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ChIP-sequencing (ChIP-seq) experiments on female ESCs expressing V5-tagged REX1 and control wild-type Embryonic stem cells (ESCs)

ABSTRACT: Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome (XCI) in all female somatic cells. Up-regulation of Xist transcription on the future inactive X chromosome (Xi) acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to XCI. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is up-regulated in differentiating mouse embryonic stem cells (ESCs) and activates Xist transcription and XCI. Here, we have identified the pluripotency factor REX1 as a key target of RNF12 in the XCI mechanism. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout mouse ESCs show an increased level of REX1. Using ChIP-seq, REX1 binding sites were detected in Xist and Tsix regulatory regions. Over-expression of REX1 in female ESCs was found to inhibit Xist transcription and XCI, whereas male Rex1+/- ESCs showed ectopic XCI. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate XCI. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and XCI.

ORGANISM(S): Mus musculus

PROVIDER: GSE36417 | GEO | 2012/06/12

SECONDARY ACCESSION(S): PRJNA153217

REPOSITORIES: GEO

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ChIP-sequencing (ChIP-seq) experiments on female ESCs expressing V5-tagged REX1 and control wild-type Embryonic stem cells (ESCs)

Project description:Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome (XCI) in all female somatic cells. Up-regulation of Xist transcription on the future inactive X chromosome (Xi) acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to XCI. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is up-regulated in differentiating mouse embryonic stem cells (ESCs) and activates Xist transcription and XCI. Here, we have identified the pluripotency factor REX1 as a key target of RNF12 in the XCI mechanism. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout mouse ESCs show an increased level of REX1. Using ChIP-seq, REX1 binding sites were detected in Xist and Tsix regulatory regions. Over-expression of REX1 in female ESCs was found to inhibit Xist transcription and XCI, whereas male Rex1+/- ESCs showed ectopic XCI. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate XCI. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and XCI. 2 (one control one pulldown) samples

2012-06-12 | E-GEOD-36417 | biostudies-arrayexpress

SPEN is required for Xist upregulation during initiation of X chromosome inactivation (RNA-seq)

Project description:At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.

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RNA-seq of mouse ES cells depleted of MOF, MSL1, MSL2 or KANSL3

2014-06-03 | GSE57698 | GEO

RNA-seq of mouse ES cells depleted of MOF, MSL1, MSL2 or KANSL3

Project description:We have studied the regulatory potential of MYST1-(MOF)-containing MSL and NSL complexes in mouse embryonic stem cells (ESCs) and neuronal progenitors. We find that both complexes influence transcription by binding to promoters as well as TSS-distal enhancer regions. In contrast to flies, the MSL complex is not enriched on the X chromosome yet it is crucial for mammalian X chromosome regulation as it specifically regulates Tsix ncRNA, the major repressor of Xist lncRNA. MSL depletion leads to severely decreased Tsix expression, reduced REX1 recruitment, and consequently accumulation of Xist RNA in ESCs. The NSL complex provides additional, Tsix-independent repression of Xist by maintaining pluripotency. MSL and NSL complexes therefore act synergistically by using distinct pathways to ensure a fail-safe mechanism for the repression of X inactivation in ESCs. We have performed ChIP-seq of KANSL3, MCRS1, MOF, MSL1 and MSL2 in mouse ESCs, and KANSL3, MOF and MSL2 in NPCs, in duplicate and normalised against their inputs. We have also performed RNA-seq following knockdown of Kansl3, Mof, Msl1 and Msl2 mouse embryonic stem cells in triplicate. NB: Kansl3 and Mof knockdown-RNAseq are analyzed against their own scrambled controls, and Msl1 and Msl2 against another scrambled control triplicate.

2014-06-03 | E-GEOD-57698 | biostudies-arrayexpress

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice.

Project description:In mice, imprinted X-chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extra-embryonic tissues is followed by X-reactivation in the epiblast precursors of the inner cell mass (ICM) of the blastocyst, to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin-ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

2018-11-15 | PXD010944 | Pride

XIST dampens X chromosome activity in a SPEN-dependent manner during early human development [RNA-seq]

Project description:XIST long non-coding RNA is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female pre-implantation embryos without triggering X chromosome silencing. The long non-coding RNA XACT co-accumulates with XIST on active Xs and may antagonize XIST function. Here we used human ES cells in a naïve state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during pre-implantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and attenuates transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional prior to XCI, confirms the existence of a transient process of X chromosome dosage compensation, and reveals that X chromosome inactivation and dampening rely on the same set of factors.

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XIST dampens X chromosome activity in a SPEN-dependent manner during early human development [RAP-seq]

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