Uterine estrogen receptor interaction with estrogen-responsive DNA sequences in vitro: effects of ligand binding on...

Interaction between the mouse estrogen receptor (ER) and its responsive element (ERE) were examined in vitro using tissue extracts and an oligonucleotide containing a Vitellogenin A2 ERE (VRE) in a gel retardation assay. Three specific complexes were formed when nuclear extracts were prepared from estrogen agonist-treated uteri or when ligand-free ER from cytosol was used. ER protein was associated with the three complexes, as demonstrated by their ability to bind anti-ER antibody H222. Specific complexes were formed only when double stranded nonheat-denatured VRE was used. Mutation of the ERE in VRE abolished specific binding. The complexes formed by nuclear extracts were qualitatively identical when obtained from mice treated with a variety of estrogenic compounds with different potencies. Nuclear extracts obtained from mice treated with an estrogen antagonist LY117018 formed complexes that migrated slower than the complexes formed by the other estrogenic compounds examined. The dissociation rates (k-1) and equilibrium dissociation constants (Kd) of the ER-VRE complexes formed by estradiol- or estriol-bound ER or ligand-free ER were measured and were found to be very similar, although the ligand-free specific ER complexes associate and dissociate less rapidly than those of estradiol- or estriol-bound ER. In addition, one of the specific complexes formed by the estriol nuclear extract dissociated more slowly than the equivalent estradiol complex. Heat activating ligand-free ER did not increase its binding to VRE. It appears that the ligand-binding domain of the ER does not exert its regulatory effects at the level of sequence-specific DNA binding, since its occupancy does not alter binding to the ERE. The subtle differences we observed in association and dissociation of multiple complexes when unoccupied ER and ER bound to ligands of varying potencies are compared may reflect differences in ER association with other protein factors that govern transcriptional activity.