Mus musculus
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA245107 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1258450_1.fastq.gz | Fastqsanger.gz | |||
| SRR1258450_2.fastq.gz | Fastqsanger.gz | |||
| SRR1258460_1.fastq.gz | Fastqsanger.gz | |||
| SRR1258460_2.fastq.gz | Fastqsanger.gz | |||
| SRR1258451_1.fastq.gz | Fastqsanger.gz |
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Project description:In mammals, dosage compensation entails inactivation of an entire X chromosome in female cells. X-chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). How PRC2 specifically interacts with Xist and other Polycomb targets remains unclear. Using XCI as a model, we take an unbiased proteomics approach to search for new Xist and PRC2 regulators. We identify ATRX, a chromatin remodeler associated with mental retardation, alpha thalassemia, and cancer in humans. We show that ATRX is a high-affinity RNA-binding protein. ATRX directly interacts with RepA/Xist RNA and is required for loading of PRC2 onto the RNA in vivo. The Xist gene is a hotspot of ATRX occupancy. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X-chromosome. Epigenomic profiling reveals a dependency of PRC2 on ATRX that extends beyond XCI. Depleting ATRX results in genome-wide disruptions to PRC2 localization and trimethylation of histone H3-lysine 27 (H3K27me3). PRC2 is displaced from genes and relocalized to intergenic space, causing reduced H3K27me3 and concomitant changes in expression of Polycomb targets. We conclude that ATRX is a required specificity determinant for PRC2 targeting and function.
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Project description:Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.
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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.
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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).
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Project description:Polycomb repressive complex 1 regulates the nucleosome landscape but not accessibility at target genes [ATAC-seq]
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Project description:Polycomb repressive complex 1 regulates the nucleosome landscape but not accessibility at target genes [RNA-seq]
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