Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation

Abstract

The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation.

Fig. 1

Dynamic change of H2A T119 phosphorylation in the cell cycle. S2 cells were immunostained with anti-dH2A-pT119 antibody. H2A T119 phosphorylation was over all chromatin in interphase (A) but enriched to centromeric regions in prophase (B) and maintained through prometaphase (C) and metaphase (E). The phosphorylation was lost in anaphase (F). The boxed region in C is magnified in D. Scale bar = 10 μm.

Fig. 2

Aurora B and Polo kinases are required for enrichment of H2A T119 phosphorylation at centromeric regions in mitosis. (A) S2 cells were immunostained using anti-dH2A-pT119 antibody after single or double depletion of Aurora B and Polo by RNAi. Aurora B is required for H2A T119 phosphorylation at centromeric regions, while Polo is required for suppressing the phosphorylation on chromosome arms. Scale bar = 10 μm. (B) Average pixel intensity of anti-dH2A-pT119 immunofluorescent signals on chromosome arms. The differences between the control and Polo or Polo/Aurora B are statistically significant (p < 0.001), while the differences between the control and Aurora B, or Polo and Polo/Aurora B are not significant (p ?>> 0.3). The H2A phosphorylation on chromosome arms in Polo-depleted cells was not dependent on Aurora B.

Fig. 3

H2A T119 phosphorylation on chromosome arms in the absence of Polo kinase depends on NHK-1 kinase. (A) S2 cells were immunostained using anti-dH2A-pT119 antibody after single or double depletion of NHK-1 and Polo by RNAi. (B) Average pixel intensity of anti-dH2A-pT119 immunofluorescent signals on chromosome arms. The differences between Polo and others are statistically significant (p < 0.001), while the differences between the control, NHK-1 and Polo/NHK-1 are not significant (p ?>> 0.2). The H2A phosphorylation on chromosome arms in Polo-depleted cells was dependent on NHK-1, which is known to phosphorylate H2A T119.

Fig. 4

Cyclin B degradation is required for the loss of H2A phosphorylation at the metaphase-anaphase transition. (A) Expression of non-degradable Cyclin B (CyclinB.TPM-GFP) prevented loss of H2A T119 phosphorylation at anaphase. Scale bar = 10 μm. (B) Frequencies of H2A T119 phosphorylation at centromeric regions amongst anaphase cells with or without expression of non-degradable Cyclin B (CyclinB.TPM) tagged with GFP. The difference is statistically significant (p < 0.001). (C) A schematic diagram for regulation of H2A phosphorylation at T119 in mitosis. In interphase, phosphorylation is found throughout the chromatin. In early mitosis, the phosphorylation is enriched at centromeric regions. This process is mediated by the concerted action of Aurora B and Polo kinases which are required for H2A T119 phosphorylation at centromeric regions and suppressing the phosphorylation on arms, respectively. At the onset of anaphase, the phosphorylation at centromeric regions is lost. Cyclin B degradation or Cdc2 inactivation is required for the loss.