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Systematic Analysis of Allelic Silencing Defines the Interplay of Key Pathways in Xist-mediated Chromosome Inactivation

ABSTRACT: Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome silencing through the stepwise recruitment of factors that modify underlying chromatin structure. Whilst considerable progress has been made towards defining key silencing factors and the elements to which they bind, their relative contribution to silencing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in ES cell-based models. We show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of low expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15-Mettl3/14 m6A-methyltransferase complex, previously proposed to have a central role, make at most a minor contribution to gene silencing. We integrate our findings in a comprehensive model for Xist-mediated chromosome silencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE119602 | GEO | 2019/06/17

REPOSITORIES: GEO

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Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

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2016-07-24 | E-GEOD-80510 | biostudies-arrayexpress

m6A RNA methylation promotes XIST-mediated transcriptional repression

Project description:XIST is a long non-coding RNA (lncRNA) that mediates transcriptional silencing of X chromosome genes. Here we show that XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues, a reversible base modification whose function in lncRNAs is unknown. We show that m6A formation in XIST, as well as cellular mRNAs, is mediated by RBM15 and its paralog RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in methylation of adenosines in adjacent m6A consensus motifs. Furthermore, knockdown of RBM15 and RBM15B, or knockdown of the m6A methyltransferase METTL3 impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTHDC1 preferentially recognizes m6A in XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression. Three to four biological HEK293T replicates were used to perform iCLIP of endogenous YTH proteins, RBM15, and RBM15B. Crosslinking induced truncations were identified using CIMS-CITS pipeline.

2016-09-07 | E-GEOD-78030 | biostudies-arrayexpress

A 5' fragment of Xist can sequester the RNA of adjacent genes on chromatin

Project description:Xist requires Repeat-A, a protein binding module in its first two kilobases (2kb), to repress transcription. We report that in mouse embryonic stem cells (ESCs), when expressed as a standalone transcript, the first 2kb of Xist (Xist-2kb) represses the expression of adjacent genes not at the transcriptional level, but by sequestering their mRNA on chromatin. Sequestration does not spread beyond adjacent genes, requires the same sequence elements in Repeat-A that full-length Xist requires to repress transcription, and can be induced by lncRNAs whose sequence composition is related to Xist-2kb. We did not detect local sequestration by full-length Xist, but did detect it by mutant forms of Xist with attenuated transcriptional silencing capability. SPEN and RBM15, Repeat-A binding proteins required for Xist-induced transcriptional silencing, also bound Repeat-A in Xist-2kb, but neither protein was necessary for sequestration. Thus, in mouse ESCs, expression of a 5¢ fragment of Xist that contains Repeat-A can repress gene expression by sequestering nearby mRNAs on chromatin, but it does not induce transcriptional silencing. Rather, Xist-induced transcriptional silencing requires synergy between Repeat-A and additional sequence elements in Xist. These results highlight an important gap in our understanding of the mechanisms through which Repeat-A functions within Xist. We discuss how the mechanisms that underpin sequestration may be relevant to the biology of Xist and of other lncRNAs.

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Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing [CLIP-seq]

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Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

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RBM15 CoIP in mouse Embryonic Stem cells

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2020-02-07 | PXD017348 | Pride

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

SPEN is a molecular integrator that bridges Xist with transcriptional and epigenetic control of X-chromosome inactivation

Project description:Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN/SHARP, whose knockdown has been associated with deficient XCI at multiple loci. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. On the other hand, SPEN is dispensable for maintenance of XCI in neural progenitor cells, although it significantly dampens expression of genes that escape from XCI. During initiation of XCI, we show by live-cell imaging and CUT&RUN approaches that SPEN is immediately recruited to the X chromosome upon Xist up-regulation, where it is targeted to enhancers and promoters of actively transcribed genes. SPEN rapidly disengages from chromatin once silencing is accomplished, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC (SPEN paralog and ortholog C-terminal) domain as a major effector of SPEN’s gene silencing function, and show that artificial tethering of SPOC to Xist RNA is sufficient to mediate X-linked gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.

2021-04-19 | PXD015699 | Pride

4sU-seq analysis of Xist-mediated chromosomal silencing

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