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Chromatin Remodeler EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation
Author(s) -
Pradhan Suman Kalyan,
Su Trent,
Yen Linda,
Jacquet Karine,
Cote Jacques,
Kurdistani Siavash,
Carey Michael
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.803.9
Subject(s) - chromatin , promoter , chia pet , chromatin remodeling , nucleosome , histone h3 , bivalent chromatin , histone code , histone , biology , microbiology and biotechnology , chromatin immunoprecipitation , enhancer , histone h1 , activator (genetics) , transcription factor , gene expression , gene , genetics
Increasing evidence indicates that gene activation in metazoans is accompanied by deposition of the histone variants H2AZ and H3.3 into promoters and enhancers. The abundance of double variant nucleosomes correlates well with the levels of gene expression. However, knowledge is limited for how variant chromatin is assembled in regulatory regions and, more importantly, whether it plays a direct role in gene activation. To unravel the mechanism, we have recreated activator‐ and HAT‐stimulated transcription from H3.3/H2AZ variant chromatin in HeLa nuclear extracts. We further employed Immobilized template assays to identify variant chromatin specific chromatin proteins. Analysis of preinitiation complex (PIC) revealed that the chromatin remodeling factor EP400 was specifically enriched on variant versus canonical chromatin. In a cell based assay, EP400 was recruited to an integrated inducible promoter in U2OS cells in response to gene activation. Moreover, siRNA knockdown of EP400 led to diminished transcription and reduced assembly of H3.3‐containing chromatin, while having little or no effect on binding of the Mediator co‐activator. Remarkably, this observation was paralleled genome‐wide at promoters and enhancers, where knockdown of EP400 significantly affected H3.3 deposition, but not Mediator recruitment. We investigated the mechanism by in vitro histone exchange assay. EP400 is known to exchange H2AZ for H2A but in biochemical histone exchange assays, it also inserted H3.3 for H3.1 in a nucleosome dependent manner. Interestingly, a side‐by‐side comparison of histone exchange assay indicates that reverse exchange, i.e. exchange of canonical histone into variant chromatin is much less efficient. However, H3.3 exchange into variant chromatin is highly efficient. We reason that this dynamic exchange of H3.3 with H3.3 may play a role in enhaced transcription. What confers this specificity remains an open question. To this end we are currently exploring the contribution of Brd8, a bromodomain containing protein that resides in the same complex and happens to interact directly with EP400. Indeed, we have preliminary evidence from modified histone peptide array experiments that suggest that Brd8‐EP400 duo is significantly enriched in H3K18ac and H3K27ac histone marks. This raises further likelihood of collaboration between EP300 and EP400 in the context of gene activation. Our data support a model in which EP400 links variant chromatin assembly directly to gene activation but downstream of PIC assembly in a pathway parallel to pre‐initiation complex assembly but necessary for transcription on chromatin. Support or Funding Information L.Y. is supported by the Ruth L. Kirschstein National Research Service Award GM007185