The transcriptional repressor GATAD2B mediates progesterone receptor suppression of myometrial contractile gene expression

Chien-Cheng Chen; Alina P. Montalbano; Imran Hussain; Wan-Ru Lee; Carole R. Mendelson

doi:10.1074/jbc.M117.791350

The transcriptional repressor GATAD2B mediates progesterone receptor suppression of myometrial contractile gene expression

UT Southwestern Medical Center, United States

↵* Corresponding author; email: carole.mendelson{at}utsouthwestern.edu

Author contributions: CCC contributed to the conceptual design, conducted the experiments, interpreted the data and wrote the paper. APM analyzed expression of GATAD2B, COX-2 and IL-6 proteins in pregnant mouse myometrium (Figure 8D - F), reviewed Mass Spectrometry data with Proteomics Core, edited manuscript, extended statistical analyses, revised figures. IH analyzed expression of PR (Fig. 2B) and performed co-immunoprecipitation assays in stably transfected HEK293 and hTERT-HM cell lines (Fig. 6A). RWL conducted the Duolink proximity ligation assays in hTERT-HM cells (Fig. 6B). CRM contributed to the conceptual design of the studies, reviewed and interpreted the data and edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Abstract

The mechanisms whereby progesterone (P₄) acting via progesterone receptor (PR) inhibits proinflammatory/contractile gene expression during pregnancy are incompletely defined. Using immortalized human myometrial (hTERT-HM) cells stably expressing wild-type PR-A or PR-B (PR_WT), we found that P₄ significantly inhibited IL-1β induction of the NF-κB target genes, COX-2 and IL-8. P₄/PR_WT transrepression occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA Pol II to COX-2 and IL-8 promoters. However, in cells stably expressing a PR-A or PR-B DNA-binding domain mutant (PR_mDBD), P₄-mediated transrepression was significantly reduced, suggesting a critical role of the PR-DBD. ChIP analysis of hTERT-HM cells stably expressing PR_WT or PR_mDBD revealed that P₄ treatment caused equivalent recruitment of PR_WT and PR_mDBD to COX-2 and IL-8 promoters, suggesting that PR inhibitory effects were not mediated by its direct DNA binding. Using immunoprecipitation, followed by MS, we identified a transcriptional repressor, GATA Zinc Finger Domain Containing 2B (GATAD2B), that interacted strongly with PR_WT, but poorly with PR_mDBD. P₄ treatment of PR_WT hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P₄/PR_WT transrepression of COX-2 and IL-8. Notably, GATAD2B expression was significantly decreased in pregnant mouse and human myometrium during labor. Our findings suggest that GATAD2B serves as an important mediator of P₄/PR suppression of proinflammatory and contractile genes during pregnancy. Decreased GATAD2B expression near term may contribute to the decline in PR function leading to labor.