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ChIPseq on H3K27me3 and H2AK119Ub on Xist-tetOP ΔB+C cell line, after 2 days of differentiation, with or without DOX induction

ABSTRACT: Xist RNA has been established as the master regulator of X-chromosome inactivation in female eutherian mammals but its mechanism of action remain unclear. By creating novel Xist mutants at the endogenous locus in mouse embryonic stem cells, we dissect the role of most of the conserved A-F repeats. We find that transcriptional silencing can be uncoupled from Polycomb repressive complex 1 and 2 (PRC1/2) recruitment, which requires repeats B and C. Xist ΔB+C specifically looses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners are largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct structural modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways, with Polycomb recruitment only being of moderate relevance in the initiation of silencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE123741 | GEO | 2019/08/28

REPOSITORIES: GEO

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RNAseq on Xist-tetOP FL, ΔA, ΔB+C cell lines, with 2 days of differentiation, without or with DOX induction

Project description:Xist RNA has been established as the master regulator of X-chromosome inactivation in female eutherian mammals but its mechanism of action remain unclear. By creating novel Xist mutants at the endogenous locus in mouse embryonic stem cells, we dissect the role of most of the conserved A-F repeats. We find that transcriptional silencing can be uncoupled from Polycomb repressive complex 1 and 2 (PRC1/2) recruitment, which requires repeats B and C. Xist ΔB+C specifically looses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners are largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct structural modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways, with Polycomb recruitment only being of moderate relevance in the initiation of silencing.

2019-08-28 | GSE123742 | GEO

4sU-seq analysis of Xist-mediated chromosomal silencing

Project description:The Polycomb repressive complexes PRC1 and PRC2 play a key role in chromosome silencing by Xist RNA. Previously we have shown that initation of Polycomb recruitment is mediated by the PCGF3/5-PRC1 complex, which catalyses chromosome-wide H2A ubiquitylation (H2AK119u1), signalling recruitment of other PRC1 complexes, and PRC2. However, the molecular basis for PCGF3/5-PRC1 recruitment by Xist RNA remains unknown. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt element encompassing the Xist B-repeat element. XR-PID is required for Polycomb recruitment by Xist RNA, Xist-mediated chromosome silencing. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking the B-repeat is sufficient for Xist-dependent Polycomb recruitment. Our findings resolve the molecular mechanism for Polycomb recruitment by Xist RNA, providing key insights into chromatin modification by non-coding RNA.

2017-12-08 | GSE99924 | GEO

RNA-seq of PCGF5 knockout and PCGF3/5 double knockout mouse embryonics stem cells bearing doxycycline-inducible Xist transgene on chromosome 16

Project description:In vertebrates, the catalytic core of PRC1 comprises RING1A/B and one of six homologues of the Polycomb group RING finger (PCGF) protein. Unique characteristics of PCGF1-6 in turn define distinct subunit assemblies, broadly subdivided into canonical and non-canonical PRC1 complexes. This RNA-seq experiment aimed to test the hypothesis that Pcgf3/5 gene knockout abrogates Xist mediated gene repression. In this experiment, Pcgf3fl/flPcgf5-/- and Pcgf3-/-Pcgf5-/- mESCs bearing Xist transgene on chromosome 16 (clone C3F8) were cultured in differentiating conditions: plated on non-gelatinized TC dish in ES medium without LIF for 72 hours with (+dox) and without doxycycline. RNA was isolated from 4 biological replicates. In analysed cells chromosome 16 bears inducible Xist and the whole study investigates role of PCGF3 and PCGF5 in Xist-dependednt gene silencing, therefore both bam files containing alignments and counts per gene were uploaded to ArrayExpress only for chromosome 16. Genome-wide data can be provided upon request.

2018-04-04 | E-MTAB-5642 | biostudies-arrayexpress

Calibrated ChIP-seq for Xist-mediated polycomb recruitment

Project description:Calibrated ChIP-seq was employed to quantitatively address the questions of how and where Xist recruits polycomb modifications (H2AK119ub and H3K27me3) to the inactive chromosome. 'Dox' samples were performed after 24 hours of induced Xist expression. Suz12 is the core enzymatic component of PRC2 while Pcgf3/5 are non-canonical PRC1 components which initiate the polycomb cascade in Xist-mediated chromosome inactivation. Xist PID region, encompassing the B-repeat and part of C-repeat, was characterized to bind hnRNPK directly, then recruit the Pcgf3/5. Notably, Suz12 KO does not affect Xist-dependent H2AK119ub deposition too much in a 24hour Xist induction.

