CRISPR activation screen to identify X-chromosomal Xist activators in male embryonic stem cells
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ABSTRACT:
PROVIDER: PRJNA798744 | ENA |
REPOSITORIES: ENA
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Project description:As the master regulator of X-chromosome inactivation (XCI), the Xist RNA is expressed nearly ubiquitously in female mice. Xist is only absent in the germ line and at the pluripotent state. Xist is generally assumed to be expressed “by default” in females, while being actively repressed in the few tissues where it is silent. Whether activating mechanisms also contribute remained largely unknown. Through a pooled CRISPR screen we identify the GATA family of transcription factors as potent direct activators of Xist. We describe a GATA-responsive regulatory element (RE79), located ~100 kb upstream of the Xist promoter. In cell lines derived from the two extraembryonic lineages, XEN and TS cells, where imprinted Xist expression is maintained, RE79 is bound by different sets of GATA factors expressed in those tissues. Here we use a CRISPR activation screen targeting X-chromosomal genes on differentiating XO mouse embryonic stem cells to identify putative Xist activators.
2022-01-26 | GSE194013 | GEO
Project description:The long noncoding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-specific XIST complexes, and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. XIST dysregylation, reflected by escape of XIST-dependent genes, occurs in CD11c+ atypical memory B cells across single-cell transcriptome data in patients with female-biased autoimmunity and COVID-19 infection. XIST inactivation with TLR7 agonism suffices to promote isotype-switched atypical B cells. These results suggest cell-type-specific diversification of lncRNA-protein complexes increase lncRNA functionalities, and expand roles for XIST in sex-differences in biology and medicine.
2021-01-12 | GSE164593 | GEO
Project description:Developmental genes are controlled by complex cis-regulatory landscapes that integrate multiple signals to ensure the correct spatio-temporal expression pattern. To investigate the underlying regulatory principles, we use the Xist locus as a model, which encodes the master regulator of X-chromosome inactivation. Xist is upregulated at the primed pluripotent state in a female-specific manner, thus integrating developmental cues and X-dosage information. It remains poorly understood how these signals are decoded by the ~800kb genomic region that controls Xist. While a series of repressive cis-regulatory elements have been identified, the distal enhancers that activate Xist transcription remain largely unknown. Here we use an inducible CRISPRi system in a non-coding CRISPR screen to profile 138 candidate regulatory elements on their effect on Xist upregulation during early differentiation of mouse Embryonic Stem Cells (mESCs).
2021-03-04 | GSE167352 | GEO
Project description:Chromosomal Silencing Induced by Xist Expression from Autosomal Transgenes
| PRJNA657761 | ENA
Project description:The Polycomb repressive complexes PRC1 and PRC2 play a key role in chromosome silencing by Xist RNA. Previously we have shown that initation of Polycomb recruitment is mediated by the PCGF3/5-PRC1 complex, which catalyses chromosome-wide H2A ubiquitylation (H2AK119u1), signalling recruitment of other PRC1 complexes, and PRC2. However, the molecular basis for PCGF3/5-PRC1 recruitment by Xist RNA remains unknown. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt element encompassing the Xist B-repeat element. XR-PID is required for Polycomb recruitment by Xist RNA, Xist-mediated chromosome silencing. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking the B-repeat is sufficient for Xist-dependent Polycomb recruitment. Our findings resolve the molecular mechanism for Polycomb recruitment by Xist RNA, providing key insights into chromatin modification by non-coding RNA.
2017-12-08 | GSE99924 | GEO
Project description:Xist transgenes (Tg) integrated into an autosome can induce transcriptional silencing of flanking genes. We investigate an autosomal integration of Xist Tg that is compatible with mouse viability but causes male sterility in homozygous transgenic mice. We show ectopic Xist expression in the transgenic male cells along with a transcriptional reduction of genes clustered in two regions of Chromosome 1. RNA-seq and DNA Fluorescent in situ Hybridization (FISH) confirms that Xist Tg is associated with Chromosome 1.
2021-04-01 | GSE156393 | GEO
Project description:Purpose: Compare the transcriptome of homogeneous XIST+ and XIST- hES cell populations. Methods: We isolated homogeneous XIST+ and XIST- cell populations. The XIST+ cells correspond to cells with a XIST cloud and one ATRX pinpoint. The XIST- cells correspond to cells with no XIST cloud and one ATRX pinpoint. Results: We took advantage of the clonal pattern of X-chromosome inactivation in H9 cells and analyzed the data in an allelic manner. By comparing the RNA-Seq data with known H9 SNPs, we identified genomic positions which were relaxed from XCI in XIST- cells compared to XIST+ cells. Conclusions: Genic as well as unannotated transcripts are massively relaxed from XCI in H9 cells when XIST expression is lost, however, this reactivation is only partial and a large region around the centromere is protected from relaxation of silencing. Total RNA (rRNA depleted) profiles of XIST+ and XIST- human embryonic stem cells
2012-12-21 | E-GEOD-39757 | biostudies-arrayexpress
Project description:The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X (Xi) and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic). Currently unclear is how Xist spreads silencing on a 150 Mb scale. Here we generate high-resolution maps of Xist binding across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism in which it initially targets gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but frequently concentrates near escapee boundaries. Xist binding was linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped of Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes on distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions. Capture hybridization analysis of RNA targets (CHART) and input samples of (differentiating) mouse embryonic stem (ES) cells and immortalized mouse embryonic fibroblasts (MEF) using paired-end 75 nt reads on Illumina HiSeq2500, with 2 replicates per sample (# of samples). RNA-seq of the same cell lines with 50 nt reads on Illumina HiSeq2000, with 2 replicates per sample (2 samples, 4 datasets total). CHIP-seq: Data from GSE36905 was aligned and processed as CHART-seq samples. Resulting coverage tracks (EZH2/K27me3) are linked directly to GSE48649 (bedGraphs linked below).
2013-11-04 | E-GEOD-48649 | biostudies-arrayexpress
Project description:The Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation (XCI), the process by which mammals compensate for unequal numbers of sex chromosomes. During XCI, Xist coats the future inactive X (Xi) and recruits Polycomb Repressive Complex 2 (PRC2) to the X-inactivation center (Xic). Currently unclear is how Xist spreads silencing on a 150 Mb scale. Here we generate high-resolution maps of Xist binding across a developmental time course using CHART-seq. In female cells undergoing XCI de novo, Xist follows a two-step mechanism in which it initially targets gene-rich islands before spreading to intervening gene-poor domains. Xist is depleted from genes that escape XCI but frequently concentrates near escapee boundaries. Xist binding was linearly proportional to PRC2 density and H3 lysine 27 trimethylation (H3K27me3), suggesting co-migration of Xist and PRC2. Interestingly, when the Xi is acutely stripped of Xist in post-XCI cells, Xist recovers quickly within both gene-rich and -poor domains on a time scale of hours instead of days, suggesting a previously primed Xi chromatin state. We conclude that Xist spreading takes on distinct stage-specific forms: During initial establishment, Xist follows a two-step mechanism, but during maintenance, Xist spreads rapidly to both gene-rich and -poor regions.
2013-11-04 | GSE48649 | GEO
Project description:Xist lacking of XR-PID (B-repeat and part of C-repeat) can't recruit the polycomb during X chromsome inactivation. The majoring of Xist-mediated chromosomal silencing wouldn’t be achieved. We found that hnRNPK was the one of main contributors for binding B-repeat of Xist. When we tethered hnRNPK to Xist which lacks of XR-PID region, the silencing and polycomb recruitment are restored.
2017-12-08 | GSE103362 | GEO