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M6A RNA methylation promotes XIST-mediated transcriptional repression

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE78030 | GEO | 2016/09/07

SECONDARY ACCESSION(S): PRJNA312381

REPOSITORIES: GEO

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m6A RNA methylation promotes XIST-mediated transcriptional repression

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

Systematic Analysis of Allelic Silencing Defines the Interplay of Key Pathways in Xist-mediated Chromosome Inactivation

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

Allelic silencing analysis for A-repeat and m6A region in mouse Xist

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

SPEN is a molecular integrator that bridges Xist with transcriptional and epigenetic control of X-chromosome inactivation

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

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [RNA-seq]

Project description:N6-methyladenosine (m6A)methylationcan be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. How the MTC is recruited to distinct genomic loci to accomplish selective transcriptional regulation remains elusive. Here we identify RBFOX2, a well-established RNA-binding protein (RBP), as a chromatin factor that recognizes m6A on caRNAs and recruits RBM15, an MTC component, to install methylation on promoter-associated RNAs (paRNAs). RBM15 also physically interacts with YTHDC1 to facilitate the recruitment of Polycomb repressive complex 2 (PRC2) to RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this m6A/RBM15/YTHDC1/PRC2 axis is essential to myeloid leukemia. Downregulation of RBFOX2 notably inhibits AML cell survival/proliferation and promotes myeloid differentiation of AML cells. RBFOX2 is also required for self-renewal of leukemia stem/initiation cells (LSCs/ LICs) and AML maintenance. Together, our study presents a clear pathway of m6A MTC recruitment and m6A deposition on caRNAs to achieve locus-specific chromatin regulation with potential therapeutic implications.

2023-06-16 | GSE229013 | GEO

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [RNA-CLIP]

2023-06-16 | GSE229012 | GEO

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [SAC-seq]

Project description:N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. How the MTC is recruited to distinct genomic loci to accomplish selective transcriptional regulation remains elusive. Here we identify RBFOX2, a well-established RNA-binding protein (RBP), as a chromatin factor that recognizes m6A on caRNAs and recruits RBM15, an MTC component, to install methylation on promoter-associated RNAs (paRNAs). RBM15 also physically interacts with YTHDC1 to facilitate the recruitment of Polycomb repressive complex 2 (PRC2) to RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this m6A/RBM15/YTHDC1/PRC2 axis is essential to myeloid leukemia. Downregulation of RBFOX2 notably inhibits AML cell survival/proliferation and promotes myeloid differentiation of AML cells. RBFOX2 is also required for self-renewal of leukemia stem/initiation cells (LSCs/ LICs) and AML maintenance. Together, our study presents a clear pathway of m6A MTC recruitment and m6A deposition on caRNAs to achieve locus-specific chromatin regulation with potential therapeutic implications.

2023-06-16 | GSE205710 | GEO

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [MeRIP-seq]

Project description:N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. How the MTC is recruited to distinct genomic loci to accomplish selective transcriptional regulation remains elusive. Here we identify RBFOX2, a well-established RNA-binding protein (RBP), as a chromatin factor that recognizes m6A on caRNAs and recruits RBM15, an MTC component, to install methylation on promoter-associated RNAs (paRNAs). RBM15 also physically interacts with YTHDC1 to facilitate the recruitment of Polycomb repressive complex 2 (PRC2) to RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this m6A/RBM15/YTHDC1/PRC2 axis is essential to myeloid leukemia. Downregulation of RBFOX2 notably inhibits AML cell survival/proliferation and promotes myeloid differentiation of AML cells. RBFOX2 is also required for self-renewal of leukemia stem/initiation cells (LSCs/ LICs) and AML maintenance. Together, our study presents a clear pathway of m6A MTC recruitment and m6A deposition on caRNAs to achieve locus-specific chromatin regulation with potential therapeutic implications.

2023-06-16 | GSE205709 | GEO

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [KAS-Seq]

Project description:N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. How the MTC is recruited to distinct genomic loci to accomplish selective transcriptional regulation remains elusive. Here we identify RBFOX2, a well-established RNA-binding protein (RBP), as a chromatin factor that recognizes m6A on caRNAs and recruits RBM15, an MTC component, to install methylation on promoter-associated RNAs (paRNAs). RBM15 also physically interacts with YTHDC1 to facilitate the recruitment of Polycomb repressive complex 2 (PRC2) to RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this m6A/RBM15/YTHDC1/PRC2 axis is essential to myeloid leukemia. Downregulation of RBFOX2 notably inhibits AML cell survival/proliferation and promotes myeloid differentiation of AML cells. RBFOX2 is also required for self-renewal of leukemia stem/initiation cells (LSCs/ LICs) and AML maintenance. Together, our study presents a clear pathway of m6A MTC recruitment and m6A deposition on caRNAs to achieve locus-specific chromatin regulation with potential therapeutic implications.

2023-06-16 | GSE205708 | GEO

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid differentiation [ChIP-Seq]

Project description:N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. How the MTC is recruited to distinct genomic loci to accomplish selective transcriptional regulation remains elusive. Here we identify RBFOX2, a well-established RNA-binding protein (RBP), as a chromatin factor that recognizes m6A on caRNAs and recruits RBM15, an MTC component, to install methylation on promoter-associated RNAs (paRNAs). RBM15 also physically interacts with YTHDC1 to facilitate the recruitment of Polycomb repressive complex 2 (PRC2) to RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this m6A/RBM15/YTHDC1/PRC2 axis is essential to myeloid leukemia. Downregulation of RBFOX2 notably inhibits AML cell survival/proliferation and promotes myeloid differentiation of AML cells. RBFOX2 is also required for self-renewal of leukemia stem/initiation cells (LSCs/ LICs) and AML maintenance. Together, our study presents a clear pathway of m6A MTC recruitment and m6A deposition on caRNAs to achieve locus-specific chromatin regulation with potential therapeutic implications.

2023-06-16 | GSE205706 | GEO

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