Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF) Carolyn M. Klinge* Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Louisville, KY 40292, USA Received 3 November 1998; accepted 17 August 1999 Abstract Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E 2 ) -liganded ER (E 2 -ER), but not by ER liganded with the antiestrogens 4- hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of puri®ed E 2 -ER, but did not aect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either puri®ed bovine or recombinant human ERa. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERa-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E 2 -induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate ¯anking regions in¯uence whether COUP-TF enhances, inhibits, or has no eect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half- sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression. # 1999 Elsevier Science Ltd. All rights reserved. 1. Introduction Estrogen receptors (ERs) a and b mediate the actions of estrogens in target tissues thus regulating es- trogenic eects on reproduction, bone homeostasis and mammary gland structure and function [1]. Estrogens play a pivotal role in the development of breast cancer. ER is a member of the steroid/thyroid superfamily of related proteins [2]. After binding its cognate ligand, e.g. estradiol (E 2 ), a series of activation events occur that increase ER binding to estrogen response elements (EREs), located in or adjacent to the coding regions of estrogen-regulated genes. The detailed mechanism accounting for estrogen-dependent transactivation remains to be fully elucidated, but recent reports indi- cate that ERE-bound ERa interacts with nuclear pro- teins including co-activator proteins, e.g., SRC-1 and components of the TFIID complex, e.g. TAF II 30, that enhance gene expression [3]. Antiestrogens are used clinically to prevent the recurrence of breast cancer [4]. 4-hydroxytamoxifen (4- OHT), a metabolite of tamoxifen (TAM), the most widely used antiestrogen, binds ER and activates ER- ERE binding [5]. However, the speci®c details of its Journal of Steroid Biochemistry & Molecular Biology 71 (1999) 1±19 0960-0760/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(99)00124-7 www.elsevier.com/locate/jsbmb * Tel.: +1-502-852-3668; fax: +1-502-852-6222. E-mail address: carolyn.klinge@louisville.edu (C.M. Klinge)