Progesterone Receptor Activation Downregulates GATA3 By Transcriptional Repression and Increased Protein Turnover Promoting Breast Tumor Growth

IZZO F, MERCOGLIANO MF, VENTURUTTI L, TKACH M, CENCIARINI ME, SCHILLACI R, CERCHIETTI L, ELIZALDE PV, PROIETTI CJ

Congreso; 97th Endocrine Society Meeting; 2015

The Progesterone Receptor (PR) exerts its functions through diverse molecular mechanisms, either rapid activation of signaling pathways or direct transcriptional regulation after binding to progesterone response elements (PREs) or tethering to other transcription factors. Although PR-mediated transcriptional activation has been extensively studied, the mechanisms and co-regulators through which PR represses transcription of target genes remain to be fully elucidated. On the other hand, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb Repressor Complex 2 (PRC2), and mediates the tri-methylation of lysine 27 of histone H3 (H3K27me3) a modification associated with chromatin compaction and transcriptional repression. One of the key aspects of PRC2 function that remains to be determined is how PRC2 is directed to its target genes in mammalian cells. Since most PRC2 target genes are involved in cell differentiation, we studied the effect of progestin on the transcription factor GATA3, which is involved in mammary gland development and the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been widely established, although insights into the mechanisms of GATA3 expression loss are still required. Our hypothesis is that PR activation promotes GATA3 downregulation through EZH2 recruitment. In the present work, we demonstrate by chromatin immunoprecipitation and DNAse sensitivity experiments that progestin-activated PR binds and co-recruits EZH2 to a potential PRE upstream of the GATA3 gene, increasing H3K27me3 and inducing chromatin compaction. These modifications result in decreased GATA3 mRNA levels as measured by q-PCR. We also show that PR ability to bind DNA is required in order to target EZH2 upstream of GATA3, and that PR and EZH2 co-immunoprecipitate upon progestin stimulation. We also show the existence of a post-translational mechanism in which progestin treatment increases the activity of cAMP-dependent Protein Kinase A which phosphorylates GATA3 at serine 308, as demonstrated by in vitro kinase phosphorylation assays. Through site-directed mutagenesis we generated a GATA3 mutant carrying a substitution of alanine instead of serine in residue 308 and showed that impeded phosphorylation of serine 308 increases the stability of GATA3 and prevents its post-translational regulation by PR activation. Finally, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and breast cancer cell in vitro proliferation and in vivo tumor growth. The results presented here address EZH2 as a player in PR-mediated transcriptional repression. In addition these findings provide insight into the molecular mechanisms of loss of GATA3 expression in breast cancer.