Estrogen Response Elements Function as Allosteric Modulators of Estrogen Receptor Conformation

ABSTRACT

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates the expression of estrogen-responsive genes. ER-mediated transcriptional changes are brought about by interaction of the ER with the estrogen response element (ERE). In this study, we examined the interaction of the Xenopus laevis ER DNA binding domain (DBD) and the intact ER with the X. laevisvitellogenin A2 ERE and the human pS2 ERE. Using gel mobility shift, DNase I footprinting, and methylation interference assays, we demonstrated that the DBD bound only as a dimer to the A2 ERE. However, the DBD bound as a monomer to the consensus pS2 ERE half site at lower DBD concentrations and then as a homodimer to the consensus and imperfect pS2 ERE half site at higher DBD concentrations. Antibody supershift experiments carried out with partially purified, yeast-expressed full-length ER demonstrated that three ER-specific antibodies interacted differentially with A2 and pS2 ERE-bound ER, indicating that receptor epitopes were differentially exposed. Furthermore, partial digestion of the A2 and pS2 ERE-bound ER with chymotrypsin or trypsin produced distinct protease cleavage patterns. Taken together, these data provide evidence that differential interaction of the DBD with the A2 and pS2 EREs brings about global changes in ER conformation. The conformational changes in ER induced by individual ERE sequences could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells.

ACKNOWLEDGMENTS

This research was supported by NIH grant R29 HD31299 (to A.M.N.) and USMRMC grant DAMD17-94-J4228 (to G.L.G.). Jennifer Wood was supported by NIH Reproductive Training Grant PHS 2T32 HD 0728-19.

We thank David Shapiro, Dean Edwards, and Robin Fuchs-Young for providing ER-specific antibodies and Varsha Likhite for additional protease sensitivity experiments. We are also grateful to David Shapiro for helpful comments during preparation of the manuscript.