Endocrine Abstracts

Progesterone receptor (PgR) is critically involved in the development of the mammary gland and the uterus in response to progesterone. It is also implicated in cancer development of these organs. An in depth understanding of how it works is pivotal for developing therapeutic interventions. PgR is a member of the nuclear receptor superfamily of transcription factors with conserved domain structures. Its transcription activity is mainly mediated by the activation function 1 (AF1) in the disordered N terminal domain (NTD) and the highly structured activation function 2 (AF2) in the ligand binding domain (LBD). It is established that AF2 forms coregulator binding interface upon ligand binding. But little is known about how AF1 works. We identified 3 critical AF1 residues K464, K481 and R492 that can be monomethylated. Mutations of these three residues to phenylalanine (KKR/FFF) largely abolished PR activity in reporter gene assay but increased AF1 interactions with coactivator SRC1 and AF2. We further determined the effect of AF1 mutations on PR regulation of cell growth, adhesion and global gene expression in MCF-7 cells stably expressing the Wt PRB or the AF1 mutant. It was found that KKR/FFF mutations significantly impaired the effect of PR on cell proliferation and cell spreading. RNA-Seq analysis revealed that the KKR/FFF mutation considerably impaired PR regulated gene expression in 40% of the genes, which included most of the well characterized PR target genes. Importantly, the impaired gene regulation by KKR/FFFF mutant is not associated with a reduced enhancer/promoter occupancy. Considering stronger interactions of AF1-KKR/FFF with AF2 and coactivator SRC1, our data lend support to the tripartite relationship among AF1, AF2 and coregulators, in which AF1 plays a key role in modulating dynamics of PgR transcription complex.