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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Eugene Dillon

LAB HEAD: Gerard Cagney

PROVIDER: PXD005381 | Pride | 2018-04-05

REPOSITORIES: Pride

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Cxorf67-FLAG co-immunoprecipitation analysis from 293T cells

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2019-04-09 | PXD013390 | Pride

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Polycomb Repressive Complex 2 methylates Elongin A to regulate transcripiton [BrU-RNA-seq]

Project description:Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of PRC2 and used the resultant data to screen for novel potential targets. The RNA polymerase II (Pol II) transcription factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of many, but not all, PRC2 target genes as measured by both steady state and nascent RNA levels. We propose that PRC2 regulates transcription of a subset of target genes in part via methylation of EloA.

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Project description:G9a/GLP and Polycomb Repressive Complex 2 (PRC2) are two major epigenetic silencing machineries, which in particular methylate histone H3 on lysines 9 and 27 (H3K9 and H3K27), respectively. Although evidence of a crosstalk between H3K9 and H3K27 methylations has started to emerge, their actual interplay remains elusive. Here, we show that PRC2 and G9a/GLP interact physically and functionally. Moreover, combining different genome-wide approaches, we demonstrate that Ezh2 and G9a/GLP share an important number of common genomic targets, encoding developmental and neuronal regulators. Furthermore, we show that G9a enzymatic activity modulates PRC2 genomic recruitment to a subset of its target genes. Taken together, our findings demonstrate an unanticipated interplay between two main histone lysine methylation mechanisms, which cooperate to maintain silencing of a subset of developmental genes. Microarray has been perform in triplicate on total extract RNA from mES cell (TT2 : Wildtype and KOs G9a-/-, GLP-/-, G9a-/- and GLP-/-)

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