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

Brittle AL, Nanba Y, Ito T, Ohkura H - Exp. Cell Res. (2007)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.

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.

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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.
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fig2: 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.

Mentions: To identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation, we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation [19]. After Aurora B was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. In Aurora B-depleted cells, the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms (Fig. 2A). However, nuclear staining in interphase cells remained high (Supplementary Fig. 5), suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms.


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

Brittle AL, Nanba Y, Ito T, Ohkura H - Exp. Cell Res. (2007)

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.
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fig2: 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.
Mentions: To identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation, we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation [19]. After Aurora B was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. In Aurora B-depleted cells, the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms (Fig. 2A). However, nuclear staining in interphase cells remained high (Supplementary Fig. 5), suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh, UK.

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.

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