Histone H1 phosphorylation during mitosis : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand

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Histone H1 phosphorylation is important for the regulation of high order chromosome organisation during mitosis. One of these phosphorylation sites in the linker histone subtype H1.4 is shown here to be phosphorylated by Aurora B kinase, a master regulator of mitosis. Altered phosphorylation of H1.4 on this phosphorylation site at serine 27 illustrated the significance of the timing of this phosphorylation. When serine 27 of H1.4 is mutated to prevent this phosphorylation chromosome congression to the equatorial plate during metaphase is hindered. In contrast, in the presence of the constitutive H1.4 serine 27 phosphorylation mimic, bridging and lagging chromosomes occurred, leading to a corresponding increase in the proportion of cells with a micronucleus. These phenotypes could be brought about through disruption of the Heterochromatin protein 1 family members bound to the adjacent methylated lysine. Such aberrations during mitosis can lead to genetic instability and ultimately aneuploidy, a hallmark of cancer. With the frequently reported over-expression of Aurora B in cancer this shows another mechanism in which this kinase, via histone H1.4 phosphorylation, can push a cell toward malignancy. Another important mitotic kinase, Cyclin dependent kinase 1 together with cyclin B, is responsible for the hyperphosphorylation of histone H1.4 during mitosis; which is required for condensing the cells genetic information into highly compact metaphase chromosomes. This vital mitotic event ensures the faithful transmission of the duplicated DNA into the dividing daughter cells. The mechanisms through which histone H1 hyperphosphorylation contribute to chromosome condensation are poorly understood. One mechanism through which this may occur is via the recruitment of condensation factors such as the condensins or Topoisomerase II. Here the interaction between the Condensin I subunit, CAPD2, and histone H1.4 is explored. CAPD2 interacts with the two most prominent linker histone subtypes, H1.4 and H1.2, through their C-terminal tails. H1.4 and CAPD2 can interact in vitro whilst each is phosphorylated by cyclin dependent kinase as they are during mitosis, in a manner dependent on RNA. Overall, these results indicate that histone H1.4 is a vital component of higher order chromatin and its phosphorylation is essential for the normal progression through mitosis.
Material removed from thesis for copyright reasons: Figure 1.1. The hierarchy of chromatin folding. From Felsenfeld, G., & Groudine, M. (2003). Controlling the double helix. Nature, 421(6921), 448-453. Figure 1.2. The structure of the nucleosome core. From Luger, K., Mäder, A. W., Richmond, R. K., Sargent, D. F., & Richmond, T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature, 389(6648), 251-260. Figure 1.3. The chromatosome. Figure retrieved from https://mbi-figure.storage.googleapis.com/figure/1389942837319.jpg. Figure 1.4. Sequence alignment of the somatic main H1 subtypes. Adapted from Hergeth, S. P., & Schneider, R. (2015). The H1 linker histones: Multifunctional proteins beyond the nucleosomal core particle. EMBO Reports, 16(11), 1439-1453. Figure 1.6. The mechanism of HP1 dislodgement from H1.4. Adapted from Fischle, W., Tseng, B. S., Dormann, H. L., Ueberheide, B. M., Garcia, B. A., Shabanowitz, J., Hunt, D. F., Funabiki, H., & Allis, C. D. (2005). Regulation of HP1–chromatin binding by histone H3 methylation and phosphorylation. Nature, 438(7071), 1116-1122. doi:10.1038/nature04219 Figure 4.1. Comparing H1.4S27 phosphorylation and the mutations that prevent or mimic this phosphorylation. Adapted from Anthis, N. J., Haling, J. R., Oxley, C. L., Memo, M., Wegener, K. L., Lim, C. J., Ginsberg, M. H., & Campbell, I. D. (2009). ß integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation. Journal of Biological Chemistry, 284(52), 36700-36710. doi: 10.1074/jbc.M109.061275
Phosphorylation, Histones, Mitosis, Biochemistry