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Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome [II]

ABSTRACT: The long non-coding RNA Xist exploits numerous effector proteins to gradually induce gene silencing across the X chromosome. Here, we show that formation of the inactive X (Xi)-compartment is induced by ~50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are dynamic structures that concentrate rapidly recycling proteins on the X by increasing their binding affinity, thereby facilitating their access to the entire chromosome. We find that gene silencing originates at Xist-SMCs and propagates across the entire X chromosome over time. The propagation of silencing is achieved by Polycomb-mediated coalescence of chromatin regions and aggregation of the critical silencing enzyme SPEN, via its intrinsically disordered domains. Our observations suggest a new model for X Chromosome Inactivation, whereby Xist RNA triggers macromolecular crowding of heterochromatinizing proteins at distinct sites to ultimately increase their occupancy throughout the X chromosome. This mechanism enables deterministic gene silencing across an entire chromosome without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene. Our findings uncover a spatial organization mechanism by which few RNA molecules can regulate a broad nuclear compartment through the recruitment and local concentration of dynamic effector proteins, and provide a quantitative framework for studying such compartments.

ORGANISM(S): Mus musculus musculus x Mus musculus castaneus

PROVIDER: GSE181235 | GEO | 2021/10/27

REPOSITORIES: GEO

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Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome [I]

Project description:The long non-coding RNA Xist exploits numerous effector proteins to gradually induce gene silencing across the X chromosome. Here, we show that formation of the inactive X (Xi)-compartment is induced by ~50 locally confined granules, where two Xist RNA molecules nucleate supra-molecular complexes (SMCs) of interacting proteins. Xist-SMCs are dynamic structures that concentrate rapidly recycling proteins on the X by increasing their binding affinity, thereby facilitating their access to the entire chromosome. We find that gene silencing originates at Xist-SMCs and propagates across the entire X chromosome over time. The propagation of silencing is achieved by Polycomb-mediated coalescence of chromatin regions and aggregation of the critical silencing enzyme SPEN, via its intrinsically disordered domains. Our observations suggest a new model for X Chromosome Inactivation, whereby Xist RNA triggers macromolecular crowding of heterochromatinizing proteins at distinct sites to ultimately increase their occupancy throughout the X chromosome . This mechanism enables deterministic gene silencing across an entire chromosome without the need for Xist ribonucleoprotein complex-chromatin interactions at each target gene. Our findings uncover a spatial organization mechanism by which few RNA molecules can regulate a broad nuclear compartment through the recruitment and local concentration of dynamic effector proteins, and provide a quantitative framework for studying such compartments.

2021-10-27 | GSE162011 | GEO

An Xist-dependent protein assembly mediates Xist localization and gene silencing

Project description:Nuclear compartments play diverse roles in regulating gene expression, yet the molecular forces and components driving compartment formation are not well understood. Studying how the lncRNA Xist establishes the inactive-X-chromosome (Xi)-compartment, we found that the Xist RNA-binding-proteins PTBP1, MATR3, TDP43, and CELF1 form a condensate to create an Xi-domain that can be sustained in the absence of Xist. The E-repeat-sequence of Xist serves a multivalent binding-platform for these proteins. Without the E-repeat, Xist initially coats the X-chromosome during XCI onset but subsequently disperses across the nucleus with loss of gene silencing. Recruitment of PTBP1, MATR3, TDP-43 or CELF1 to E-Xist rescues these phenotypes, and requires both self-association of MATR3 and TDP-43 and a heterotypic PTBP1-MATR3-interaction. Together, our data reveal that Xist sequesters itself within the Xi-territory and perpetuates gene silencing by seeding a protein-condensate. Our findings uncover an unanticipated mechanism for epigenetic memory and elucidate the interplay between RNA and RNA-binding-proteins in creating compartments for gene regulation.

2020-09-03 | GSE137305 | 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

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

Project description:Xist orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the 3-dimensional structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here we show that Xist directly interacts with the Lamin B Receptor (LBR), an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing. We show that this interaction recruits the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the accessibility of DNA thereby enabling Xist, and its silencing proteins, to spread across the X to silence transcription. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in male mouse ES cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33) containing 1) wild-type Xist (WT), 2) A-repeat deletion Xist (dA), 3) LBR binding site deletion Xist (dLBS), 4) dLBS-Xist rescued with LMNB1 (LMNB1Res), 5) LBR CRISPRi knock down (LBRKD), or 6) SHARP CRISPRi knock down (SHARPKD).

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

Xist exploits three-dimensional chromosome architecture to spread across the X-chromosome

2013-05-30 | GSE46918 | GEO

Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

Project description:Xist-seeded nucleation sites form local concentration gradients of silencing proteins to inactivate the X-chromosome

| PRJNA751089 | ENA

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

Xist exploits three-dimensional chromosome architecture to spread across the X-chromosome

Project description:Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. Here we investigate the localization mechanisms of Xist during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X-chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X-chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X-chromosome. Xist initially accumulates on the periphery of actively transcribed regions and requires its silencing domain to spread across active regions. This suggests a model where Xist coats the entire X-chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in multiple cell contexts: 1) differentiated female cells (MLFs); 2) a time-course of Xist localization in male embryonic stem (ES) cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33); 3) a time-course of Xist localization in differentiating female ES cells (F1 2-1); and 4) wild-type (delXF6) and A-repeat deletion (delSXC9) Xist transgenes incorporated into the Hprt locus under the control of a tet-inducible promoter.

2013-05-30 | E-GEOD-46918 | biostudies-arrayexpress

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

2016-09-02 | GSE86250 | GEO

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

2016-07-24 | GSE80510 | GEO