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Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells.

ABSTRACT: Polycomb repressive complex 2 (PRC2) silences expression of developmental transcription factors in pluripotent stem cells by methylating lysine 27 on histone H3. Two mutually exclusive subcomplexes, PRC2.1 and PRC2.2, are defined by the set of accessory proteins bound to the core PRC2 subunits. Here we introduce separation-of-function mutations into the SUZ12 subunit of PRC2 to drive it into a PRC2.1 or 2.2 subcomplex in human induced pluripotent stem cells (iPSCs). We find that PRC2.2 occupies polycomb target genes at low levels and that homeobox transcription factors are upregulated when this complex is exclusively present. In contrast with previous studies, we find that chromatin occupancy of PRC2 increases drastically when it is forced to form PRC2.1. Additionally, several cancer-associated mutations also coerce formation of PRC2.1. We suggest that PRC2 chromatin occupancy can be altered in the context of disease or development by tuning the ratio of PRC2.1 to PRC2.2.

SUBMITTER: Youmans DT

PROVIDER: S-EPMC7867654 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells

Project description:Polycomb repressive complex 2 (PRC2) silences expression of developmental transcription factors in pluripotent stem cells by methylating lysine 27 on histone H3. Two mutually exclusive subcomplexes, PRC2.1 and PRC2.2, are defined by the set of accessory proteins bound to the core PRC2 subunits. Here we introduce separation-of-function mutations into the SUZ12 subunit of PRC2 to drive it into a PRC2.1 or 2.2 subcomplex in human induced pluripotent stem cells (iPSCs). We find that PRC2.2 occupies polycomb target genes at low levels and that homeobox transcription factors are upregulated when this complex is exclusively present. In contrast with previous studies, we find that chromatin occupancy of PRC2 increases drastically when it is forced to form PRC2.1. Additionally, several cancer-associated mutations also coerce formation of PRC2.1. We suggest that PRC2 chromatin occupancy can be altered in the context of disease or development by tuning the ratio of PRC2.1 to PRC2.2.

2020-12-17 | GSE150588 | GEO

Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells

Project description:Competition between PRC2.1 and 2.2 subcomplexes regulates PRC2 chromatin occupancy in human stem cells

| PRJNA632872 | ENA

RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells.

Project description:Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, the biological importance of this RNA binding remains unsettled. Here, we tackle this question in human induced pluripotent stem cells by using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNase A, by a chemical inhibitor of transcription or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome wide. The physiological relevance of PRC2-RNA interactions is further underscored by a cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.

| S-EPMC10353856 | biostudies-literature

LncRNA HBL1 is required for genome-wide PRC2 occupancy and function in cardiogenesis from human pluripotent stem cells.

Project description:Polycomb repressive complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of the RNA-PRC2 interaction in human cardiogenesis remains elusive. Here, we found that human-specific heart brake lncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss of JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. In addition, ChIP-seq revealed reduced EED occupancy on 62 overlapped cardiogenic genes in HBL1-/- and JARID2-/- hPSCs, indicating that the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurs, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved 'microRNA-1-JARID2' axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis.

| S-EPMC8276986 | biostudies-literature

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

Project description: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 this 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.

| S-EPMC7994559 | biostudies-literature

Interactions between JARID2 and noncoding RNAs regulate PRC2 recruitment to chromatin.

Project description:JARID2 is an accessory component of Polycomb repressive complex-2 (PRC2) required for the differentiation of embryonic stem cells (ESCs). A role for JARID2 in the recruitment of PRC2 to target genes silenced during differentiation has been put forward, but the molecular details remain unclear. We identified a 30-amino-acid region of JARID2 that mediates interactions with long noncoding RNAs (lncRNAs) and found that the presence of lncRNAs stimulated JARID2-EZH2 interactions in vitro and JARID2-mediated recruitment of PRC2 to chromatin in vivo. Native and crosslinked RNA immunoprecipitations of JARID2 revealed that Meg3 and other lncRNAs from the imprinted Dlk1-Dio3 locus, an important regulator of development, interacted with PRC2 via JARID2. Lack of MEG3 expression in human induced pluripotent cells altered the chromatin distribution of JARID2, PRC2, and H3K27me3. Our findings show that lncRNAs facilitate JARID2-PRC2 interactions on chromatin and suggest a mechanism by which lncRNAs contribute to PRC2 recruitment.

| S-EPMC4026005 | biostudies-literature

RNA is essential for PRC2 chromatin occupancy and function in human pluripotent stem cells

Project description:Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, whether this RNA binding has any biological importance remains unsettled. Here we tackle this question in human induced pluripotent stem cells using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNaseA, by a chemical inhibitor of transcription, or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome-wide. The physiological relevance of PRC2-RNA interactions is further underscored by the dramatic cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.

