Mitotic histone phosphorylation and the complexities of histone covalent modifications

Eukaryotic chromosomes are highly compacted during mitosis thus ensuring faithful segregation of genetic information in each nuclear division. Phosphorylation of many histone and non-histone proteins is closely associated with mitotic chromosome condensation. To analyze the function of histone phosphorylation during mitosis, I chose mammalian cells as a model system. In addition to well-studied H3 phosphorylation on serine 10 (Ser10), I demonstrate that an H3 variant, CENP-A, which localizes to the centromere, is also phosphorylated on serine 7 (Ser7). This phosphorylation occurs during a discrete window of mitosis and displays a pattern that is similar to, but different from, H3 Ser10 phosphorylation. Aurora B kinase has been shown to be the mitotic kinase for both H3 and CENP-A. Aurora B exists in a complex with INCENP and survivin. This complex displays a dynamic localization pattern as the cell progresses through mitosis. The proteins or events responsible for this localization are unknown. Relatively weak interactions have been observed between survivin and H3 phosphorylated on Ser10 and Ser28 and with the unmodified CENP-A N-terminus. These findings have led to the formulation of a model for the interaction between the aurora B mitotic complex and chromatin. In addition, my work has provided new insights into the complexities of histone phosphorylation, identifying Ser1 of H4 and H2A as new mitotic sites and S-phase modifications.; Numerous insights into histone modifications in vivo have been established using modification specific antibodies. My work has demonstrated that H3 K9 methylation and H3 Serl0 phosphorylation exist on the same H3 N-terminus in vivo. Therefore the possibility exists for epitope disruption or occlusion. In order to properly interrupt results obtained from the use of modification-specific antibodies, it is imperative to know the modification state of surrounding residues. To circumvent this problem, I have been involved in an alternative collaborative approach. By combining purification of histones from HeLa cells with mass spectrometry, we have been able to identify a complex array of modifications present on histones. Understanding the patterns of modifications and complexity on the histones remains an important challenge of future studies.