Estrogen receptor alpha phosphorylation in promoter recruitment and transcription of estrogen-dependent genes
Description
Promoter recruitment to genes representing different paradigms of ERalpha promoter interaction was assessed to test the hypothesis that gene specific transcription by ERalpha is regulated by receptor phosphorylation that will, in turn, direct recruitment of specific coregulator proteins needed for gene expression. Using chromatin immunoprecipitation (ChIP), it was determined that there is a gene-specific and ligand-dependent recruitment of coregulators to ERalpha target genes. Increased recruitment of AIB-1 and NCoR was detected only at the c-myc AP-1 in response to 17beta-estradiol and at the pS2 ERE in response to 4-hydroxytamoxifen respectively. In addition, GRIP-1 recruitment was increased to cyclin D1 CRE only in response to 4-hydroxytamoxifen, but to the c-myc AP-1 in response to both 17beta-estradiol and 4-hydroxytamoxifen. Furthermore, siRNA-mediated down regulation of SRC-1, GRIP-1 and AIB-1, revealed the importance of these transcriptional coactivators in 4-hydroxytamoxifen-mediated transcriptional repression. For example, incubation of MCF-7 cells with 4-hydroxytamoxifen results in a significant decrease in gene expression. Downregulation of GRIP-1 by siRNA results in an increase in cyclin D1 gene expression levels in response to tamoxifen similar to that of cells incubated with vehicle Novel ERalpha phospho-specific antibodies and ERalpha phosphorylation site mutants were used to determine the impact of 17beta-estradiol and 4-hydroxytamoxifen on several aspects of ERalpha function. In response to 17beta-estradiol, increased phosphorylation at ERalpha Ser 118 and 305 and decreased phosphorylation of Ser 167 were detected. In response to 4-hydroxytamoxifen, increased ERalpha Ser 118, 167 and 236 phosphorylation was detected. An examination of ERalpha recruitment to target genes revealed that phosphorylation of the sites in the absence of ligand were retained in ligand-dependent ERalpha recruitment to target genes. For example in the absence of ligand and in the presence of 17beta-estradiol and 4-hydroxytamoxifen, ERalpha recruited to c-myc AP-1 is phosphorylated at Ser 167 and 305. The addition of 17beta-estradiol and 4-hydroxytamoxifen, however, resulted in the ligand-specific phosphorylation of additional sites. Importantly, effects of individual phosphorylation sites on ER(+) breast cancer cell proliferation were detected using transient transfection of single phosphorylation site mutants despite the fact that phosphoproteins, such as ERalpha, are often phosphorylated at multiple sites at the same time. Taken together, these results suggest that ERalpha phosphorylation directs a gene-specific recruitment of ERalpha and transcriptional coregulators to ERalpha target gene promoters