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The deubiquitinase Usp9x regulates PRC2-mediated chromatin reprogramming during mouse development

ABSTRACT: Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of the global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of “stemness” and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE146800 | GEO | 2021/01/25

REPOSITORIES: GEO

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Loss of Usp9x increases Wnt and Notch signaling in neural progenitors

Project description:Correct neural progenitor fate determination requires the coordination of extrinsic fate determinant signals with intrinsic responses. Post-translational modifications dynamically alter protein function and so are ideally situated to regulate development. Here we show that the deubiquitylaying enzyme, Usp9x modulates both intrinsic and extrinsic regulators of mouse neural progenitors. Nestin-cre mediated deletion of Usp9x from neural progenitors results in a transient disruption of cell adhesion and apical-basal polarity as well as the premature differentiation of intermediate neural progenitors. Ablation of Usp9x also significantly increased Î²-catenin protein levels, especially S33/S37/T41 phospho-Î²-catenin, and Wnt signalling. Usp9x was found to be part of the Î²-catenin destruction complex and loss of Usp9x affects destruction complex composition. Notch signalling was also increased in Usp9x ablated neural progenitors, coinciding with decreased Itch and Numb, and increased Notch intracellular domain protein levels. Usp9x co-localized and immunopreciptiated with Numb from neural progenitors suggesting it is required for Numb stabilisation. These data suggest Usp9x plays a role in coordinating intrinsic responses to extrinsic signals during neural development.

2017-08-18 | E-MTAB-4150 | biostudies-arrayexpress

Integrative proteomics reveals a protective role of PRC2 for the ground state pluripotent epigenome

Project description:The ground state of pluripotency is defined as a basal proliferative state free of epigenetic restriction, represented by mouse embryonic stem cells (ESCs) cultured with two kinase inhibitors (so-called “2i”). Through comparison with serum-grown ESCs, we identify epigenetic features characterizing 2i ESCs by proteome profiling of chromatin including post-translational histone modifications. The most prominent difference is H3K27me3 and its enzymatic writer complex PRC2 that are highly abundant on eu- and heterochromatin in 2i ESCs, with H3K27me3 redistributing outside canonical PRC2 targets in a CpG-dependent fashion. Using PRC2-deficient 2i ESCs, we identify epigenetic crosstalk with H3K27me3, including significant increases in H4 acetylation and DNA methylation. This suggests that the unique H3K27me3 configuration protects 2i ESCs from preparation to lineage priming. Interestingly, removal of DNA methylation in PRC2-deficient 2i ESCs lacking H3K27me3 using 5-azacytidine hardly affected ESC viability and transcriptome, showing that ESCs are independent of both major repressive epigenetic marks.

2018-11-21 | PXD007154 | Pride

The PRC2-associated factor C17orf96 is a novel CpG island regulator in mouse ES cells

Project description:CpG islands (CGIs) are key DNA regulatory elements in the vertebrate genome and are often found at gene promoters. In mammalian embryonic stem (ES) cells, CGIs are decorated by either the active or repressive histone marks, H3K4me3 and H3K27me3, respectively, or by both modifications (‘bivalent domains’), but their precise regulation is incompletely understood. Remarkably, we find that the polycomb repressive complex 2 (PRC2)-associated protein C17orf96 (a.k.a. esPRC2p48 and E130012A19Rik) is present at most CGIs in mouse ES cells. At PRC2-rich CGIs, loss of C17orf96 results in an increased chromatin binding of Suz12 and elevated H3K27me3 levels concomitant with gene repression. In contrast, at PRC2-poor CGIs, located at actively transcribed genes, C17orf96 colocalizes with RNA polymerase II and its depletion leads to a focusing of H3K4me3 in the core of CGIs. Our findings thus identify C17orf96 as a novel context-dependent CGI regulator. ChIP-seq of C17orf96, H3K4me3 and H3K27me3 in mouse ES cells (E14).

2015-04-29 | E-GEOD-63491 | biostudies-arrayexpress

Novel cell lines from mouse epiblast share defining features with human embryonic stem cells

Project description:The application of human embryonic stem (ES) cells has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo prior to implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells (PGCs). The post-ES cell lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, post-ES and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development. This SuperSeries is composed of the SubSeries listed below.

