Proper centromere function is essential for the faithful segregation of chromosomes during mitosis. The histone variant H2A.Z is present at centromeres, and we have previously shown that H2A.Z enrichment occurs during M phase. Its exact function and deposition mechanism at centromeres is unknown, however the p400 complex is known to deposit H2A.Z into nucleosomes, and the p400 subunit TIP60 has been implicated to function at the centromere during early M phase. Therefore our aim was to uncover the dynamic roles of H2A.Z and the p400 complex in centromere function.
To identify potential cell cycle roles, we investigated effects of p400 or TIP60 depletion by siRNA. Interestingly, TIP60 but not p400 depletion causes an almost complete loss of mitotic cells, suggesting they play different functions during M phase. In contrast to TIP60, we show by immunofluorescence that p400 does not localise to centromeres during early M phase. Instead, it becomes enriched at centromeres during early G1. Although p400 is thought to mainly mediate H2A.Z deposition, its function at centromeres is H2A.Z deposition-independent, as p400 depletion does not alter H2A.Z abundance at centromeres. Instead, H2A.Z is required for recruiting p400, as H2A.Z depletion impedes the centromeric enrichment of p400 during early G1. Therefore, we identify novel roles for both H2A.Z and p400.
Thus, our results demonstrate that p400 and TIP60, although known components of the same complex, are also likely to form sub-complexes with different mitotic functions. Furthermore, our data supports the existence of mechanisms whereby H2A.Z recruits the p400 complex to chromatin rather than vice versa, specifically at centromeres.