2020-05-31 | GSE128135 | GEO

MNase titration reveals differences between nucleosome occupancy and chromatin accessibility.

Project description:Chromatin accessibility plays a fundamental role in gene regulation. Nucleosome placement, usually measured by quantifying protection of DNA from enzymatic digestion, can regulate accessibility. We introduce a metric that uses micrococcal nuclease (MNase) digestion in a novel manner to measure chromatin accessibility by combining information from several digests of increasing depths. This metric, MACC (MNase accessibility), quantifies the inherent heterogeneity of nucleosome accessibility in which some nucleosomes are seen preferentially at high MNase and some at low MNase. MACC interrogates each genomic locus, measuring both nucleosome location and accessibility in the same assay. MACC can be performed either with or without a histone immunoprecipitation step, and thereby compares histone and non-histone protection. We find that changes in accessibility at enhancers, promoters and other regulatory regions do not correlate with changes in nucleosome occupancy. Moreover, high nucleosome occupancy does not necessarily preclude high accessibility, which reveals novel principles of chromatin regulation.

| S-EPMC4859066 | biostudies-literature

The influence of transcription factor competition on the relationship between occupancy and affinity.

Project description:Transcription factors (TFs) are proteins that bind to specific sites on the DNA and regulate gene activity. Identifying where TF molecules bind and how much time they spend on their target sites is key to understanding transcriptional regulation. It is usually assumed that the free energy of binding of a TF to the DNA (the affinity of the site) is highly correlated to the amount of time the TF remains bound (the occupancy of the site). However, knowing the binding energy is not sufficient to infer actual binding site occupancy. This mismatch between the occupancy predicted by the affinity and the observed occupancy may be caused by various factors, such as TF abundance, competition between TFs or the arrangement of the sites on the DNA. We investigated the relationship between the affinity of a TF for a set of binding sites and their occupancy. In particular, we considered the case of the transcription factor lac repressor (lacI) in E.coli, and performed stochastic simulations of the TF dynamics on the DNA for various combinations of lacI abundance and competing TFs that contribute to macromolecular crowding. We also investigated the relationship of site occupancy and the information content of position weight matrices (PWMs) used to represent binding sites. Our results showed that for medium and high affinity sites, TF competition does not play a significant role for genomic occupancy except in cases when the abundance of the TF is significantly increased, or when the PWM displays relatively low information content. Nevertheless, for medium and low affinity sites, an increase in TF abundance (for both cognate and non-cognate molecules) leads to an increase in occupancy at several sites.

| S-EPMC3785477 | biostudies-literature

Engaging chromatin: PRC2 structure meets function.

Project description:Polycomb repressive complex 2 (PRC2) is a key epigenetic multiprotein complex involved in the regulation of gene expression in metazoans. PRC2 is formed by a tetrameric core that endows the complex with histone methyltransferase activity, allowing it to mono-, di- and tri-methylate histone H3 on lysine 27 (H3K27me1/2/3); H3K27me3 is a hallmark of facultative heterochromatin. The core complex of PRC2 is bound by several associated factors that are responsible for modulating its targeting specificity and enzymatic activity. Depletion and/or mutation of the subunits of this complex can result in severe developmental defects, or even lethality. Furthermore, mutations of these proteins in somatic cells can be drivers of tumorigenesis, by altering the transcriptional regulation of key tumour suppressors or oncogenes. In this review, we present the latest results from structural studies that have characterised PRC2 composition and function. We compare this information with data and literature for both gain-of function and loss-of-function missense mutations in cancers to provide an overview of the impact of these mutations on PRC2 activity.

| S-EPMC7000746 | biostudies-literature

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