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[Preprint]. 2024 Sep 17:2024.09.17.613553.
doi: 10.1101/2024.09.17.613553.

RNA Polymerase II coordinates histone deacetylation at active promoters

Jackson A Hoffman  1 , Kevin W Trotter  1 , Trevor K Archer  1
Affiliations

RNA Polymerase II coordinates histone deacetylation at active promoters

Jackson A Hoffman et al. bioRxiv. .

Update in

Abstract

Nucleosomes at actively transcribed promoters have specific histone post-transcriptional modifications and histone variants. These features are thought to contribute to the formation and maintenance of a permissive chromatin environment. Recent reports have drawn conflicting conclusions about whether these histone modifications depend on transcription. We used triptolide to inhibit transcription initiation and degrade RNA Polymerase II and interrogated the effect on histone modifications. Transcription initiation was dispensable for de novo and steady-state histone acetylation at transcription start sites (TSSs) and enhancers. However, at steady state, blocking transcription initiation increased the levels of histone acetylation and H2AZ incorporation at active TSSs. These results demonstrate that deposition of specific histone modifications at TSSs is not dependent on transcription and that transcription limits the maintenance of these marks.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Figure 1:
Figure 1:. Transcription is dispensable for steady-state and de novo H3K27 acetylation.
A) Differential heatmap of K27ac ChIP-seq signal (log2 Dex/Veh) over Class III GR peaks +/− 2 hours triptolide. B) Browser image of K27ac ChIP-seq and RNAP2 ChIP-seq over ZBTB16 gene. C) Browser image of K27ac ChIP-seq and RNAP2 ChIP-seq over GLUL gene. D) Western blot of whole cell lysates from 4 biological replicates each of A1–2 cells +/− 2 hours triptolide. E) Meta-profiles of representative ChIP-seq and Cut&Tag replicates over Refseq TSSs. F) Meta-profiles of representative RNAP2 ChIP-seq +/− 2 hours triptolide over Start-seq defined active, inactive, and enhancer TSSs. G) Meta-profiles of representative K27ac Cut&Tag +/− 2 hours triptolide over Start-seq defined active, inactive, and enhancer TSSs.
Figure 2:
Figure 2:. Transcription initiation suppresses acetylation and H2AZ incorporation at active TSSs.
A-J) Meta-profiles of representative Cut&Tag replicates +/− 2 hours triptolide over active TSSs. K-M) Heatmaps depicting relative change ((Triptolide – control) / max(control)) in Cut&Tag signal over Refseq TSSs in indicated cell lines.
Figure 3:
Figure 3:. RNAP2 degradation reproduces the effect of inhibiting transcription inhibition.
A-D) Meta-profiles of ChIP-seq and Cut&Tag signal +/− 2 hours flavopiridol over active TSSs. E) Western blots of whole cell lysate from HCT116-mAID-POLR2A cells +/− Auxin or triptolide. F-H) Heatmaps depicting relative change ((auxin – control) / max(control)) in Cut&Tag signal for indicated marks.
Figure 4:
Figure 4:. HDAC inhibition masks the effect of inhibiting transcription initiation.
A) Western blots of whole cell lysates from A1–2 cells treated +/− triptolide, A485, and TSA. B-D) Meta-profiles of Cut&Tag signal in A1–2 cells treated +/− triptolide, A485, and TSA.
See this image and copyright information in PMC

References

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