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Non-core subunits of the PRC2 complex are collectively required for its target site specificity

ABSTRACT: The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation across the genome, which through involvement in transcriptional regulation is critical in establishment of cell identity. Because of its essential function during development and in cancer, understanding the establishment of genome-wide H3K27 methylation patterns has been the focus of intense investigation. PRC2 methylation activity is abundant and dispersed throughout the genome, but highest activity is specifically directed to a subset of target regions that are stably occupied by the complex and highly enriched for H3K27me3. Here we show, by systematically knocking out single and multiple non-core subunits of the PRC2 complex in mouse embryonic stem cells (mESCs), that they each contribute in directing PRC2 activity to targets sites. Furthermore, combined knockout of six non-core subunits reveal that, while dispensable for global H3K27 methylation levels, the non-core PRC2 subunits are collectively required for focusing H3K27me3 activity to specific sites in the genome.

ORGANISM(S): Mus musculus

PROVIDER: GSE127804 | GEO | 2019/09/11

REPOSITORIES: GEO

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Physical and functional screening of Polycomb Repressor Complex 2 highlights novel regulators of histone H3 lysine 27 methylation status

Project description:Polycomb Repressor Complex 2 (PRC2) is a multi-protein epigenetic regulator complex that plays critical roles in early development and tissue differentiation. The complex catalyzes the methylation of histone H3 lysine 27 (H3K27). The tri-methyl state (H3K27me3) is well studied as an agent of gene repression, but more recently distinct roles for the mono- and di-methyl states have been discovered. In order to more fully understand how PRC2-associated factors might influence H3K27 status, we screened for physical associations of PRC2 subunits. Knockdown of these factors was used to produce functional interaction maps focused on H3K27 methylation, including proteins that have positive and negative effects on the levels each histone mark.

2018-04-05 | PXD005381 | Pride

Accurate H3K27 methylation patterns can be established de novo by SUZ12-directed PRC2

Project description:The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation and is required for maintaining transcriptional patterns and cellular identity, but the specification and maintenance of genomic PRC2 binding and H3K27 methylation patterns remain incompletely understood. Epigenetic mechanisms have been proposed, wherein pre-existing H3K27 methylation directs recruitment and regulates the catalytic activity of PRC2 to support its own maintenance. Here we investigate if such mechanisms are required for specifying H3K27 methylation patterns in mouse embryonic stem cells. Through genetic knockouts of subunits, we find that PRC2 is responsible for all levels of H3K27 methylation, and that methylation patterns can be accurately established de novo, when a subunit is reintroduced in the absence of pre-existing H3K27 methylation and PRC2 chromatin binding. Furthermore, we find that the core-subunit SUZ12 directs genomic binding of PRC2, which is essential for establishing correct methylation patterns.

2018-01-12 | GSE103685 | GEO

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Project description:We report different roles of three PRC2 components, KMT6, P55 and SIN3 played in distribution of genome-wide H3K27 tri-methylation of Pyricularia oryzae. Polycomb repressive complex 2 (PRC2), which is composed of Ezh(Kmt6), Su(z)12, Esc(Eed) and accessory subunits RbAp48/Nurf55(P55), catalyzes the methylation of histone H3 at lysine 27. Loss of P55 caused severe global defects in the normal distribution of H3K27me3 and transcriptional reprogramming on the H3K27me3-occupied genes. Furthermore, we found that the Sin3 histone deacetylase complex was required to sustain H3K27me3 occupancy and stably maintain gene repression by directly interacting with P55.

2022-02-20 | GSE195863 | GEO

Paramecium Polycomb Repressive Complex 2 physically interacts with the small RNA binding PIWI protein to repress transposable elements

Project description:Polycomb Repressive Complex 2 (PRC2) maintains transcriptionally silent genes in a repressed state via deposition of histone H3 K27 trimethyl (me3) marks. PRC2 has also been implicated in silencing transposable elements (TEs) yet how PRC2 is targeted to TEs remains unclear. To address this question, we performed tandem affinity purification combined with mass spectrometry and identified proteins that physically interact with the Paramecium Enhancer-of-zeste Ezl1 enzyme, which catalyzes H3K9me3 and H3K27me3 deposition at TEs. We show that the Paramecium PRC2 core complex comprises four subunits, each required in vivo for catalytic activity. We also identify PRC2 cofactors, including the RNA interference (RNAi) effector Ptiwi09, which are necessary to target H3K9me3 and H3K27me3 to TEs. We find that the physical interaction between PRC2 and the RNAi pathway is mediated by a RING finger protein and that small RNA recruitment of PRC2 to TEs is analogous to the small RNA recruitment of H3K9 methylation SU(VAR)3-9 enzymes.

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Cxorf67-FLAG co-immunoprecipitation analysis from 293T cells

Project description:The regulation of gene expression is controlled in part by post-translational modifications to histone proteins. Methylation at histone H3, lysine 27 (H3K27), which is catalyzed by Polycomb repressive complex 2 (PRC2), is associated with silenced chromatin. Previous studies have identified dysregulation of H3K27 methylation in pediatric diffuse intrinsic pontine gliomas (DIPGs), the majority of which feature mutation of lysine 27 to methionine. This “oncohistone” potently inhibits PRC2 activity and leads to a global reduction in H3K27 methylation. Similar to DIPG, posterior fossa type A (PFA) ependymomas also show low levels of H3K27 methylation. Although PFAs do not possess the H3K27M oncohistone mutation, they do show increased expression of Cxorf67. Interestingly, Cxorf67 contains a C-terminal sequence that resembles the sequence surrounding H3K27, and we find that this portion of Cxorf67 inhibits PRC2 activity to an even greater extent than the H3K27M oncohistone. Thus, we suggest re-naming Cxorf67 as EZHIP (Enhancer of Zeste Homologs Inhibitory Protein). Furthermore, when expressed in 293T cells, Cxorf67 interacts with several members of PRC2 and induces changes in H3K27 methylation patterns that mirror the changes in H3K27 methylation induced by expression of H3K27M. We propose that PFAs have dysregulated H3K27 methylation by a mechanism that involves inhibition of PRC2 by Cxorf67, which could drive tumorigenesis.

