p27-induced Down-Regulation of Estrogen Receptor a Transcriptional Activity is Mediated by Inhibition of Estrogen Receptor α Nuclear Localization and Modulation of Estrogen Receptor α

Abstract

Estrogen receptor α (ERα) is a DNA binding transcription factor which regulates reproductive function and transcription of its target genes. A member of cip/kip family of cyclin-dependent kinase inhibitors, p27 is responsible for cell cycle arrest at G1 stage. To investigate the effect of p27 on ERα-mediated transcription, we generated p27 knock-down MCF-7 breast cancer cells (MCF-7 hp27shRNA) via retroviral delivery of p27 shRNA. Knock-down of p27 in MCF-7 cells resulted in up-regulation of ERα-mediated transcription by estradiol as compared to control MCF-7 cells (MCF-7 mp27shRNA). Accordingly, transient transfection studies revealed that overexpression of p27 in 293T cells reduced ERα-mediated transcription. It appeared that the effect of p27 on ERα transcriptional activity is independent from cell cycle arrest by p27 since cell cycle arrest induced by serum starvation did not significantly affect ERα-mediated transcription. We found that p27 inhibited proper nuclear localization of ERα and association of p27 with ERα in the cytoplasm as revealed by subcellular fractionation and subsequent co-immunoprecipitation assays. Because significant portion of ERα was still localized in the nucleus upon p27 overexpression, we also investigated the role of p27 in the modulation of ERα transcriptional activity in the nucleus. We found that p27 negatively modulated ERα transcriptional activity by inhibiting association of cyclin D1 with ERα and recruiting BRCA1 to ERα transcriptional complex. Taken together, these data suggest that p27 inhibited ERα transcriptional activity by two independent mechanisms, 1) physical nuclear exclusion of ERα and 2) modulation of transcriptional complex.