An Xist-dependent protein assembly mediates Xist localization and gene silencing
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ABSTRACT:
PROVIDER: PRJNA565139 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10102807.fastq.gz | Fastqsanger.gz | |||
| SRR10102808.fastq.gz | Fastqsanger.gz | |||
| SRR10102809.fastq.gz | Fastqsanger.gz | |||
| SRR10102810.fastq.gz | Fastqsanger.gz | |||
| SRR10102811.fastq.gz | Fastqsanger.gz |
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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
Project description: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 proteins3-5 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.
| S-EPMC7644664 | biostudies-literature
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
Project description:Xist RNA, which directs the process of X chromosome inactivation in mammals, localizes in cis over the chromosome from which it is transcribed, recruiting chromatin modifiers to silence underlying genes. To analyze cis-limited Xist RNA localization we have developed time-resolved super-resolution imaging of individual Xist RNA molecules in single cells. Using this approach, we quantify fundamental parameters underpinning Xist RNA dynamics, demonstrating a feedback mechanism linking Xist RNA synthesis and degradation, and an unusual coupling behavior that physically links temporally separated Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a separable function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique and unusual behavior of Xist RNA.
2021-06-12 | GSE154656 | GEO
Project description:Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes (~1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: specificity to the X and chromosome-wide gene silencing, and suggest a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets.
2022-01-21 | GSE192572 | GEO
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.
2015-09-02 | E-GEOD-68109 | biostudies-arrayexpress
Project description:Although thousands of lncRNAs are encoded in mammalian genomes, their mechanisms of action are largely uncharacterized because they are often expressed at significantly lower levels than their proposed targets. One such lncRNA is Xist, which mediates chromosome-wide gene silencing on one of the two X chromosomes to achieve gene expression balance between males and females. How a limited number of Xist molecules can mediate robust silencing of a significantly larger number of target genes (~1 Xist RNA: 10 gene targets) while maintaining specificity to genes on the X within each cell is unknown. Here, we show that Xist drives non-stoichiometric recruitment of the essential silencing protein SHARP to amplify its abundance across the inactive X, including at regions not directly occupied by Xist. This amplification is achieved through concentration-dependent homotypic assemblies of SHARP on the X and is required for chromosome-wide silencing. We find that expressing Xist at higher levels leads to increased localization at autosomal regions, demonstrating that low levels of Xist are critical for ensuring its specificity to the X chromosome. We show that Xist (through SHARP) acts to suppress production of its own RNA which may act to constrain overall RNA levels and restrict its ability to spread beyond the X. Together, our results demonstrate a spatial amplification mechanism that allows Xist to achieve two essential but countervailing regulatory objectives: specificity to the X and chromosome-wide gene silencing, and suggest a more general mechanism by which other low abundance lncRNAs can balance specificity to, and robust control of, their regulatory targets.
2022-01-21 | GSE192573 | GEO
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
2015-09-02 | GSE68109 | GEO
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
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