2007-06-27 | GSE7902 | GEO

Effects of Brca1 knock-down on Ezh2 targeting

Project description:The Polycomb repressive complex 2 (PRC2) is important for the maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and silencing expression of genes regulating differentiation. Here we show decreased expression of BRCA1 promotes EZH2 re-targeting genome wide and enhances H3K27me3 levels at PRC2 target loci in mouse ES cells.

2013-07-01 | GSE37321 | GEO

PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (ChIP-Seq)

Project description:Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation, an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, which presents a major roadblock for dissecting the precise roles of PRC2 activity during lineage commitment. While recent studies suggest that loss of H3K27me3 leads to changes in DNA methylation in ESCs, how these two pathways coordinate to regulate gene expression programs during lineage commitment is poorly understood. Here, we analyzed gene expression and DNA methylation levels in several PRC2 mutant ESC lines that maintain varying levels of H3K27me3. We found that maintenance of intermediate levels of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). However, genes that function to specify other lineages failed to be repressed, suggesting that PRC2 activity is necessary for lineage fidelity. We also found that H3K27me3 is antagonistic to DNA methylation in cis. Furthermore, loss of H3K27me3 leads to a gain in promoter DNA methylation in developmental genes in ESCs and in lineage genes during differentiation. Thus, our data suggest a role for PRC2 in coordinating dynamic gene repression while protecting against inappropriate promoter DNA methylation during differentiation. Embryonic Stem Cell (ESC) lines mutant for PRC2 core components Suz12 (Suz12GT and Suz12delta) and Eed (Eednull) were subjected to in vitro directed differentiation down the spinal motor neuron lineage. ESCs and day 5 differentiated cells from the three mutant lines and wild-type were used for H3K27me3 ChIP-seq.

2014-11-17 | E-GEOD-53505 | biostudies-arrayexpress

PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (RRBS)

2014-11-17 | E-GEOD-53507 | biostudies-arrayexpress

PRC2 coordinates lineage fidelity and DNA methylation during ESC differentiation (RNA-Seq)

2014-11-17 | E-GEOD-53506 | biostudies-arrayexpress

USP9X stabilizes EWS-FLI1 to promote cancer cell growth in Ewing’s sarcoma

Project description:Ewing’s sarcoma (EWS) is a cancer of the bones or soft tissues in children and adolescents. EWS-FLI1 is a transcriptional factor and the key driver of EWS. To characterize the changes of downstream transcriptional profiles of EWS-FLI1 in A673 cells upon knockdown of EWS-FLI1 and deubiquitinase USP9X, RNA-seq was performed in A673 cells transfected with EWS-FLI1 esiRNA, USP9X esiRNA or GFP esiRNA as control. The data indicates that USP9X regulates transcriptional profiles of EWS through mediating EWS-FLI1.

2022-01-01 | GSE185429 | GEO

Phf19 links methylated lysine 36 of histone H3 to regulation of Polycomb activity [ChIP-Seq]

Project description:Polycomb group proteins are transcriptional repressors that play essential roles in regulating genes required for differentiation and embryonic development. The Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for lysine 27 on histone 3 (H3K27me3), which is a docking site for the PRC1 complex and leads to gene repression. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of histone H3 methylated at lysine 36 (H3K36me), an mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. We provide structural evidence for this interaction by nuclear magnetic resonance spectroscopy (NMR). Using genome-wide chromatin binding analyses and expression analyses, we show that Phf19 binds to a subset of PRC2 targets in embryonic stem (ES) cells, and that this is required for their repression and for H3K27me3 deposition. These findings reveal that the H3K36me2/3-Phf19 interaction is essential for PRC2 complex activity and for proper regulation of gene repression in ES cells. We determined the genome binding/occupancy profile of Phf19, H3K36me3, H3K36me2, H3K27me3 and Suz12 by high throughput sequencing in mouse embryonic stem cells. For Phf19 two independent biological replicas were performed and Phf19 binding sites were defined as those sites (ChIP-seq peaks) present in both replicas. H3K27me3 was evaluated in control ES cells and cells depleted of Phf19 (shRd and shPhf19 respectively).

2012-10-16 | E-GEOD-41589 | biostudies-arrayexpress

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