Abstract Hormone-activated steroid receptors (SR) bind to specific regulatory elements on DNA and recruit coregulators which remodel chromatin, regulate assembly of the transcription complex, and regulate transcription of their target genes. As for many coregulators, the lysine methyltransferases G9a and GLP function in both activation and repression of transcription depending on the target gene. Indeed, G9a and GLP catalyze methylation of H3K9, a well-known repressive mark, but can also act as coactivators of SR (e.g. estrogen receptor α and glucocorticoid receptor or GR). G9a and GLP can form homo- and heterodimers via interactions between their C-terminal domains. At the molecular level, we established that GLP, as well as G9a, is methylated on the N terminal domain of the protein, which is the region implicated in coactivator function. This event is required for the recruitment of HP1g, while HP1g binding to G9a and GLP is inhibited by aurora B-mediated phosphorylation of G9a or GLP adjacent to the methylation site. In transient reporter gene assays, mutations of G9a and GLP methylation sites significantly decreased their coactivator activity with GR. Inversely, inhibition of Aurora kinase B activity increased the reporter gene activation. In addition, inhibition of the kinase activity of Aurora B increased the expression of endogenous GR target genes positively regulated by G9a and GLP, while depletion of HP1g inhibited their expression. Moreover methylated G9a or GLP can form a ternary complex with HP1g and GR in cell lines, and HP1g was recruited to the GR binding sites of the target genes positively regulated by G9a and GLP (but not on GR target genes that do not require G9a or GLP). Interestingly, the subset of GR target genes positively regulated by G9a and GLP contains some genes involved in cellular movement. At the physiological level, we established that G9a and GLP act as coactivators of GR target genes that mediate glucocorticoid repression of cell migration, which accompanies epithelial-to-mesenchymal transition associated with cancer progression. In this study we demonstrated that G9a and GLP methylation is required for their coactivator function with GR, because it facilitates recruitment of HP1g which functions selectively as a coactivator for genes positively regulated by G9a, GLP and GR. Inversely, G9a and GLP phosphorylation antagonizes these effects. This competition could be involved in the switch to determine whether G9a and GLP function as coactivator or corepressor on specific target genes. Defining the specific molecular functions of SR coregulators and how their recruitment and actions are coordinated is key to understanding how transcription is regulated and will suggest new targets in steroid hormone regulated cancer and in other steroid hormone-related diseases. Citation Format: Coralie Poulard, Michael Stallcup. Crosstalk between automethylation and phosphorylation tightly regulates G9a and GLP coactivator function with steroid hormone receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3622. doi:10.1158/1538-7445.AM2017-3622
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