The role of histone phosphorylations in chromatin regulation during mitosi

Abstract

A network of epigenetic components tightly regulates chromatin structure and accessibility, including histone modifications, transcription factors and reader proteins. The epigenetic landscape changes dramatically during mitotic chromosome condensation. Notably, histone phosphorylations increase significantly, contributing to chromosome segregation and reader protein recruitment. However, the functional role of many mitotic histone phosphorylations remains unclear. Histone H3 Serine 10 phosphorylation (H3S10ph), the most frequently studied mitotic phosphorylation, begins to enrich at the centromeric regions in late G2 of the cell cycle, before spreading along chromosome arms by prometaphase. However, whether significant H3S10ph enrichment occurs at specific loci remains unclear, and different studies have reached contrasting conclusions regarding the functions of H3S10ph. One possible issue is that traditional chromatin immunoprecipitation techniques struggle to distinguish subtle enrichments of abundant marks, preventing understanding of the detailed distribution of mitotic histone phosphorylations. To address this, we develop the CUT+RUN technique for use in mitotic HeLa cells. We also validate a quantitative ChIP approach, MINUTE-ChIP, targeting H3S10ph and H3S28ph in mouse embryonic stem cells. We develop an integrative analysis approach, combining MINUTE-ChIP-sequencing with chromatin epigenetic state data, to produce a high-resolution map of mitotic H3S10ph and H3S28ph enrichment in the context of the chromatin regulatory landscape. For the first time, we reveal significant mitotic enrichment peaks in both H3S10ph and H3S28ph at promoters in specific regulatory states, and identify gene ontology functional pathways correlating with these enrichments. We also analyse ChIP-seq of H3 threonine 3 phosphorylation (H3T3ph) and identify significant H3T3ph peaks at promoters in the absence of inhibitory adjacent marker H3 lysine 4 trimethylation (H3K4me3). We conclude that histone phosphorylations are significantly enriched at promoters in mitosis, dependent on the local chromatin state, and propose that previous findings linking histone phosphorylation to interphase gene regulation should be expanded to consider the mitotic gene regulatory context.