Estrogen receptor α requires no accessory factors for high-affinity binding to a consensus response element

Estrogen receptor (ER) alpha is commonly thought to bind to a consensus estrogen response element (ERE) as a homodimer, but previous experiments have not ruled out the presence of other proteins in the ER alpha/ERE complex. To characterize this interaction in more detail, we overexpressed mouse (m) ER alpha in a baculovirus system, using the selective advantage of the apoptosis inhibitor p35. Recombinant mER alpha possesses the predicted molecular weight and binds 17 beta-estradiol and an oligonucleotide containing a consensus vitellogenin ERE with high affinity. Over a wide concentration range of mER alpha protein (0.1-50 nM), only one complex was detected between mER alpha and vitellogenin ERE in gel shift assays. The ratio of E-2:vitellogenin ERE bound by mER alpha was close to 2.1, and each complex contained only one ERE. The molecular weight of the complex was determined to be 160000, very close to that predicted for two mER alpha proteins and one ERE oligonucleotide, therefore providing strong evidence that no other proteins were present. Recombinant mER alpha was purified such that it was the only protein observable by silver stain. Purified mER alpha and mER alpha in a nuclear extract behaved identically in Ferguson analysis, providing more evidence that only mER alpha was binding to the ERE. Purified mER alpha bound vitellogenin ERE with high affinity (K-d = 0.92 +/- 0.20 nM), indicating that no other proteins are necessary for high affinity mER alpha interaction with a consensus ERE. To determine whether ER alpha in an estrogen-responsive mammalian tissue behaves the same as the overexpressed mER alpha, we tested rat uterine cytosol by Ferguson analysis. ER alpha in rat uterine cytosol behaved identically to overexpressed mER alpha, suggesting that ER alpha in the uterine extract also binds to DNA predominantly as a homodimer with no additional proteins.