Product binding enforces the genomic specificity of a yeast Polycomb repressive complex
Ontology highlight
ABSTRACT:
ORGANISM(S): Cryptococcus neoformans
PROVIDER: GSE61550 | GEO | 2014/11/10
SECONDARY ACCESSION(S): PRJNA261445
REPOSITORIES: GEO
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Project description:Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate impacts on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Importantly, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro while HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.
2023-04-03 | GSE195824 | GEO
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.
2021-09-22 | PXD020684 | Pride
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
Project description:The Polycomb repressive complexes PRC1 and PRC2 act non-redundantly at target genes to maintain transcriptional programs and ensure cellular identity. PRC2 methylates lysine 27 on histone H3 (H3K27me), while PRC1 mono-ubiquitinates histone H2A at lysine 119 (H2Aub1). Here we present engineered mouse embryonic stem cells (ESCs) targeting the PRC2 subunits EZH1 and EZH2 to discriminate between contributions of distinct H3K27 methylation states and the presence of PRC2/1 at chromatin. We generate catalytically inactive EZH2 mutant ESCs, demonstrating that H3K27 methylation, but not recruitment to the chromatin, is essential for proper ESC differentiation. We further show that EZH1 activity is sufficient to maintain repression of Polycomb targets by depositing H3K27me2/3 and preserving PRC1 recruitment. This occurs in the presence of altered H3K27me1 deposition at actively transcribed genes and by a diffused hyperacetylation of chromatin that compromises ESC developmental potential. Overall, this work provides insights for the contribution of diffuse chromatin invasion by acetyltransferases in PRC2-dependent loss of developmental control.
2019-02-28 | GSE116603 | GEO
Project description:Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.
2024-06-27 | GSE246527 | GEO
Project description:In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes: the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes. Keywords: cell type comparison Suz12, Ezh2, Ring1b ChIP-Seq in singlicate from mouse embryonic stem (mES) cells. H3K4me3, H3K27me3, H3K36me3, Ezh2 and Ring1b ChIP-Seq in singlicate from human embyonic stem cells (hES; H9).
2008-11-01 | E-GEOD-13084 | biostudies-arrayexpress
Project description:Heterochromatic histone H3 methylation at lysine 9 (H3K9me2/3) and lysine 27 (H3K27me3) is generally catalyzed by SUV39 and EZH family methyltransferases, respectively, and has distinct functions in most eukaryotes. In contrast, the ciliated protozoan Tetrahymena lacks the SUV39 enzyme, and the EZH enzyme Ezl1p is responsible for the deposition of both H3K9me3 and H3K27me3 during Piwi-directed DNA elimination. Understanding how both modifications are catalyzed by the EZH enzyme should shed light on the mechanism and the evolution of the division of labor between the two heterochromatic histone methyltransferase families in eukaryotes. Here, we show that loss of the novel protein Ded2p abolishes the accumulation of H3K9me3 but not H3K27me3 in Tetrahymena. The phenotypes of DED2 KO cells revealed that H3K27me3 is sufficient for promoting secondary small RNA biogenesis but not for suppressing TEs, assembling heterochromatin bodies and completing DNA elimination. Ded2p associates with Ezl1p through Rnf2p, a component of Tetrahymena Polycomb Repressive Complex 2 (PRC2) to promote the accumulation of H3K9me3. Our results indicate that there are functional distinctions between H3K9me3 and H3K27me3 in different steps of the DNA elimination process and the specificity of the methyltransferase activity of PRC2 can be modulated by its associated factor to catalyze H3K9me3.
2023-04-01 | GSE190308 | GEO
Project description:Methylation of histone H3 lysine 9 (H3K9me) and small RNA are associated with constitutively silent chromatin in diverse eukaryotes including plants. In plants, silent transposons are also marked by cytosine methylation, especially at non-CpG sites. The transposon-specific non-CpG methylation in plants is controlled by small RNA and H3K9me. Although it is often assumed that small RNA directs H3K9me, interaction between small RNA and H3K9me has not been directly shown in plants. We have previously shown that a mutation in a chromatin remodeling gene DDM1 (decrease in DNA methylation) induces a global decrease as well as local increase of cytosine methylation and accumulation of small RNA in a locus called BONSAI. Here we show that the de novo BONSAI methylation does not depend on RNAi but depends on H3K9me. Notably, in mutant of H3K9 methylase gene KRYPTONITE or H3K9me-dependent DNA methylase gene CHROMOMETHYALSE3, the ddm1-induced de novo cytosine methylation was abolished for all three contexts, CpG, CpHpG, and CpHpH. Furthermore, RNAi mutants showed strong developmental defects when combined with ddm1 mutation. Our results revealed unexpected interactions of epigenetic modifications, which could be conserved among diverse eukaryotes. comparison of DNA methylation between WT, 2G ddm1 (2 replications), 8G ddm1 (2 replications), and 8G ddm1 kyp
2011-12-25 | E-GEOD-34222 | biostudies-arrayexpress
Project description:Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit the spread of inappropriate H3K9me. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me—which would lead to aberrant gene silencing and epigenetic instability—is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Together, conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue suggest that the mechanism described here is broadly conserved.
2018-07-17 | GSE102905 | GEO
Project description:Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin, like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells silence genes in traditional Polycomb domains and in generally inactive chromatin like embryonic somatic cells. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly-induced Scm concentrates PRC2 activity on traditional Polycomb domains. Our results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.
2020-08-10 | GSE145282 | GEO