Modular structure and function of the XIST RNP in X chromosome inactivation
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PROVIDER: PRJNA523034 | ENA |
REPOSITORIES: ENA
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Project description:Long noncoding RNAs (lncRNAs) are an important class of transcripts that regulate gene expression on many levels, yet their mechanisms of action remain poorly understood. Previous studies have indicated that these RNAs can serve as modular scaffolds to recruit a variety of protein complexes and coordinate their distinct functions. XIST, a founding member of the lncRNA family, controls the inactivation of an entire X chromosome in placental mammals. Here we develop and integrate several orthogonal structure-interaction analysis methods to demonstrate that XIST RNA-protein complex folds into a modular architecture that is conserved in evolution. The discrete XIST RNA domains interact with distinct sets of effector proteins to orchestrate the X chromosome inactivation (XCI). The modular architecture plays an essential role, in addition to the sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and establishes a paradigm for mechanistic studies of lncRNA functions.
2020-04-23 | GSE126715 | GEO
Project description:Xist-mediated Chromosome Inactivation
| PRJNA489702 | ENA
Project description:Long noncoding RNAs (lncRNAs) are an important class of transcripts that regulate gene expression on many levels, yet their mechanisms of action remain poorly understood. Previous studies have indicated that these RNAs can serve as modular scaffolds to recruit a variety of protein complexes and coordinate their distinct functions. XIST, a founding member of the lncRNA family, controls the inactivation of an entire X chromosome in placental mammals. Here we develop and integrate several orthogonal structure-interaction analysis methods to demonstrate that XIST RNA-protein complex folds into a modular architecture that is conserved in evolution. The discrete XIST RNA domains interact with distinct sets of effector proteins to orchestrate the X chromosome inactivation (XCI). The modular architecture plays an essential role, in addition to the sequence motifs, in determining the specificity of RBP binding and m6A modification. Together, this work builds a comprehensive structure-function model for the XIST RNA-protein complex, and establishes a paradigm for mechanistic studies of lncRNA functions.
2020-04-23 | GSE126716 | GEO
Project description:The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation
2019-08-29 | PXD014287 | Pride
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
Project description:RNA-seqs followed by whole and segmental deletions of XIST genes in human K562 cells. The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and the XCI-involving RNA elements remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, ten smaller XIST segments, including repeats A, D, and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and fluorescence in-situ hybridization experiments on XIST RNA.
2019-01-25 | GSE65830 | GEO
Project description:The long non-coding RNA (lncRNA) Xist is a master regulator of X-chromosome inactivation in mammalian cells. Models for how Xist and other lncRNAs function depend on thermodynamically stable secondary and higher-order structures that RNAs can form in the context of a cell. Probing accessible RNA bases can provide data to build models of RNA conformation that provide insight into RNA function, molecular evolution, and modularity. To study the structure of Xist in cells, we built upon recent advances in RNA secondary structure mapping and modeling to develop Targeted Structure-Seq, which combines chemical probing of RNA structure in cells with target-specific massively parallel sequencing. By enriching for signals from the RNA of interest, Targeted Structure-Seq achieves high coverage of the target RNA with relatively few sequencing reads, thus providing a targeted and scalable approach to analyze RNA conformation in cells. We use this approach to probe the full-length Xist lncRNA to develop new models for functional elements within Xist, including the repeat A element in the 5'-end of Xist. This analysis also identified new structural elements in Xist that are evolutionarily conserved, including a new element proximal to the C repeats that is important for Xist function. Examination of dimethylsufate reactivity of Xist lncRNA and 18S rRNA in cells using targeted reverse transcription to determine reactivity, and comparisons with untreated control samples.
2016-02-09 | E-GEOD-67667 | biostudies-arrayexpress
Project description:At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.
2021-09-30 | GSE163320 | GEO
Project description:At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.
2021-09-30 | GSE163319 | GEO
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