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Polycomb Repressive Complex 2 methylates Elongin A to tune transcript levels of targeted genes [ChIP-seq]

ABSTRACT: Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27). PRC2 is required to maintain gene repression during development and differentiation and misregulation of PRC2 is linked to a range of neoplastic malignancies, activities that are believed to involve H3K27 methylation. The full spectrum of non-histone substrates of PRC2, however, is not known, and it is not known which other substrates might also contribute to the biological functions of PRC2. We characterized the target recognition specificity and substrate diversity of PRC2, and identified more than one hundred potential novel nuclear targets of PRC2. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is an in vivo target of PRC2. Mutation of the EloA residue that is methylated by PRC2 decreases repression of numerous PRC2 target genes. We propose that this functional crosstalk between PRC2 and EloA tunes the level of repression of targeted genes and contributes to the biological functions of PRC2.

ORGANISM(S): Mus musculus

PROVIDER: GSE104659 | GEO | 2017/11/16

SECONDARY ACCESSION(S): PRJNA413411

REPOSITORIES: GEO

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Project description:Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. In an effort to explore regulatory roles for Elongin A, we performed IP-MS. We constructed a DLD1 cell line expressing Flag-tagged Elongin A at endogenous levels and performed anti-Flag immuno-purification of solubilized chromatin followed by analysis of binding partners by mass spectrometry in triplicates. We identified a large group of Elongin A-associated proteins. We confirmed a subset of possible interacting proteins by Co-IP and western blotting. Consistent with previous studies, we identified RNA Pol II subunits and proteins related to transcription elongation and RNA processing. Among the top hits, we identified nearly all subunits of the PAF1 complex, except for RTF1, which does not stably associate with mammalian PAF1. Concordantly, in our previous PAF1 proteomic study, Elongin A was also among the top hits. We also identified several subunits of the Integrator complex, a multi-subunit complex that has been shown to participate in enhancer RNA (eRNA) processing, a finding consistent with our conclusion that Elongin A localizes to potential enhancers. Collectively, our results suggest that Elongin A stably interacts with RNAPII and the transcription machinery on chromatin. Taken together, our studies suggest that Elongin A associates with the transcription machinery at actively transcribed genomic regions and may be involved in the release of paused RNAPII. However, Elongin A does not appear to be critical for maintaining transcription elongation rates in vivo.

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Polycomb Repressive Complex 2 methylates Elongin A to tune transcript levels of targeted genes [ChIP-seq]

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