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. 2008 Nov 28;283(48):33585-90.
doi: 10.1074/jbc.M803312200. Epub 2008 Oct 3.

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Qing Duan  1 , Haobin Chen, Max Costa, Wei Dai
Affiliations

Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis

Qing Duan et al. J Biol Chem. .

Abstract

Post-translational modifications of histones play a critical role in regulating genome structures and integrity. We have focused on the regulatory relationship between covalent modifications of histone H3 lysine 9 (H3K9) and H3S10 during the cell cycle. Immunofluorescence microscopy revealed that H3S10 phosphorylation in HeLa, A549, and HCT116 cells was high during prophase, prometaphase, and metaphase, whereas H3K9 monomethylation (H3K9me1) and dimethylation (H3K9me2), but not H3K9 trimethylation (H3K9me3), were significantly suppressed. When H3S10 phosphorylation started to diminish during anaphase, H3K9me1 and H3K9me2 signals reemerged. Western blot analyses confirmed that mitotic histones, extracted in an SDS-containing buffer, had little H3K9me1 and H3K9me2 signals but abundant H3K9me3 signals. However, when mitotic histones were extracted in the same buffer without SDS, the difference in H3K9me1 and H3K9me2 signals between interphase and mitotic cells disappeared. Removal of H3S10 phosphorylation by pretreatment with lambda-phosphatase unmasked mitotic H3K9me1 and H3K9me2 signals detected by both fluorescence microscopy and Western blotting. Further, H3S10 phosphorylation completely blocked methylation of H3K9 but not demethylation of the same residue in vitro. Given that several conserved motifs consisting of a Lys residue immediately followed by a Ser residue are present in histone tails, our studies reveal a potential new mechanism by which phosphorylation not only regulates selective access of methylated lysines by cellular factors but also serves to preserve methylation patterns and epigenetic programs during cell division.

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Figures

FIGURE 1.
FIGURE 1.
H3K9me2 was barely detectable in mitotic cells until anaphase. HeLa cells were stained with antibodies to phosphorylated serine 10 of histone H3 (p-H3S10) (red) and to dimethylated lysine of histone H3 (H3K9me2) (green). DNA was stained with DAPI (blue). Pro, Prometa, Meta, and Ana represent prophase, prometaphase, metaphase, and anaphase, respectively. Representative images are shown.
FIGURE 2.
FIGURE 2.
H3K9me1 signals were also low in early mitosis. A, HeLa cells were stained with antibodies to p-H3S10 (red) and to H3K9me2 (green). DNA was stained with DAPI (blue). Representative images are shown. B, HeLa cells were stained with an antibody to H3K9me3 (green). DNA was stained with DAPI (blue). Pro, Prometa, Meta, and Ana represent prophase, prometaphase, metaphase, and anaphase, respectively. Representative images are shown.
FIGURE 3.
FIGURE 3.
H3K9me1 and H3K9me2 signals were suppressed in mitotic A549 cells. A549 cells were stained with antibodies to H3K9me1 (green) and to H3K9me2 (green). DNA was stained with DAPI (blue). Representative images are shown.
FIGURE 4.
FIGURE 4.
H3S10 phosphorylation blocks the accessibility of specific antibodies to H3K9. A, HeLa cells were treated with nocodazole overnight. Mitotic cells were collected via shake-off. Interphase cells were collected from HeLa cell culture after removing mitotic cells. Histones were extracted from interphase and mitotic cells with a RIPA supplemented with or without SDS. Approximately equal amounts of extracted histones were blotted for H3K9me1, H3K9me2, H3K9me3, and histones. B, HeLa cells were fixed and then treated with or without λ-phosphatase (λ-PPAse) before staining with antibodies to p-H3S10 (red) and to H3K9me2 (green). DNA was stained with DAPI (blue). Representative images are shown.
FIGURE 5.
FIGURE 5.
Dephosphorylation of H3S10 unmasks H3K9me1 and H3K9me2 signals. A, A549 cells were fixed and then treated with or without λ-phosphatase (λ-PPAse) before staining with the antibody to H3K9me2 (green). DNA was stained with DAPI (blue). Pro, Prometa, Meta, and Ana represent prophase, prometaphase, metaphase, and anaphase, respectively. Representative cells of various mitotic stages are shown. B, histones were extracted from interphase and mitotic cells with a RIPA containing SDS. Duplicate blots treated briefly with λ-phosphatase or left untreated as control were blotted for H3K9me2 and histones.
FIGURE 6.
FIGURE 6.
H3S10 phosphorylation selectively blocks the access of H3K9 modification enzymes in vitro. A, methylation of H3K9 by G9a was determined by an in vitro histone methyl transfer assay using either histone H3 peptide or phospho(Ser-10)-histone H3 peptide as substrate. Each bar represents the mean incorporation of radioactivity per 10 μl of sample ± standard deviation from three samples. B, histones extracted from interphase and mitotic cells were incubated with JHDM2A in a reaction buffer supplemented with or without EDTA. After the demethylation reaction, both treated histones, as well as the control ones, were blotted for H3K9me1, H3K9me2, p-H3S10, and histones.
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