CHICKEN OVALBUMIN UPSTREAM PROMOTER TRANSCRIPTION FACTOR INTERACTS WITH ESTROGEN-RECEPTOR, BINDS TO ESTROGEN RESPONSE ELEMENTS AND HALF-SITES, AND INHIBITS ESTROGEN-INDUCED GENE-EXPRESSION

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) was identified as a low abundance protein in bovine uterus that co-purifi ed with estrogen receptor (ER) in a ligand-independent manner and was separated from the ER by its lower retention on estrogen response elem ent (ERE)-Sepharose. In gel mobility shift assays, COUP-TF bound as an apparent dimer to ERE and ERE half-sites. COUP-TF bound to an ERE hal f-site with high affinity, K-d = 1.24 nM. In contrast, ER did not bind a single ERE half-site. None of the class II nuclear receptors analyz ed, i.e. retinoic acid receptor, retinoid X receptor, thyroid receptor , peroxisome proliferator-activated receptor, or vitamin D receptor, w ere constituents of the COUP-TF.DNA binding complex detected in gel mo bility shift assays. Direct interaction of COUP-TF with ER was indicat ed by GST ''pull-down'' and co-immunoprecipitation assays. The nature of the ER ligand influenced COUP-TF-ERE half-site binding. When ER was liganded by the antiestrogen 4-hydroxytamoxifen (4-OHT), COUP-TF-half -site interaction decreased. Conversely, COUP-TF transcribed and trans lated in vitro enhanced the ERE binding of purified estradiol (E-2)-li ganded ER but not 4-OHT-liganded ER. Co-transfection of ER-expressing MCF-7 human breast cancer cells with an expression vector for COUP-TFI resulted in a dose-dependent inhibition of E-2-induced expression of a luciferase reporter gene under the control of three tandem copies of EREc38. The ability of COUP-TF to bind specifically to EREs and half- sites, to interact with ER, and to inhibit E-2-induced gene expression suggests COUP-TF regulates ER action by both direct DNA binding compe tition and through protein-protein interactions.