title: Roles of nucleosome asymmetry and KAT6B-mediated histone acetylation in the regulation of bivalent promoters
Creators: name: Valsakumar, Devisree
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: epigenetics
subject: chromatin
subject: histone modifications
subject: bivalent nucleosome
subject: chromatin remodellers

description: Histone modifications are key regulators of gene expression. Nucleosomes can be modified both symmetrically and asymmetrically, carrying a specific modification on one or both sister histones within a nucleosome. Bivalent nucleosomes are a special case of asymmetric nucleosomes, carrying the active mark histone H3 lysine 4 trimethylation (H3K4me3) and the repressive mark H3K27me3 in an asymmetric fashion. Bivalency is postulated to fine-tune gene expression during development, however, the mechanisms by which bivalent genes are poised for expression are still not well understood. To determine how nucleosomal asymmetry regulates histone mark readout, I generated asymmetric nucleosomes and performed nucleosome pulldown assays. H3K4me3 binders exhibited differential binding to symmetric and asymmetric nucleosomes, whereas more abundant H3K27me3 binders such as Polycomb complex subunits were recruited similarly to both symmetric and asymmetric nucleosomes. While nucleosomal asymmetry affects reader domains for H3K4me3 and H3K27me3 similarly, differential reader abundance and basal nucleosome affinity of repressive binders drive differential enrichment of active and repressive binders on asymmetric nucleosomes in embryonic stem cells. Accordingly, asymmetric bivalent nucleosomes attract H3K27me3 binders but fail to enrich activating H3K4me3 binders. These findings show that nucleosomal asymmetry is critical for histone mark readout and function, providing a mechanism for differential recruitment of active and repressive complexes at bivalent domains. Bivalent nucleosomes also attract specific binders that are not enriched by the individual marks, including the histone acetyltransferase complex KAT6B. This finding suggests that KAT6B recruitment involves a combinatorial readout of H3K4me3, unmodified H3K4 (H3K4un), and H3K27me3. However, KAT6B does not possess any known binding domains for H3K27me3. I tested whether the catalytic MYST domain interacts with H3K27me3 in the context of acetylating H3K23 or whether KAT6B interacts with PRC1. MYST domain indeed exhibited a modestly increased affinity for nucleosomes in the presence of H3K27me3. In addition, proximity labelling suggests that KAT6B interacts with PRC1, providing an additional mechanism for interaction recruitment of KAT6B to bivalent nucleosomes. This interaction was further confirmed via immunoprecipitation. These observations suggests that a combination of direct KAT6B interaction with H3K27me3 and indirect interaction via PRC1 mediates its enhanced recruitment at bivalent genes. The presence of KAT6B at bivalent genes suggests that its marks H3K23ac and H3K14ac regulate readout of bivalent nucleosomes. Nucleosome pulldown-based proteomics analysis revealed that H3K23ac enriches the chromatin remodellers SWI/SNF and ISWI at bivalent nucleosomes, suggesting that H3K23ac promotes resolution of bivalency towards activation during differentiation via recruitment of chromatin remodellers. While further investigation is required to understand the underlying mechanisms, these studies explore how nucleosome asymmetry, histone acetylation and chromatin remodellers work in concert to shape the regulatory landscape at bivalent domains.