Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation

Abstract

The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs) 1 – 3 . While recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions 4 – 8 , the function of H3.3 in transcription regulation has been less clear 9 – 16 . Here, we find that H3.3-specific phosphorylation 17 – 19 stimulates activity of the acetyltransferase p300 in trans , suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Cells lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and globally impact genome regulation, which may help better understand how mutations in these proteins contribute to human cancers 20 , 21 .

Differential contribution of p300 and CBP to regulatory elements in mESCs

“…Overall, and in agreement with our previous observations and others (Raisner et al 2018;Martire et al 2019), these data demonstrate that maintenance of the open chromatin state in mESCs is independent of the high levels of H3K27ac enrichment observed at these regions in wild-type mESCs.…”

“…Given the high correlation between the "open" chromatin state and histone acetylation, we were surprised that reduced p300 and the subsequent reduction in H3K27 acetylation did not affect chromatin accessibility at acetylated regions. Nevertheless, this data aligns with our previous observation that a significant reduction of p300 HAT activity (i.e., H3K27ac) fails to "close" chromatin at p300 targets (Martire et al 2019). It is also supported by a recent study in which small-molecule inhibition of the CBP/p300 bromodomain reduced global H3K27ac without changing the open chromatin state (Raisner et al 2018).…”

“…Although acetylation has long been correlated with gene expression, we and others find that reduced acetylation is well-tolerated in mESCs (Dorighi et al 2017;Rickels et al 2017;Martire et al 2019). It is possible that under culture conditions regulatory elements are acetylated more robustly than is required for a functional readout of this modification.…”

“…Chromatin Immunoprecipitation (ChIP). Native and crosslinking (X) ChIP were performed with 5×10 6 and 1×10 8 cells, respectively, as previously described (Martire et al 2019) with slight modifications. A spike-in normalization strategy was used to normalize all ChIP-seq data to reduce the effects of technical variation and sample processing bias.…”

Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells

“…Replacing lysine 27 with a nonmodifiable residue in one of the two genes encoding H3.3 in Drosophila causes male lethality and infertility and reduced global H3K27ac levels [25]. A similar reduction in H3K27ac levels was also observed in mouse embryonic stem cells (ESCs) depleted of H3.3 [26]. Therefore, we reasoned that targeting enhancer H3K27ac might be achievable by mutating both H3.3encoding genes (H3f3a and H3f3b) and producing a homogenous population of H3.3 lysine 27-to-arginine (K27R) mutant histones in mouse ESCs.…”