Contributors: Daniel A. Gilchrist, David C. Fargo, Karen Adelman

Date: 2009-08-01

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...NimbleGen HD2 Drosophila whole genome arrays) or ChIP-seq (Illumina Genome...ChIP-chip and ChIP-seq), or unmappable regions (ChIP-seq). ...or ChIP-seq reads sequenced with the Illumina Genome Analyzer (green line...and ChIP-seq experiments. Following experimental manipulation (yellow ...ChIP-seq experiments. Following experimental manipulation (yellow boxes...ChIP-seq...Drosophila whole genome arrays) or ChIP-seq (Illumina Genome Analyzer ...ChIP-seq. Drosophila S2 cells were crosslinked, sonicated, and total Pol...ChIP-seq to study the regulation of gene expression: Genome-wide localization...ChIP-seq with genetic, chemical, and experimental manipulation to obtain...and ChIP-seq. Drosophila S2 cells were crosslinked, sonicated, and total...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: Leighton J. Core, Joshua J. Waterfall, Daniel A. Gilchrist, David C. Fargo, Hojoong Kwak, Karen Adelman, John T. Lis

Date: 2012-10-25

Drosophila....and C) Pol II ChIP-seq and NELF ChIP-chip data (Gilchrist et al., 2010...ChIP-seq for total Pol II (α-Rpb3) is shown in green (reads/25 bp bin)...showing Pol II Chip-seq (green) and GRO-seq (red) with y axis in reads...Drosophila putative enhancers. ...Drosophila promoters is transcriptionally engaged; very little exists ...and Pol II ChIP-seq, (B) small-RNA-seq to ChIP-seq, and (C) GRO-seq to...II-bound by ChIP-seq (n = 3,168) in red. rho is Spearman's correlation...Drosophila, and this specificity in orientation correlates with directional...ChIP-seq and NELF ChIP-chip data (Gilchrist et al., 2010), respectively...ChIP-seq, (B) small-RNA-seq to ChIP-seq, and (C) GRO-seq to small-RNA-seq.... ChIP-seq for total Pol II (α-Rpb3) is shown in green (reads/25 bp bin...ChIP-seq (n = 3,168) in red. rho is Spearman's correlation coefficient ... Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.

Contributors: Jian Zhou, Wangjie Yu, Paul E. Hardin

Date: 2015-01-01

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: Georgi K. Marinov, Jie Wang, Dominik Handler, Barbara J. Wold, Zhiping Weng, Gregory J. Hannon, Alexei A. Aravin, Phillip D. Zamore, Julius Brennecke, Katalin Fejes Toth

Date: 2015-03-23

ChIP-seq dataset and high enrichment of H3K9me3 (from Muerdter et al.,...ChIP-seq and background (input) data from Huang et al. showing (1) unique...Piwi ChIP-seq dataset. (B) Strong apparent enrichment over individual ...ChIP-seq enrichment observed over some individual transposable elements...ChIP-seq and input RPM levels over the transposon consensus sequences ...Drosophila. Their study also reported that loss of Piwi causes widespread...ChIP-seq data from Huang et al. (red) and H3K9me3 data from Muerdter et...ChIP-seq) reveals the genome-wide sites of occupancy by Piwi, a piRNA-guided ... Huang et al. (2013) recently reported that chromatin immunoprecipitation sequencing (ChIP-seq) reveals the genome-wide sites of occupancy by Piwi, a piRNA-guided Argonaute protein central to transposon silencing in Drosophila. Their study also reported that loss of Piwi causes widespread rewiring of transcriptional patterns, as evidenced by changes in RNA polymerase II occupancy across the genome. Here we reanalyze their data and report that the underlying deep-sequencing dataset does not support the authors’ genome-wide conclusions.

Contributors: Rajprasad Loganathan, Joslynn S. Lee, Michael B. Wells, Elizabeth Grevengoed, Matthew Slattery, Deborah J. Andrew

Date: 2016-01-01

salivary gland based on ChIP-Seq. ...ChIP-seq analysis were linked to changes in gene expression, we performed...ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically...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 and microarray (774 targets activated and 1176 repressed by ... on ChIP-Seq. ...Drosophila embryonic salivary gland through transcriptional activation...ChIP-seq analysis identifies Rib binding sites in salivary gland cells... ChIP-seq and microarray data confirms significant expression change in...Drosophila salivary gland via a diverse array of targets through both ...ChIP-Seq. ...sites. ChIP-seq datasets were obtained from samples using two different...Drosophila...Drosophila salivary gland (SG). Notably, the morphogenetic changes in ... 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: David A. Orlando, Mei Wei Chen, Victoria E. Brown, Snehakumari Solanki, Yoon J. Choi, Eric R. Olson, Christian C. Fritz, James E. Bradner, Matthew G. Guenther

