Liu Y, Chiu J F
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405.
Nucleic Acids Res. 1994 Mar 25;22(6):1079-86. doi: 10.1093/nar/22.6.1079.
Retinoic acid (RA) is widely involved in the control of cell proliferation and differentiation, as well as embryo pattern formation. Transcription of the oncodevelopmental protein, alpha-fetoprotein (AFP), is stimulated by retinoic acid (RA) in neoplastic cells. To study RA regulation of AFP gene expression, the 5'-flanking region of AFP gene was cloned and analyzed. In the present study, transfection of deletion mutants and sequence analysis revealed a retinoid X receptor response element (AFP-RXRE) located at position -139 to -127 of the AFP promoter. Synthetic AFP-RXRE was ligated into a reporter construct with the heterologous promoter and chloramphenicol acetyltransferase (CAT). AFP-RXRE conferred a marked RA responsiveness in the cotransfection with retinoid X receptor (RXR), but not with retinoic acid receptors (RARs). Consistent with these data, only RXR bound to AFP-RXRE with high affinity in the mobility shift assays. Chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of the steroid/thyroid hormone superfamily, also demonstrated specific binding activity to AFP-RXRE in vitro. In cotransfection assays, COUP-TF dramatically repressed the transactivation of RXR on AFP-RXRE. The mechanism of repression by COUP-TF may involve the mutual occupancy of the AFP-RXRE binding site between RXR and COUP-TF.
维甲酸（RA）广泛参与细胞增殖与分化的调控以及胚胎模式形成。在肿瘤细胞中，维甲酸（RA）可刺激肿瘤发生发展蛋白甲胎蛋白（AFP）的转录。为研究RA对AFP基因表达的调控，对AFP基因的5'侧翼区进行了克隆与分析。在本研究中，缺失突变体的转染及序列分析揭示了位于AFP启动子-139至-127位的类视黄醇X受体反应元件（AFP-RXRE）。将合成的AFP-RXRE与异源启动子及氯霉素乙酰转移酶（CAT）连接到报告基因构建体中。在与类视黄醇X受体（RXR）共转染时，AFP-RXRE表现出显著的RA反应性，但与维甲酸受体（RARs）共转染时则无此反应。与这些数据一致，在凝胶迁移试验中，只有RXR能以高亲和力结合到AFP-RXRE上。鸡卵清蛋白上游启动子转录因子（COUP-TF）是类固醇/甲状腺激素超家族的一个孤儿成员，在体外也显示出对AFP-RXRE的特异性结合活性。在共转染试验中，COUP-TF显著抑制RXR对AFP-RXRE的反式激活。COUP-TF的抑制机制可能涉及RXR与COUP-TF之间对AFP-RXRE结合位点的相互占据。
