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Dynamic phosphorylation of G9a by Plk1 and PP2A modulates its catalytic activity and chromosome accessibility during mitosis

ABSTRACT: Phosphorylation of Ser10 of histone H3 (H3S10p), together with the adjacent dimethylation of Lys9 (H3K9me2), has been proposed to function as a ‘phospho-methyl switch’ to regulate mitotic chromosome dynamics and control peripheral heterochromatin localization (1, 2). Despite of immensely understanding the regulation of H3S10 phosphorylation, how this modification temporarily masks the H3K9me2 mark during mitosis is poorly understood. Herein, we report that the master mitotic kinase Plk1 phosphorylates G9a at Thr1045 (pT1045) in vitro and in vivo at early mitosis. T1045p inhibits G9a catalytic activity, leading to decrease H3K9me2 levels. RNA-sequencing analysis showed that the phospho-mimic T1045 mutant of G9a (T1045E) compromised its repressive activity on several targeted genes, which was confirmed by luciferase reporter assays. Using genome-wide epigenomic technologies, we found that T1045E mutant displayed reduced H3K9me2 level, accompanied with increased chromatin accessibility, compared to the wild-type G9a cells. Finally, we found that T1045 phosphorylation could be removed by the phosphatase PP2A at late mitosis, which inversely reactivated G9a activity on H3K9me2 level, correlated with lower levels of H3S10p. Therefore, our result may provide a mechanistic explanation for ‘phospho-methyl switch’ and highlight the importance of Plk1 and PP2A-mediated dynamic regulation of G9a during mitosis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE223359 | GEO | 2023/07/17

REPOSITORIES: GEO

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Project description:Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in chromatin-dependent gene silencing and maintenance of genome stability by repressing repetitive DNA elements and recruiting downstream factors essential for faithful chromosome segregation. Fundamental principles of these processes have been elucidated in the fission yeast Schizosaccharomyces pombe (S. pombe), in which Clr4, the homologue of mammalian SUV39H, is the sole H3K9 methyltransferase. Although H3K9 methylation has been intensely studied in mitotic cells, occurrence and regulation during sexual differentiation remain largely unknown. Whether distinct H3 methylation states are relevant for gametogenesis is also not known. Here, using diploid S. pombe cells, we map H3K9 methylation genome-wide during the meiotic program and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells exit mitosis and commit to the meiotic life cycle. Cells lacking the ability to tri-methylate H3K9 exhibit severe meiotic chromosome segregation defects, which does not occur in mitotic cells. Artificially increasing the affinity of the fission yeast heterochromatin protein 1 (HP1) homologue Swi6 towards H3K9me2 rescues chromosome segregation in the absence of H3K9me3. Thus, the H3K9me2 to H3K9me3 switch during early meiosis serves to retain Swi6 at centromeres, which is necessary for cohesin recruitment and kinetochore assembly. Finally, we show that the H3K9 methylation switch is regulated by differential phosphorylation of Clr4 by the cyclin-dependent kinase (CDK) Cdk1. Our results reveal how a highly conserved master regulator of the cell cycle regulates the specificity of the H3K9 methyltransferase and thereby controls the ratio of H3K9me2 to H3K9me3 to prevent unwanted gene silencing and to ensure faithful meiotic chromosome segregation. High degree of conservation of the proteins involved in our study and CDK-dependent phosphorylation of the mammalian SUV39H1 homologue suggest broad conservation of the mechanisms described here.

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Dynamic phosphorylation of G9a by Plk1 and PP2A modulates its catalytic activity and chromosome accessibility during mitosis

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