Ligand-Dependent and -Independent Functions of Activation Function 1 of Progesterone Receptor in Genome-Wide Gene Regulation and in Cell Proliferation and Apoptosis of Breast Cancer Cells

TL;DR

This study explores how the progesterone receptor's AF1 domain regulates gene activity and cell behavior in breast cancer, revealing the role of methylation in modulating its function.

Contribution

The study identifies specific methylation sites in AF1 and demonstrates their impact on ligand-dependent gene regulation and cell proliferation in breast cancer cells.

Findings

AF1-FFF mutation reduced PR regulation of cell proliferation and apoptosis in response to progestin.

AF1-FFF mutation affected ~60% of PR target genes, including those involved in cell proliferation and signaling pathways.

Impaired PRB-FFF activity correlated with increased chromatin binding and stronger association with SRC-1 coactivator.

Abstract

Progesterone receptor (PR) regulates gene expression through recruiting coregulators and general transcription factors by activation functions AF1 and AF2. AF1 localizes to the non-conserved and disordered N-terminal domain and is believed to facilitate tissue- and gene-specific activity. Our previous proteomic analysis identified three key residues (K464, K481 and R492) in AF1 that are monomethylated. Methylation mimic mutations KKR → FFF created hypoactive PR, whereas the KKR → QQQ mutation generated hyperactive PR in gene reporter assays. The current study used these mutants to determine the roles of AF1 in PR regulation of cellular activities and global gene regulation in breast cancer cells MCF-7. AF1-FFF mutation attenuated PR regulation of cell proliferation and apoptosis in response to progestin, whereas AF1-QQQ mutation enhanced these effects. AF1-FFF mutation attenuated gene regulation by progestin in ~60% of PR target genes, including genes involved in cell proliferation, hypoxia and TNFα signaling. However, the AF1-FFF mutation had little effect on ligand-independent gene regulation, suggesting distinct mechanisms of gene regulation by liganded and unliganded PR. Intriguingly, impaired activity of methylation mimic mutant PRB-FFF is associated with greater chromatin binding in ChIP-Seq analysis, corresponding to a stronger association between PRB-FFF and Steroid Receptor Coactivator-1 (SRC-1), a member of the p160 family of nuclear receptor coactivators, as was previously reported. In conclusion, PR AF1 is important for the core activities of liganded PR in regulating ~half of target genes and cell proliferation. AF1 monomethylation may modulate PR-chromatin interactions through stronger association with coregulators, thereby decelerating chromatin binding kinetics. This is supported by PRODIGY’s prediction of higher binding affinities of monomethylated AF1 and methylation mimic mutant with SRC-1.

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