2019-06-17 | GSE119599 | GEO

ChIPseq on H3K27ac, H3K27me3 and H2AK119Ub on TXY cell line

Project description:During development, changes in gene transcription are accompanied by changes in chromatin modification but the order and causality of events often remain unclear. Here we address this question using X-chromosome inactivation (XCI), which entails chromosome-wide gene silencing and heterochromatin formation. We initiate XCI in female, mouse embryonic stem cells by inducing Xist expression and monitor subsequent changes in transcription and chromatin modification by allele-specific TTseq and ChIPseq respectively. An unprecedented temporal resolution has enabled us to define early alterations in chromatin that are induced upon Xist RNA coating. Xist-induced repression begins with histone deacetylation, which involves the histone deacetylase HDAC3 and occurs before efficient loss of H3K4me3 and H3K4me1 modifications. Polycomb-associated repressive histone marks accumulate rapidly, starting with PRC1-associated H2AK119Ub and followed by PRC2-associated H3K27me3. However, polycomb accumulates initially at large premarked domains, some of which correspond to Xist entry sites, and then spreads into genes. We also show that spreading can only ensue when transcriptional silencing has occurred. These results establish a detailed epigenomic time course for XCI and reveal a hierarchy of events with chromatin playing an important role in transcriptional silencing of the X chromosome.

2019-01-01 | GSE116477 | GEO

TTseq on TX1072 during time course of DOX induction

2019-01-01 | GSE116479 | GEO

Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X

Project description:During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/ Polycomb spreading, silencing, and changes in chromosome architecture.

2019-03-01 | GSE107217 | GEO

Functional Landscape of PCGF Proteins Reveals Both RING1A/B Dependent and Independent Specific Activities

Project description:Polycomb repressive complexes 1 and 2 (PRC1 and PCR2) control cell identity by establishing facultative heterochromatin repressive domains at common sets of target genes. PRC1, which deposits H2Aub1 through the E3 ligases RING1A/B, forms six biochemically distinct subcomplexes containing one type of PCGF protein (PCGF1–6); it is unclear whether these subcomplexes also have specific activities. Here we show that PCGF1 and PCGF2 largely compensate for each other, but that the other PCGF proteins have high levels of specificity for distinct target genes, with little functional crosstalk. PCGF2 associates with transcription repression, while PCGF3 and PCGF6 associate with actively transcribed genes. Notably, PCGF3 and PCGF6 complexes can assembly and be recruited to several active sites independently of RING1A/B activity (therefore, of PRC1). For chromatin recruitment, the PCGF6 complex requires the combinatorial activities of its MGA-MAX and E2F6-DP1 subunits, while PCGF3 requires an interaction with the USF1 DNA binding transcription factor.

2019-04-29 | GSE122715 | GEO

Xist Repeats A and B account for two distinct phases of X-inactivation establishment

Project description:X-chromosome inactivation (XCI) is a global silencing mechanism by which XX and XY mammals equalize X-linked gene dosages. XCI begins with an establishment phase during which Xist RNA spreads and induces de novo heterochromatinization across a female X chromosome, and is followed by a maintenance phase when multiple epigenetic pathways lock down the inactive X (Xi) state. Involvement of Polycomb repressive complexes 1 and 2 in XCI has been intensively studied, but with conflicting conclusions regarding their recruitment and role in Xi silencing. Here we reveal that establishment of XCI has two phases and reconcile the roles that Xist Repeats A and B play in gene silencing and Polycomb recruitment. Repeat A initiates both processes, whereas Repeat B bolsters or stabilizes them thereafter. Once established, XCI no longer requires Repeat A during maintenance. These findings integrate disparate studies and present a unified view of Xist?s role in Polycomb-mediated silencing.

2020-06-15 | GSE135389 | GEO

VAL1 acts as an assembly platform co-ordinating co-transcriptional repression and chromatin 2 regulation at Arabidopsis FLC

Project description:• Polycomb (PcG) regulation is crucial for development across eukaryotes, but how PcG targets are specified is still incompletely understood. The slow timescale of cold-induced Polycomb Repressive Complex 2 silencing during vernalization at Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate the sequence of events. Association of the DNA binding protein VAL1 to an FLC intronic RY motif within the PcG nucleation region is an important step. VAL1 is associated in vivo with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and Polycomb Repressive Complex 1 (PRC1). Here, we show that ASAP and PRC1 functions are necessary for co-transcriptional repression and chromatin regulation during FLC silencing. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulation increases at the nucleation region upon exposure to cold, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2Aub is thus involved in the transition to epigenetic silencing at FLC facilitating H3K27me3 accumulation, which in turn is necessary for long-term epigenetic memory. Overall, our work highlights the importance of the DNA sequence-specific binding protein VAL1 as an assembly platform coordinating the co-transcriptional repression and chromatin regulation necessary for the epigenetic silencing of FLC.

2022-10-13 | PXD036548 | Pride

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