ChIP-Seq of chromatin marks at distal enhancers in Mouse Embryonic Stem Cells and adult tissues.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE24164 | GEO | 2010/10/31
SECONDARY ACCESSION(S): PRJNA133681
REPOSITORIES: GEO
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Project description:Enhancers are enriched by histone H3 lysine 4 mono methylation (H3K4me1). To gain insight into the relation between enhancers and developmental state, chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) was performed in several cell types to determine the genome-wide binding targets of H3K4me1 and several other possible enhancer associated modifications. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A chromatin IP against histone H3 or whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed mainly using antibodies against the chromatin marks H3K4me1 and H3K27ac.
2010-10-31 | E-GEOD-24164 | biostudies-arrayexpress
Project description:Enhancers act to regulate cell type specific gene expression by facilitating the transcription of target genes. In mammalian cells active or primed enhancers are commonly marked by monomethylation of Histone H3 at lysine 4 (H3K4me1) in a cell-type specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals has been unclear. In the present study, we conducted SILAC Mass-spec experiments with mono-nucleosomes and identified multiple H3K4me1 associated proteins, including proteins involved in chromatin remodeling. We demonstrate that H3K4me1 augments the association of the chromatin remodeling complex BAF to enhancers in vivo. Furthermore we show that in vitro, H3K4me1 nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of a BAF component BAF45c further reveal that monomethylation, but not trimethylation, is accommodated in this protein’s H3K4 binding site. Our results suggest that H3K4me1 plays an active role at enhancers by facilitating the binding of the BAF complex and possibly other chromatin regulators.
2018-10-23 | PXD007942 | Pride
Project description:FOXA1 is a pioneer factor that is important in hormone dependent cancer cells to stabilise nuclear receptors, such as estrogen receptor (ER) to chromatin. FOXA1 binds to enhancers regions that are enriched in H3K4mono- and dimethylation (H3K4me1, H3K4me2) histone marks and evidence suggests that these marks are requisite events for FOXA1 to associate with enhancers to initate subsequent gene expression events. However, exogenous expression of FOXA1 has been shown to induce H3K4me1 and H3K4me2 signal at enhancer elements and the order of events and the functional importance of these events is not clear. We performed a FOXA1 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells in order to identify FOXA1 interacting partners and we found histone-lysine N-methyltransferase (MLL3) as the top FOXA1 interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition, in line with our observation that MLL3 associates with an enhancer specific protein. We performed MLL3 ChIP-seq in breast cancer cells and unexpectedly found that MLL3 binds mostly at non-promoter regions enhancers, in contrast to the prevailing hypothesis. MLL3 was shown to occupy regions marked by FOXA1 occupancy and as expected, H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. Motif analysis and subsequent genomic mapping revealed a role for Grainy head like protein-2 (GRHL2) which was shown to co-occupy regions of the chromatin with MLL3. Regions occupied by all three factors, namely FOXA1, MLL3 and GRHL2, were most enriched in H3K4me1. MLL3 silencing decreased H3K4me1 at enhancer elements, but had no appreciable impact on H3K4me3 at enhancer elements. We identify a complex relationship between FOXA1, MLL3 and H3K4me1 at enhancers in breast cancer and propose a mechanism whereby the pioneer factor FOXA1 can interact with a chromatin modifier MLL3, recruiting it to chromatin to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.
2016-12-12 | PXD005253 | Pride
Project description:Enhancers act to regulate cell type specific gene expression by facilitating the transcription of target genes. In mammalian cells active or primed enhancers are commonly marked by mono-methylation of Histone H3 at lysine 4 (H3K4me1) in a cell-type specific manner. While this histone modification regulates enhancer-dependent transcription programs in mammals, the exact mechanism has remained unclear. In the current study, we conducted SILAC Mass-spec experiments with mono-nucleosomes and identified multiple H3K4me1 associated proteins, including the mediator complex, Cohesin complex, as well as proteins involved in chromatin remodeling. We demonstrate that binding of these protein complexes to enhancers in vivo depends on H3K4me1. We have characterized the PHD domains of DPF1-3 and PHF10 and identified the amino acids involved in binding to H3K4me1. In addition, we discovered that H3K4me1 nucleosomes are more efficiently remodeled by BAF complex in vitro. Our results suggest that H3K4me1 plays an active role at enhancers through binding of a diverse set of chromatin regulators.
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Project description:Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation. Chromatin immuno-precipitations of p300, PU.1 and mono-methylated H3 lysine 4 followed by multiparallel sequencing were performed in bone marrow-derived macrophages. Experiments for p300 and PU.1 were also carried out in cells treated for 2hrs with lipopolysaccharide (LPS). In case of p300, reads obtained from two different biological replicates were merged.
2010-03-05 | E-GEOD-19553 | biostudies-arrayexpress
Project description:We used genome-wide sequencing methods to study stimulus-dependent enhancer function in neurons. We identified ~12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Remarkably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 that is mono-methylated at lysine 4 (H3K4me1). The level of eRNA expression at neuronal enhancers positively correlates with the level of mRNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis. Examination of genome-wide binding of three types of modified histones, four transcription factors, and RNA polymerase II (ChIP-Seq), as well as RNA expression (RNA-Seq) before and after membrane depolarization via application of extracellular potassium.
