Multimodal Regulation of E2F1 Gene Expression by Progestins

Abstract

An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.

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This work was supported by DOD grant W81XWH-06-1-0745 (H.E.W.) and by NIH grants DK048807 (D.P.M.); EY13499 (D.C.O.); and DK074967, CA089393, and CA080111 (M.B.).

We thank the members of the McDonnell laboratory for critical review of the manuscript and Ganesan Sathya for construction of the pcDNA3-hPR-B plasmid. We are grateful to Dean Edwards at Baylor College of Medicine in Houston, TX, for PR antibodies and T47D:C42 cell lines; to Craig Jordan at the Fox Chase Cancer Center in Philadelphia, PA, for T47D:A18 cells; and to Kathryn Horwitz at University of Colorado in Denver, CO, for hPR-Bcys. We also thank the NIEHS Microarray Core for technical assistance related to the gene expression microarray work.