Estrogen receptor binding to estrogen response elements slows ligand dissociation and synergistically activates reporter gene expression

Estradiol (E-2)-liganded estrogen receptor (ER) bound to three or four tand em copies of a consensus ERE (EREc38) in a cooperative manner. E-2-ER bindi ng to one or two EREs was non-cooperative. When ER was liganded by the anti estrogen 4-hydroxytamoxifen (4-OHT), ER-ERE binding was not cooperative, re gardless of the number of EREs. Here we evaluated how binding to EREc38 aff ects ER conformation in the ligand binding domain (LBD) as reflected in the dissociation kinetics of [H-3]ligand from the ER. Binding of ER to EREc38 slowed the rate of dissociation of either E-2 or 4-OHT, indicating that DNA allosterically modulates the LED conformation creating a tighter fit betwe en the ligand and the ER. Conformational differences in ER induced by E-2 v ersus antiestrogen were not reflected in differences in E-2 or 4-OHT dissoc iation parameters under these conditions. No difference in the association rate of E-2- versus 4-OHT-liganded ER binding to EREc38 was detected in ele ctrophoretic mobility shift assay (EMSA). Synergistic, E-2-dependent activa tion of a reporter gene was detected from three and four, but not one or tw o, tandem copies of EREc38. These observations suggest that cooperative bin ding of E-2-ER to multiple copies of EREc38 is likely responsible for trans criptional synergy and that cooperativity may not involve direct interactio n between the LBDs of ERE-bound ER. Since the number of copies of EREc38 di d not alter E-2 dissociation kinetics, functional synergy must involve cell ular factors in addition to the ER ligand. (C) 1999 Elsevier Science Irelan d Ltd. All rights reserved.