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

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. Overall design: Comparative gene expression profiling analysis of RNA-seq data for HEK293T cells and its overexpression derivatives (T1045E G9a and WT G9a). Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for the histone modifications H3K9me2 in HeLa S3 cells. Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) for the different treatments in HeLa S3 cells.