Klinge C M
Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, KY 40292, USA.
J Steroid Biochem Mol Biol. 1999 Nov;71(1-2):1-19. doi: 10.1016/s0960-0760(99)00124-7.
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 (E2) -liganded ER (E2-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 purified E2-ER, but did not affect 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 purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-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 E2-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 flanking regions influence whether COUP-TF enhances, inhibits, or has no effect 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.
雌激素反应基因通过改变雌激素受体（ER）与转录激活因子和抑制剂之间的平衡来进行调控。在此，我们研究了ER配体在ER与鸡卵清蛋白上游启动子转录因子（COUP-TF）孤儿核受体相互作用中的作用。添加雌二醇（E2）配体化的ER（E2-ER）可增加COUP-TF与半位点雌激素反应元件（ERE）的结合，但与抗雌激素4-羟基他莫昔芬（4-OHT-ER）或他莫昔芬氮丙啶（TAz-ER）配体化的ER则不会增加这种结合。ER不会与单个半位点结合。相反，COUP-TF增强了纯化的E2-ER与ERE的结合，但不影响TAz-ER与ERE的结合。相比之下，通过GST下拉实验评估，只有抗雌激素增强了ER与COUP-TF之间的直接相互作用。使用纯化的牛源或重组人ERα均获得了相同的结果。免疫共沉淀实验表明，在MCF-7细胞提取物以及转染了ERα的MDA-MB-231细胞中，ER与COUP-TF相互作用。在此我们证明，ER配体影响COUP-TF与ER的相互作用。COUP-TF的相互作用由ER的DNA结合域和配体结合域介导。我们认为，DNA结合诱导的ER构象变化会降低ER与COUP-TF的相互作用。用人COUP-TFI表达载体瞬时转染人MCF-7乳腺癌细胞，可抑制来自单个或多个串联拷贝共有ERE的E2诱导的荧光素酶报告基因表达。COUP-TFI刺激了来自最小ERE的4-OHT诱导的荧光素酶活性。单独存在时，COUP-TFI以序列依赖的方式增加了ERE半位点或单个ERE的转录。这些数据提供了证据，表明ERE序列及其紧邻的侧翼区域会影响COUP-TF是增强、抑制还是不影响ER配体诱导的ERE报告基因表达，并且COUP-TFI可激活来自ERE半位点的基因转录。我们认为COUP-TFI在减轻雌激素反应基因表达中发挥作用。