2010-04-14 | E-GEOD-21161 | biostudies-arrayexpress
Project description:Combinations of post-translational histone modifications shape the chromatin landscape during cell development in eukaryotes. However, little is known about the modifications exactly delineating functionally engaged regulatory elements. For example, although histone H3 lysine 4 mono-methylation (H3K4me1) indicates the presence of transcriptional gene enhancers, it does not provide clear-cut information about their actual position and stage-specific activity. Histone marks were, therefore, studied here at genomic loci differentially expressed in early stages of T-lymphocyte development. The concomitant presence of the three H3K4 methylation states (H3K4me1/2/3) was found to clearly reflect the activity of bona fide T-cell gene enhancers. Globally, gain or loss of H3K4me2/3 at distal genomic regions correlated with, respectively, the induction or the repression of associated genes during T-cell development. In the Tcrb gene enhancer, the H3K4me3-to-H3K4me1 ratio decreases with the enhancer’s strength. Lastly, enhancer association of RNA-polymerase II (Pol II) correlated with the presence of H3K4me3 and Pol II accumulation resulted in local increase of H3K4me3. Our results suggest the existence of functional links between Pol II occupancy, H3K4me3 enrichment and enhancer activity. ChIP-seq for H3K4me1, H3K4me3 and Pol II was performed from DRag thymocytes or DRag thymocytes from CD3 stimulated mice
2014-03-07 | E-GEOD-55635 | biostudies-arrayexpress
Project description:Primed enhancers are marked by histone H3K4 mono-methylation (H3K4me1), and the conversion to active enhancers involves acetylation of histone H3K27 (H3K27Ac). However, whether active enhancers are regulated remains unclear. Here we report a biochemical complex consisting of a potential chromatin reader (RACK7) and a histone demethylase (KDM5C) that occupies many active enhancers in a breast cancer cell line. Loss of RACK7 or KDM5C results in hyperactive enhancers marked by H3K4me3 and H3K27Ac, and characterized by an increased eRNA transcription and elevated expression of nearby genes. Loss of RACK7 or KDM5C also leads to increased cell invasion and migration, and enhanced tumor growth. We propose that RACK7/KDM5C functions as an enhancer âbrakeâ to ensure appropriate enhancer activities in the cell. Our findings provide important insight into histone H3K4 methylation dynamics at enhancers and reveal a molecular mechanism that controls the activities of active enhancers, which when compromised, can contribute to tumorigenesis. mRNA-seq of parental and RACK7-KO ZR-75-30 cells
2016-04-07 | E-GEOD-71325 | biostudies-arrayexpress
Project description:Primed enhancers are marked by histone H3K4 mono-methylation (H3K4me1), and the conversion to active enhancers involves acetylation of histone H3K27 (H3K27Ac). However, whether active enhancers are regulated remains unclear. Here we report a biochemical complex consisting of a potential chromatin reader (RACK7) and a histone demethylase (KDM5C) that occupies many active enhancers in a breast cancer cell line. Loss of RACK7 or KDM5C results in hyperactive enhancers marked by H3K4me3 and H3K27Ac, and characterized by an increased eRNA transcription and elevated expression of nearby genes. Loss of RACK7 or KDM5C also leads to increased cell invasion and migration, and enhanced tumor growth. We propose that RACK7/KDM5C functions as an enhancer âbrakeâ to ensure appropriate enhancer activities in the cell. Our findings provide important insight into histone H3K4 methylation dynamics at enhancers and reveal a molecular mechanism that controls the activities of active enhancers, which when compromised, can contribute to tumorigenesis. nascent RNA-seq of parental and RACK7-KO cells
2016-04-07 | E-GEOD-71326 | biostudies-arrayexpress
Project description:Mono-methylation of histone H3 on lysine 4 (H3K4me1) and acetylation of histone H3 on lysine 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and enhancers. Although in yeast all H3K4 methylation patterns including H3K4me1 are implemented by Set1/COMPASS, there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of mammalian Mll3/4, can function as a major H3K4 mono-methyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac in various tissues. Assays with the cut wing margin enhancer imply a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrates that Trr is required for H3K4me1 and H3K27ac on chromatin signatures that resemble the histone modification patterns described for enhancers. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 mono-methyltransferase Trr, and the H3K27 demethylase, UTX, cooperate to regulate the transition from inactive/poised to active enhancers. ChIP-seq of Trr, LPT, UTX in Drosophila S2 Cells. ChIP-seq of H3K4me1, H3K4me3, H3K27ac, H3K27me3 in WT and Trr knock-down Drosophila S2 cells. ChIP-seq of H3K4me1, H3K27me3 in LPT knock-down Drosophila S2 cells. ChIP-seq of LPT and UTX in Trr knock-down Drosophila S2 cells. ChIP-seq of H3K4me1 and H3K27me3 in MLL1(+/+), MLL1(-/-), MLL3(+/+), and MLL3(-/-) Mouse Embryonic Fibroblasts (MEFs).
2012-12-01 | E-GEOD-41440 | biostudies-arrayexpress