Allelic silencing analysis for A-repeat and m6A region in mouse Xist
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ABSTRACT:
PROVIDER: PRJNA596451 | ENA |
REPOSITORIES: ENA
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Project description:We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing.
2020-02-01 | GSE142270 | GEO
Project description:By m6A-seq, we examined the m6A pattern on Xist and Xist_Del_A-repeat, which immediately after the A-repeat region. A-repeat deletion abolished the m6A methylation downstream the A-repeat but not the m6A peak located in Xist exon VII.
2020-02-01 | GSE142271 | GEO
Project description:Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome silencing through the stepwise recruitment of factors that modify underlying chromatin structure. Whilst considerable progress has been made towards defining key silencing factors and the elements to which they bind, their relative contribution to silencing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in ES cell-based models. We show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of low expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15-Mettl3/14 m6A-methyltransferase complex, previously proposed to have a central role, make at most a minor contribution to gene silencing. We integrate our findings in a comprehensive model for Xist-mediated chromosome silencing.
2019-06-17 | GSE119602 | 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:m6A-seq profiling in Xist and Xist_Del_A-repeat cell lines
| PRJNA596450 | ENA
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.
2016-09-07 | GSE78030 | GEO
Project description:By m6A-seq, we examined the m6A pattern on Xist, which immediately after the A-repeat region. The cell line, integrating multiple copy of Xist on chr3, with m6A core machinery knockout (Wtap and Mettl3) were generated. We also profiled m6A-seq in transgenic Xist line, and observed a global reduction of m6A level, validated by m6A-LC-MS/MS. Unexpectedly, we haven’t observed reduced m6A level on Xist m6A region downstream of A-repeat in transgenic Xist line.
2019-06-17 | GSE119487 | GEO
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: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: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