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A protein assembly mediates Xist localization and gene silencing.

ABSTRACT: Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear¹. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus² and binding diverse proteins^3-5 to achieve X-chromosome inactivation (XCI)^6,7. The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off⁸. Here we show that the Xist RNA-binding proteins PTBP1⁹, MATR3¹⁰, TDP-43¹¹ and CELF1¹² assemble on the multivalent E-repeat element of Xist⁷ and, via self-aggregation and heterotypic protein-protein interactions, form a condensate¹ in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase⁸, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing.

SUBMITTER: Pandya-Jones A

PROVIDER: S-EPMC7644664 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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

Project description:An Xist-dependent protein assembly mediates Xist localization and gene silencing

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The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene.

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Project description:We performed RNA-seq and ChIP-seq on clones of human cell lines carrying an inducible XIST transgene on 1p, 8p, or 12q to study the effects of allelic silencing in cis Total gene expression and allelic changes were examined in HT1080 clones carrying an inducible XIST transgene on 1p, 8p, or 12q after induction by doxycycline. A replicate was done for the 8p clone treated with DOX. An additional 1p clone integrated with an empty vector, and an 1p, 8p, and 12q clone without induction were included as controls. ChIP was performed on the 8p clone to investigate the changes in H3K27 acetylation and trimethylation.

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Active chromatin marks are retained on X chromosomes lacking gene or repeat silencing despite XIST/Xist expression in somatic cell hybrids.

Project description:X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.

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