Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor α

Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of breast cancer by activating estrogen receptors (ERs) alpha and beta. Ligand-activated ER stimulates the expression of target proteins by interacting with specific DNA sequences: estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact ER alpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates ER alpha conformation by measuring changes in sensitivity to protease digestion. ER alpha, occupied by estradiol (E-2) or 4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by chymotrypsin followed by a Western analysis with antibodies to ER alpha. ERE binding increased the sensitivity of ER alpha to chymotrypsin digestion. We found both ligand-specific and ERE-specific differences in ER alpha sensitivity to chymotrypsin digestion. The ERE-mediated increase in ER alpha sensitivity to chymotrypsin digestion correlates with E-2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of ER alpha -ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in ER alpha conformation modulate interaction with co-regulatory proteins. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.