Fuernkranz H A, Wang Y, Karathanasis S K, Mak P
Department of Cardiovascular Molecular Biology, Lederle Laboratories, American Cyanamid Co., Pearl River, NY 10965.
Nucleic Acids Res. 1994 Dec 25;22(25):5665-71. doi: 10.1093/nar/22.25.5665.
Hepatocyte Nuclear Factor 4 (HNF-4), a liver-enriched orphan receptor of the nuclear receptor superfamily, is required for the expression of a wide variety of liver-specific genes including apoAI. To explore the possibility that site A of the apoAI gene enhancer might also be the target for HNF-4 without the interference of endogenous mammalian cell proteins that also bind to site A, we tested the ability of HNF-4 to activate transcription from site A in yeast cells. Electrophoretic mobility shift assays (EMSA) and Scatchard plot analysis demonstrated that yeast produced HNF-4 binds to site A with an affinity two times higher than that of yeast produced RXR alpha. Mapping analysis indicated that the 5' portion of site A containing two imperfect direct repeats (TGAACCCTTGACC) and the sequence of the trinucleotide spacer (CCT) between these imperfect repeats are critical determinants for selective binding and transactivation by HNF-4. Similar observations were obtained when these mutated versions of site A were evaluated by transient cotransfection assays in CV1 cells. We conclude that the unique structural determinants of site A in conjunction with the differential binding affinity of HNF-4 for site A may play a fundamental role in apoAI gene regulation.
肝细胞核因子4（HNF - 4）是核受体超家族中一种肝脏富集的孤儿受体，它是包括载脂蛋白AI（apoAI）在内的多种肝脏特异性基因表达所必需的。为了探究apoAI基因增强子的A位点在不受同样结合A位点的内源性哺乳动物细胞蛋白干扰的情况下，是否也可能是HNF - 4的作用靶点，我们检测了HNF - 4在酵母细胞中激活A位点转录的能力。电泳迁移率变动分析（EMSA）和Scatchard图分析表明，酵母产生的HNF - 4与A位点的结合亲和力比酵母产生的视黄酸X受体α（RXRα）高两倍。定位分析表明，A位点的5'部分包含两个不完全直接重复序列（TGAACCCTTGACC）以及这些不完全重复序列之间的三核苷酸间隔序列（CCT），是HNF - 4选择性结合和反式激活的关键决定因素。当在CV1细胞中通过瞬时共转染试验评估这些A位点的突变版本时，也得到了类似的结果。我们得出结论，A位点独特的结构决定因素以及HNF - 4对A位点的不同结合亲和力可能在apoAI基因调控中起重要作用。