Date: 2014-01-01

orange). ChIP-seq signals were calculated based on traditional or Drosophila-reference-normalized... of ChIP-Seq Data (A) Schematic representation of a typical ChIP-seq data...ChIP-Seq Normalization Reveals Global Modulation of the Epigenome...ChIP-seq peaks (blue). A comparison of the peaks as a percentage of the...ChIP-seq in the presence of the reference Drosophila epigenome (orange...ChIP-Seq Data (A) Schematic representation of a typical ChIP-seq data ...ChIP-seq) is a prevailing methodology used to investigate chromatin-based...subjected to ChIP-seq in the presence of the reference Drosophila epigenome...reveals ChIP-seq peaks (blue). A comparison of the peaks as a percentage...ChIP-seq experiments enables the discovery of disease-relevant changes...ChIP-seq signals were calculated based on traditional or Drosophila-reference-normalized...ChIP-seq reads aligning to either test (human, blue) or Drosophila (reference ... 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: Jun-ichi Satoh

Date: 2014-01-01

ChIP-Seq...ChIP-Seq-based NF-κB p65 target genes involves diverse immune functions...ChIP-Seq-based NF-κB p65 target genes. Entrez Gene IDs of 918 ChIP-Seq-based...ChIP-Seq-based NF-κB p65 genes. ...of ChIP-Seq-based NF-κB p65 target genes. Entrez Gene IDs of 918 ChIP-Seq-based...version of this article.) ...networks of ChIP-Seq-based NF-κB p65 target genes. Entrez Gene IDs of 9...article.) ...ChIP-Seq peaks in the intronic region of target genes. The genomic location... 918 ChIP-Seq-based NF-κB p65 genes. ... 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: Sarah E. Lacher, Joslynn S. Lee, Xuting Wang, Michelle R. Campbell, Douglas A. Bell, Matthew Slattery

Date: 2015-01-01

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...Nrf2 ChIP-seq peak and ARE sequence at the SQSTM1 locus; gene models and...ChIP-seq peak and ARE sequence at the VCP locus. Bottom: Nrf2 ChIP-seq...Drosophila to human. The ancient network consists of canonical antioxidant...ChIP-seq signal from LCL cells treated with DMSO or SFN as indicated. ...Nrf2 ChIP-seq peak and ARE sequence at the Keap1 locus; gene models and...cells. (A) Human Nrf2 ChIP-seq signal from LCL cells treated with DMSO...human cells. (A) Nrf2 ChIP-seq data from human LCL cells treated with SSFN. Top: Nrf2 ChIP-seq peak and ARE sequence at the PSMA3 locus. Middle...Drosophila...Drosophila and humans. These data have allowed us to construct the deeply...ChIP-seq data from human LCL cells treated with SFN. Top: Nrf2 ChIP-seq ... 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: Yingchun Liu, Zhen Shao, Guo-Cheng Yuan

Date: 2010-07-01

Drosophila but their promoter sequences show different properties. (A)...and Drosophila but their promoter sequences show different properties....ChIP-chip/seq experiments in mouse ESCs. The statistically significant...by ChIP-chip/seq experiments in mouse ESCs. The statistically significant...Drosophila PcG targets are associated with different sequence signatures...ChIP-chip/seq data. The enrichment score is defined as the ratio of the...information is based on ChIP-chip/seq data. The enrichment score is defined...Drosophila and found that they are strikingly different. Our predictions...legend. ...T...ChIP-seq...Drosophila. ...PcG status in Drosophila. ... Polycomb group (PcG) proteins are important epigenetic regulators, yet the underlying targeting mechanism in mammals is still poorly understood. We have developed a computational approach to predict genome-wide PcG target genes in mouse embryonic stem cells. We use TF binding and motif information as predictors and apply the Bayesian Additive Regression Trees (BART) model for classification. Our model has good prediction accuracy. The performance can be mainly explained by five TF features (Zf5, Tcfcp2l1, Ctcf, E2f1, Myc). Our analysis of H3K27me3 and gene expression data suggests that genomic sequence is highly correlated with the overall PcG target plasticity. We have also compared the PcG target sequence signatures between mouse and Drosophila and found that they are strikingly different. Our predictions may be useful for de novo search for Polycomb response elements (PRE) in mammals.

Contributors: Yan Zheng, Yuanyuan Zha, Robbert M. Spaapen, Rebecca Mathew, Kenneth Barr, Albert Bendelac, Thomas F. Gajewski

Date: 2013-10-01

of two ChIP-Seq analyses. (B) Consensus sequence of Egr2 binding sites...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...ChIP-Seq reveal novel biologic targets in T cell anergy...identified by ChIP-Seq analysis. (A) Distribution of the types of Egr2...ChIP-Seq number one and two respectively. The locations confirmed by ChIP ... 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.