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SETDB1/HUSH modulates Xist RNA levels during establishment of X chromosome inactivation [4sU-seq]

ABSTRACT: The non-coding RNA Xist mediates X chromosome inactivation (XCI) in mammals, functioning in cis to direct heterochromatin formation over a single X chromosome in cells of early female embryos. Prior studies have determined that Xist transcription and turnover are modulated to maintain the correct level of Xist RNA to inactivate a single X chromosome. The underlying molecular mechanisms are poorly understood. In this study we demonstrate that RNA-dependent recruitment of the H3K9me3 methyltransferase SETDB1 and the HUSH complex across the transcribed Xist locus functions to suppress Xist transcription during establishment of XCI. Thus, degron-mediated acute depletion of SETDB1 or core HUSH subunits abrogates allelic H3K9me3 deposition, increasing Xist transcription, accumulation of Xist RNA, and the rate of X chromosome gene silencing. Our findings support a model in which the SETDB1/HUSH pathway coordinates Xist transcription rates to maintain optimal Xist RNA levels, ensuring the silencing of a single X chromosome.

ORGANISM(S): Mus musculus

PROVIDER: GSE309421 | GEO | 2026/03/12

REPOSITORIES: GEO

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SETDB1/HUSH modulates Xist RNA levels during establishment of X chromosome inactivation [ChIP-Seq]

Project description:The non-coding RNA Xist mediates X chromosome inactivation (XCI) in mammals, functioning in cis to direct heterochromatin formation over a single X chromosome in cells of early female embryos. Prior studies have determined that Xist transcription and turnover are modulated to maintain the correct level of Xist RNA to inactivate a single X chromosome. The underlying molecular mechanisms are poorly understood. In this study we demonstrate that RNA-dependent recruitment of the H3K9me3 methyltransferase SETDB1 and the HUSH complex across the transcribed Xist locus functions to suppress Xist transcription during establishment of XCI. Thus, degron-mediated acute depletion of SETDB1 or core HUSH subunits abrogates allelic H3K9me3 deposition, increasing Xist transcription, accumulation of Xist RNA, and the rate of X chromosome gene silencing. Our findings support a model in which the SETDB1/HUSH pathway coordinates Xist transcription rates to maintain optimal Xist RNA levels, ensuring the silencing of a single X chromosome.

2026-03-12 | GSE309423 | GEO

SETDB1/HUSH modulates Xist RNA levels during establishment of X chromosome inactivation [RNA-Seq]

2026-03-12 | GSE310108 | GEO

SETDB1/HUSH modulates Xist RNA levels during establishment of X chromosome inactivation [ChIP-Seq, 2]

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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.

2021-04-19 | PXD015699 | Pride

RNA binding by Periphilin plays an essential role in initiating silencing by the HUSH complex

Project description:The human silencing hub (HUSH) complex is a transcription-dependent, epigenetic repressor complex that provides a genome-wide immunosurveillance system for the recognition and silencing of newly-integrated retroelements. The core HUSH complex of TASOR, MPP8 and Periphilin, represses these retroelements through SETDB1-mediated H3K9me3 deposition and MORC2-dependent chromatin compaction. HUSH-dependent silencing is RNA-mediated, yet no HUSH components encode any RNA-binding domain. Here we used an unbiased approach to identify which HUSH component was able to bind RNA and determine whether RNA-binding was essential for HUSH function. We identify Periphilin as the major RNA-binding component of the HUSH complex and show that Periphilin’s N-terminal domain is essential for both RNA binding and HUSH function. Periphilin binding to RNA was independent of its interaction with TASOR or MPP8, as its N-terminal domain was sufficient for RNA targeting. The artificial tethering of Periphilin, to a HUSH-insensitive, nascent transcript, enabled the HUSH-dependent silencing of the transcript. This tethering of Periphilin allowed the RNA-binding region of Periphilin to be removed such that only its C-terminal domain was required, for oligomerisation and interaction with TASOR. We therefore show that Periphilin is the predominant RNA-binding protein of the HUSH complex and this RNA-binding is essential for HUSH activity.

