Regulation and Pathophysiology of H2B Serine 6 Phosphorylation

Abstract

Histone modifications play a vital role in controlling the function of the genome. Some histone phosphorylations function as important signals during mitosis. Previous work showed transient phosphorylation of H2B S6 at the inner centromeres during mitosis between metaphase and early anaphase. To understand the molecular mechanisms restricting the occurrence of this histone modification in space and time, the responsible phosphatase complex and its regulation were identified by in vitro and in vivo experiments. CDK1 phosphorylates H2B upon mitotic entry, but this phosphorylation is counteracted by the phosphatase activity of PP1 on the chromosome arms. Two catalytic phosphatase subunits, PP1α and PP1 γ , associate with the phosphatase regulator RepoMan. The local inhibition of the PP1/RepoMan complex by Aurora B through phosphorylation of PP1/RepoMan preserves H2B S6ph at the centromeres. The motor protein Mklp2 contributes to the re-localisation of Aurora B from chromatin to the central spindle during anaphase, which relieves Aurora B-dependent inhibition of the PP1/RepoMan complex to enable dephosphorylation of this histone modification. This relocalisation is observed to be dysregulated in some tumour cells. The resulting deregulation of the Aurora B/PP1/RepoMan axis can lead to delayed H2B S6 dephosphorylation, which might contribute to chromosomal instability. To understand the physiological function of H2B S6ph (and other histone PTMs), this study initiated the development of a novel inducible histone replacement system in vertebrate cells. Towards this goal, the theoretical framework for a tetracycline-inducible histone replacement system based on shRNA-mediated knock-down and re-expression was developed. Chicken cells were identified as a suitable tetrapod model system due to their limited number of histone genes. The first version of this system (V1) optimised the shRNA design strategy to ensure efficient knock-down of endogenous H2B and further histones. Stable cell clones were generated that allowed efficient knocked-down of H2B expression, resulting in drastically reduced histone expression and cell death. Simultaneous re-expression of H2B rescued the knock-down cells and allowed robust re-expression of H2B in cell pools. As random chromatin integration of this system compromises the comparability of different cell clones, a second version of this system (V2) was developed. This employs the integration of genomic landing pads to allow Bxb1 recombinase-mediated cassette exchange of integrated histone cassettes with mutant histones. Maximal protein expression using this system was limited, requiring further analysis and optimisation. Together, this study provides a proof-of-concept for such a histone replacement system in tetrapods. Such a system will in the future allow for the investigation of the function of various histone PTMs.