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P300/CBP sustains Polycomb silencing by non-enzymatic functions (ChIP-seq on RNAi-treated Drosophila S2 cells)

ABSTRACT: Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here we show that the p300/CBP co-activator is associated with two-thirds of PcG-regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE186721 | GEO | 2022/10/06

REPOSITORIES: GEO

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p300/CBP sustains Polycomb silencing by non-enzymatic functions (ChIP-seq on inhibitor-treated Drosophila S2 cells)

Project description:Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here we show that the p300/CBP co-activator is associated with two-thirds of PcG-regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

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p300/CBP sustains Polycomb silencing by non-enzymatic functions (ChIP-seq on inhibitor-treated mouse ES cells)

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Project description:The Polycomb group (PcG) and Trithorax group (TrxG) proteins are key epigenetic regulators controlling silenced and active states of genes in multicellular organisms, respectively. In Drosophila PcG/TrxG proteins are recruited to chromatin via binding to specialized DNA-elements termed Polycomb Response Elements (PREs). While precise mechanisms of PcG/TrxG proteins recruitment remains unknown, the important role is suggested to belong to sequence specific DNA-binding factors. Here, using affinity purification coupled with high throughput mass spectrometry (IP/MS), we isolated factors associated with Combgap, Psq, Zeste and Adf1 PRE DNA-binding Drosophila proteins. As a control we used unspecific IgG. For affinity purification the nuclear extract from Drosophila S2 cells and polyclonal antibodies against Combgap, Psq, Zeste and Adf1 were used.

2024-05-22 | PXD029459 | Pride

The Polycomb group mutant esc leads to augmented the levels of paused Pol II in the Drosophila embryo.

Project description:The role of Polycomb group (PcG) was studied on RNA Polymerase II (Pol II) pausing phenomenon. Wild type and Polycomb mutant (esc) embryos were used for ChIP-Seq experiments using Pol II and histone methylation antibodies. Enhanced Pol II occupancy was observed for thousands of genes in esc mutant embryos, including genes not known to be bound by PRC2 or having H3K27me3 associated. Most of these genes exhibit a concomitant reduction in H3K27me3 and increase in H3K4me3 at promoter-proximal regions. Silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into âpoisedâ genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants. ChIP-Seq data was generated for antibodies against Pol-II, H3K4me3, H3K27me3 in both wild type and esc mutant Drosophila embryos (0-2hr, 8-12hr, and 18-24hr embryos). In addition GRO-Seq data was generated for 18-24hr esc mutant embryos.

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The Polycomb group mutant esc leads to augmented the levels of paused Pol II in the Drosophila embryo.

2011-06-15 | GSE28833 | GEO

Context dependent role of Pho binding sites in Polycomb complex recruitment in Drosophila

Project description:Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, “PcG recruiters”, including Pho, Spps, Cg, GAF and many others. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here we directly addressed the importance of Pho binding sites in two engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, two PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho.

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A PRE loop at the dac locus acts as a topological chromatin structure that restricts and specifies enhancer promoter communication

Project description:3D genome folding plays a fundamental role for the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CREs involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TADs) called Polycomb domains. PREs can establish looping contacts that stabilize gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine tune gene expression is unknown. Using CRISPR/Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C, and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function: it restricts enhancer promoter communication and contributes to enhancer specificity.

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