2024-11-11 | GSE272315 | GEO

The role of early chromatin changes in X chromosome inactivation

Project description:During development, transcriptional and chromatin modification changes co-occur but the order and causality of events often remain unclear. We explore the interrelationship of these processes using the paradigm of X-chromosome inactivation (XCI). We initiate XCI in female, mouse embryonic stem cells by inducing Xist expression and monitor changes in transcription and chromatin by allele-specific TT-seq and ChIP-seq respectively. An unprecedented temporal resolution enabled identification of the earliest chromatin alterations during XCI. We demonstrate that HDAC3 interacts with both NCOR1 and NCOR2 and is pre-bound on the X chromosome where it deacetylates histones to promote efficient gene silencing. We also reveal the choreography of polycomb accumulation following Xist RNA coating, with PRC1-associated H2AK119Ub preceding PRC2-associated H3K27me3. Furthermore, polycomb-associated marks accumulate initially at large, intergenic domains and then spreads into genes but only in the context of gene silencing. Our results provide the hierarchy of chromatin events during XCI and demonstrate that some chromatin changes play key roles in mediating transcriptional silencing.

2019-01-17 | PXD011344 | Pride

Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype

Project description:The X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts examines the role of microRNA (miRNAs) in XCI. A striking finding is the identification of miR106a among the top candidates from 46 the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. The XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the genetic inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.

2026-02-09 | PXD064749 | Pride

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

The HUSH-SETDB1-MORC2 epigenetic repressor complex restricts herpesvirus infection in association with PML nuclear bodies

Project description:The establishment of latent herpes simplex virus 1 (HSV-1) infection is controlled by promyelocytic leukemia nuclear bodies (PML NBs). Viral genomes are recruited to PML NBs structures and chromatinized by repressive H3.3K9me3 modified H3.3 histone variant to form viral DNA-containing PML-NBs (vDCP NBs). Exactly how this occurs is unclear. Here we identify an essential role for the HUSH complex and its SETDB1 and MORC2 effectors in the establishment and maintenance of latent herpes simplex virus 1 (HSV-1) infection. ChIP-seq analyses conducted on latently/quiescently infected primary human fibroblasts highlight the association of the H3K9me3 mark with the entire viral genome. We demonstrate the necessity of HUSH, SETDB1, and MORC2 in the establishment of repressive heterochromatin in vDCP NBs. Depletion of these components prior to viral infection results in reduced H3K9me3 levels across the entirety of latent/quiescent HSV-1 genomes, with minimal impact on the cellular genome as a whole. Once latency is established, depletion of HUSH, SETDB1, or MORC2 by shRNAs or the HIV-2ROD Vpx protein, induces the reactivation of HSV-1 in infected primary human fibroblasts as well as in human induced pluripotent stem cell-derived sensory neurons (hiPSDN). We discovered that the viral protein ICP0 induces MORC2 degradation via the proteasome machinery. This process is concurrent with ICP0 capability to initiate full HSV-1 reactivation, as well as the significant reactivation of HSV-1 upon MORC2 depletion in hiPSDN. Our data demonstrate the potent antiviral restriction activity of the HUSH/SETDB1/MORC2 complex to a non-integrated human herpesvirus, its close association with PML NBs, and introduces a new target for anti-herpesvirus therapy.

2024-09-24 | GSE263436 | GEO

The efficiency of Xist-mediated silencing of X-linked and autosomal genes is determined by the genomic environment

Project description:Xist is indispensable for X chromosome inactivation (XCI) in female mammalian cells. However, how Xist RNA directs chromosome-wide transcriptional inactivation of the X chromosome is largely unknown. Here, to study chromosome inactivation by Xist, we generated a system where ectopic Xist expression can be induced from several genomic contexts in aneuploid mouse ES cells. We found that ectopic Xist expression from any location on the X chromosome faithfully recapitulated endogenous XCI, showing the potency of Xist to initiate XCI. Genes that escape XCI remain consistently transcriptionally active upon ectopic XCI, regardless of their position relative to Xist transgenes, and the enrichment of CTCF at their promoters is implicated in directing XCI escape. Xist expression from autosomes facilitates their transcriptional silencing to different degrees, and gene density in proximity of the Xist transcription locus plays a central role in determining the efficiency of gene inactivation. We also show that the enrichment of LINE elements together with a specific chromatin environment facilitates Xist-mediated silencing of both X-linked and autosomal genes. These findings provide new insights into the epigenetic mechanisms that mediate XCI and identify genomic features that promote Xist-mediated chromosome-wide gene inactivation

2017-07-04 | GSE92894 | GEO

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