ChIP for H3K27me3 in Murine ES Cells: wild type and Ring1B-/- cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE20213 | GEO | 2010/05/14
SECONDARY ACCESSION(S): PRJNA125727
REPOSITORIES: GEO
Similar Datasets
Project description:Native ChIP on chip for H3K27me3 in murine ES cells comparing WT and Ring1B-/- cells. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo. Biological replicates: 3 independently grown, harvested,preplated, micrococcal nuclease digested and ChIP for H3K27me3. 5 Technical replicates.
2010-05-14 | E-GEOD-20213 | biostudies-arrayexpress
Project description:ChIP on chip for H3K27me3 in murine ES cells comparing Undifferentiated and Day 3 differentiated. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo. Biological replicates: 3 independently grown, harvested, micrococcal nuclease digested and ChIP for H3K27me3. 6 Technical replicates.
2010-05-14 | E-GEOD-20201 | biostudies-arrayexpress
Project description:ChIP on chip for H3K27me3 in murine ES cells comparing Undifferentiated and Day 3 differentiated. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.
2010-05-14 | GSE20201 | GEO
Project description:Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3) respectively. Compared to H3K27me3, localization and role of H2AK119ub1 is not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation. This SuperSeries is composed of the following subset Series: GSE38224: Expression data from Ring1A(-/-);Ring1B(fl/fl);R26::CreERT2 ES cells expressing either of mock, WT or mutant Ring1B construct before or after OHT treatment GSE38504: ChIP-on-chip analysis of Ring1B, Ring1A, H2AK119u1 and H3K27me3 in mouse ES cells Total RNAs were extracted from the respective ES cells, and were subjected to microarray analysis using Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using various antibodies. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions.
2012-06-11 | E-GEOD-38650 | biostudies-arrayexpress
Project description:Polycomb repressive complex PRC1 is essential for gene regulation in numerous cell fate decisions. We show that RING1B (RING2, RNF2) and canonical PRC1 (cPRC1) genes are amplified and overexpressed in breast cancer (BC). Moreover, cPRC1 complexes functionally associate with genes regulated by cell type specific key transcription factors such as estrogen receptor (ER) in ER+ tumor cells and BRD4 in triple negative BC cells. cPRC1 is recruited to active enhancers in a manner independent of PRC2 and RING1B enzymatic activity. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of BC oncogenes but also by regulating chromatin accessibility for oncogenic transcription factors. RING1B recruitment, and thus PRC1 association, to active enhancers occurs in multiple cancers.
2018-10-19 | PXD009570 | Pride
Project description:In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes: the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes. Keywords: cell type comparison Suz12, Ezh2, Ring1b ChIP-Seq in singlicate from mouse embryonic stem (mES) cells. H3K4me3, H3K27me3, H3K36me3, Ezh2 and Ring1b ChIP-Seq in singlicate from human embyonic stem cells (hES; H9).
2008-11-01 | E-GEOD-13084 | biostudies-arrayexpress
Project description:The Polycomb group (PcG) gene products mediate heritable silencing of developmental regulators in metazoans, participating in one of two distinct multimeric protein complexes, the Polycomb repressive complexes-1 (PRC1) and -2 (PRC2). PRC2 catalyses trimethylation of histone H3 at lysine 27 (H3K27) which in turn is thought to provide a recruitment site for PRC1. Recent studies demonstrate that mono-ubiquitylation of histone H2A at lysine 119 is important in PcG mediated silencing with the core PRC1 component Ring1A/B functioning as the E3 ligase8. PRC2 has been shown to share target genes with the core transcription network to maintain embryonic stem (ES) cells including Oct4 and Nanog. Here we identify an essential role for PRC1 in repressing developmental regulators in ES cells, and thereby in maintaining ES cell pluripotency. A significant proportion of the PRC1 target genes are also repressed by Oct4. We demonstrate that engagement of PRC1 and PRC2 at target genes is Oct4-dependent and moreover that Ring1B interacts with Oct4. Collectively these results show that PcG complexes are instrumental in Oct4-dependent repression required to maintain pluripotency of ES cells. This study provides a first functional link between a core ES cell regulator and global epigenetic regulation of the genome. Experiment Overall Design: Eed is a protein included in PRC2 (Polycomb repressor complex). Experiment Overall Design: We generated constitutive Eed KO mouse ES cells and observed gene expression using Affymetrix MOE430.2 microarray. Experiment Overall Design: These results were compared with other KO cells of PRC1 proteins (Ring1A, Ring1B) and other proteins in our study.
2008-04-07 | E-GEOD-10574 | biostudies-arrayexpress
Project description:The chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on the prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to the recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. Genetic ablation of catalytic subunit of the PRC1 complex (RINGA/B) and ChIP-seq analysis of PRC1 and PRC2 components confirmed genome-wide decreases in PRC2 occupancy and H3K27me3 levels at PRC target sites. This activity is restricted to variant PRC1 complexes and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for polycomb domain formation and normal development. Together these observations provide a surprising new PRC1-dependent logic for PRC2 occupancy and polycomb domain formation. RING1A-/-;RING1Bfl/fl ES cells were treated with 800M-BM-5M tamoxifen for 48hours and compared to untreated control cells by ChIP-seq for RING1B, SUZ12, EZH2 and H3K27me3.
2014-05-27 | E-GEOD-55697 | biostudies-arrayexpress
Project description:The established hierarchical model explaining co-occupancy of Polycomb repressor complexes 1 and 2 (PRC 1 and 2) at target loci proposes that the chromodomain of the polycomb protein, a core PRC1 subunit, recognises the H3K27me3 histone modification catalysed by PRC2. We used chromatin immunoprecipitation to analyse PRC1 occupancy at target loci in Eed-/- mouse embryonic stem cells (ESCs) that lack H3K27me3. Occupancy of the core PRC1 proteins Ring1B and Mel18 was strongly reduced, consistent with the hierarchical model. However, levels of H2A ubiquitylation (H2AK119u1), the histone modification catalysed by PRC1, were similar to wild-type cells, suggesting PRC1 recruitment is independent of H3K27me3. ChIP-sequencing analysis of Ring1B occupancy genome wide substantiated this conclusion, demonstrating significant Ring1B levels at polycomb target loci in Eed-/- ESCs. Thus PRC1 and PRC2 are recruited independently to sites that they co-occupy. We conclude that the primary function of H3K27me3 is to increase the residency of PRC1 at target loci and thereby to contribute to the stability of PRC1 mediated silencing. Examination of Ring1B binding in WT, Eed ko and Input of ESCs Examination of CBX7 in WT and Eed ko of ESCs
2011-11-30 | E-GEOD-23716 | biostudies-arrayexpress
Project description:Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3) respectively. Compared to H3K27me3, localization and role of H2AK119ub1 is not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation. This SuperSeries is composed of the SubSeries listed below.
2012-06-12 | GSE38650 | GEO