIP) studies, which can provide spatial and temporal resolution of protein–DNA binding events. The coupling of ChIP with DNA microarray and high-throughput sequencing technologies (ChIP-chip and ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide maps of Pol II or elongation factor binding sites can now be readily produced. However, while ChIP-chip/ChIP-seq data allow for high-resolution localization of protein–DNA binding sites, they are not sufficient to dissect protein function. Here we describe techniques for coupling ChIP-chip/ChIP-seq with genetic, chemical, and experimental manipulation to obtain mechanistic insight from genome-wide protein–DNA binding studies. We have employed these techniques to discern immature promoter-proximal Pol II from productively elongating Pol II, and infer a critical role for the transition between initiation and full elongation competence in regulating development and gene induction in response to environmental signals.

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: 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: 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: 2013-03-04

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: unknown

Date: 2010-04-06

Drosophila....Drosophila Third Instar Larval Polytene Chromosomes...ChIP-Seq Analysis Reveals that NSL1 and MCRS2 Bind to Promoters Genome...Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed...Drosophila ... Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.

Contributors: unknown

ChIP-Seq...ChIP-Seq data derives novel biological knowledge on multiple levels, from...Drosophila melanogaster genome. Three series of tracks for three TFs are...ChIP-Seq base coverage profiles; (middle) MEME, an EM-based conventional...ChIP-Seq data processed for HOCOMOCO. The existing JASPAR models are shown...ChIP-Seq captures highly specific protein–DNA interactions, such as transcription...ChIP-Seq, has become an industry standard to study a landscape of DNA–protein...ChIP-Seq data set (Guillon et al., 2009). From top to bottom: the results...ChIP-Seq peaks truncated to a certain percent of their lengths around ...ChIP-Seq data. We discuss numerous tasks starting from basic DNA motif ... Nowadays, chromatin immunoprecipitation followed by next-generation sequencing, often referred to as ChIP-Seq, has become an industry standard to study a landscape of DNA–protein interactions in vivo. ChIP-Seq captures highly specific protein–DNA interactions, such as transcription factors (TFs) bound to appropriate binding sites, and sparse patterns formed by different histone marks. In this review, we focus on DNA sequence analysis methods adequate for TF ChIP-Seq data. We discuss numerous tasks starting from basic DNA motif finding and motif discovery as is, further applied to explore various features of experimental data. We show how sequence analysis of ChIP-Seq data derives novel biological knowledge on multiple levels, from individual transcription factor binding sites to genome segments operating as regulatory modules. Finally, we provide an overview of existing software in the field.

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.