2019-04-09 | PXD013390 | Pride

Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development

Project description:Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis. Examination of two different types of histone modifications (H3K27me3 and H3K4me3) in spermatocytes at two different stages of development (P12 and P17) and the DNA binding protein, EED, in spermatocytes at one stage of development, P17. At P12, there was a single replicate of H3K27me3, H3K4me3 and input. At P17, there were two replicates of H3K27me3, H3K4me3 and input and a single replicate for EED.

2014-10-01 | E-GEOD-61902 | biostudies-arrayexpress

Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

Project description:Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by catalysing trimethylation of histone H3 lysine 27 (H3K27me3), resulting in gene repression. PRC2 consists of two sub-complexes, PRC2.1 and PRC2.2, in which the PRC2 core associates with distinct ancillary subunits such as MTF2 and JARID2, respectively. Both MTF2, present in PRC2.1, and JARID2, present in PRC2.2, play a role in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). However, it remains unclear how these distinct sub-complexes cooperate to establish H3K27me3 domains. Here, we combine a range of Polycomb mutant mESCs with chemical inhibition of PRC2 catalytic activity, to systematically dissect their relative contributions to PRC2 binding to target loci. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, with mutually reinforced binding. Part of the cross-talk between PRC2.1 and PRC2.2 occurs via their catalytic product H3K27me3, which is bound by the PRC2 core-subunit EED, thereby mediating a positive feedback. Strikingly, removal of either JARID2 or H3K27me3 only has a minor effect on PRC2 recruitment, whereas their combined ablation largely attenuates PRC2 recruitment. This strongly suggests an unexpected redundancy between JARID2 and EED-H3K27me3-mediated recruitment of PRC2. Furthermore, we demonstrate that all core PRC2 recruitment occurs through the combined action of MTF2-mediated recruitment of PRC2.1 to DNA and PRC1-mediated recruitment of JARID2-containing PRC2.2. Both axes of binding are supported by EED-H3K27me3 positive feedback, but to a different degree. Finally, we provide evidence that PRC1 and PRC2 mutually reinforce reciprocal binding. Together, these data disentangle the interdependent and cooperative interactions between Polycomb complexes that are important to establish Polycomb repression at target sites.

2020-08-07 | PXD014290 | Pride

Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

2021-09-22 | PXD020684 | Pride

EZH1 in Germ Cells Safeguards the Function of PRC2 during Spermatogenesis

Project description:We previously reported the requirement of Polycomb Repressive Complex 2 (PRC2) for spermatogenesis through transcriptional repression of somatic genes and meiosis-specific genes. To characterize how PRC2's two methyltransferase subunits, EZH1 and EZH2, regulate histone H3 lysine 27 (H3K27) methylation during germ cell development, we generated mouse models with a germline ablation of EZH1 and/or EHZ2. Only the combined loss of EZH1 and EZH2 caused a depletion of global H3K27me3 marks and meiotic arrest in spermatocytes. Genome-wide analysis of H3K27me3 in spermatogenic cells revealed that a noncanonical EZH1-PRC2 could establish and maintain this histone mark on somatic genes and certain meiotic genes. Consistent with it having active enhancers in testis, Ezh1 was not only abundant in highly differentiated spermatocytes but also in actively proliferating progenitor and stem germ cells. Taken together, our findings suggest that the expression level of Ezh1 determines the restoration of H3K27 methylation in the absence of the canonical EZH2-PRC2.

2017-03-07 | GSE95225 | GEO

Gemone-wide maps of PRC2 complex components and chromatin states in prostate cancer cell lines

Project description:We report the high-throughput profiling of PRC2 core subunits chromatin states and histone modifications in two prostate cancer cell lines, androgen-dependent cells LNCaP and androgen-independent cells LNCaP-abl (abl). By obtaining over 1 billion bases of sequence from chromatin immunoprecipitated DNA, we generated the genome-wide localizations of EZH2, SUZ12 and H3K27 trimethylation in both cell lines, and found that EZH2 can be localized to a subsets of chromating sites that don't have H3K27me3 or SUZ12 signals nearby. Interestingly, these sites are enriched for the active histone marks H3K4 di/trimethylation as well as the RNA polymerase II, which suggest the potential function of these peaks in gene activation. Indeed, these sites are enriched near the transcription start sites of EZH2-activated genes. Further analysis of the transcription factor motifs enriched at these peaks revealed the enrichement of androgen receptor motif, suggesting a co-activator role for EZH2 in concert with AR. Our work demonstrated a novel funtion of EZH2 in transcriptional activation by directly binding to the chromatin sites that cooperate with AR. Study of the chromatin localizations of PRC2 complex core subunits and different histone marks in 2 cell types

2012-11-30 | E-GEOD-39459 | biostudies-arrayexpress

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