Defects in centromeric/pericentromeric histone H2A T120 phosphorylation by hBUB1 cause chromosome missegregation producing multinucleated cells.

ABSTRACT

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.