Investigation of the kinase haspin and mitosis specific histone H3 threonine 3 phosphorylation

Abstract

Histones are subject to a wide range of modifications which are differentially regulated throughout the cell cycle to modulate protein recruitment, chromatin structure, and gene expression. During mitosis, several prominent histone phosphorylation events occur. One such event is histone H3 threonine 3 phosphorylation (H3T3ph), which is enriched at centromeres to recruit the chromosomal passenger complex, a key mitotic regulator. In addition, deposition of H3T3ph on chromatin arms during mitosis is proposed to displace transcription factors that recognise the adjacent histone modification, H3 lysine 4 trimethylation (H3K4me3). This mechanism is known as a phospho-methyl switch. However, the exact localisation of H3T3ph is unclear and whether these two modifications actually colocalise in vivo is unknown. Questions also remain regarding the H3T3 kinase, haspin, such as how its kinase activity is regulated to act during mitosis and whether it may have other functions during interphase. Haspin is also known to protect centromeric cohesion during mitosis via an interaction with the cohesin regulator PDS5. However, whether both PDS5A and PDS5B are equally important in this role is unclear. To address some of these questions we defined the localisation of H3T3ph and other histone marks at the sequence level using ChIP-seq. We show that H3T3ph and H3K4me3 do not colocalise, arguing against the proposed phospho-methyl switch. We also use proximity-based labelling (BioID) to assess the environment in which haspin is found both in interphase and mitosis. Our data suggest that it may specifically be PDS5B which regulates haspin and that this also occurs during interphase, implicating haspin in cohesin regulation throughout the cell cycle. We also identify proteins involved in chromatin organisation and nuclear formation. Overall, our results suggest that haspin is enriched in heterochromatic regions at centromeres and plays a role in the regulation of these chromatin domains in both interphase and mitosis.