Chromatin Remodeler EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation
Bibliographic record
Abstract
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
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".