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Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization (RNA-SPLIT)

ABSTRACT: Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization (RNA-SPLIT)

PROVIDER: PRJNA646962 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12235200_1.fastq.gz	Fastqsanger.gz
	SRR12235200_2.fastq.gz	Fastqsanger.gz
	SRR12248800_1.fastq.gz	Fastqsanger.gz
	SRR12248800_2.fastq.gz	Fastqsanger.gz
	SRR12235201_1.fastq.gz	Fastqsanger.gz

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Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization [ChrRNA-seq]

Project description:Xist RNA, which directs the process of X chromosome inactivation in mammals, localizes in cis over the chromosome from which it is transcribed, recruiting chromatin modifiers to silence underlying genes. To analyze cis-limited Xist RNA localization we have developed time-resolved super-resolution imaging of individual Xist RNA molecules in single cells. Using this approach, we quantify fundamental parameters underpinning Xist RNA dynamics, demonstrating a feedback mechanism linking Xist RNA synthesis and degradation, and an unusual coupling behavior that physically links temporally separated Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a separable function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique and unusual behavior of Xist RNA.

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Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization [ChrRNA-seq]

Project description:Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization [ChrRNA-seq]

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Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization (RNA-SPLIT)

Project description:This SuperSeries is composed of the SubSeries listed below.

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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

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Project description:Identification of cell type-specific XIST co-factors during maintenance of X chromosome inactivation by XIST CHIRP-MS

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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.

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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.

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