Li Y, Shen R F, Tsai S Y, Woo S L
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Mol Cell Biol. 1988 Oct;8(10):4362-9. doi: 10.1128/mcb.8.10.4362-4369.1988.
The human alpha-1-antitrypsin (AAT) gene is expressed in the liver, and its deficiency causes pulmonary emphysema. We have demonstrated that its 5'-flanking region contains cis-acting elements capable of directing proper transcription in the presence of rat liver nuclear extract. The in vitro transcription system is tissue-specific in that the AAT promoter is functional in nuclear extracts prepared from the liver but not from HeLa cells. Experiments in which rat liver and HeLa nuclear extracts were mixed suggested the presence of a specific activator(s) in hepatocytes rather than a repressor(s) in nonproducing cells. Two protected regions were detected in the promoter by DNase I footprinting analysis with rat liver nuclear extracts. Region one spanned -78 to -52 and region two spanned -125 to -100 in the 5'-flanking sequence of the gene. By gel retardation assays with synthetic oligonucleotides, at least two distinct liver nuclear factors were identified, HNF-1 and HNF-2 (hepatocyte nuclear factors), which bound specifically to the first and second region, respectively. We present evidence that HNF-1 and HNF-2 are positively acting, tissue-specific transcription factors that regulate hepatic expression of the human AAT gene.
人类α-1-抗胰蛋白酶（AAT）基因在肝脏中表达，其缺乏会导致肺气肿。我们已经证明其5'-侧翼区域含有能够在大鼠肝核提取物存在的情况下指导正确转录的顺式作用元件。体外转录系统具有组织特异性，因为AAT启动子在从肝脏制备的核提取物中起作用，但在从HeLa细胞制备的核提取物中不起作用。将大鼠肝脏和HeLa核提取物混合的实验表明，肝细胞中存在特异性激活剂，而非产生细胞中存在阻遏物。用大鼠肝核提取物进行DNase I足迹分析在启动子中检测到两个受保护区域。区域一在基因的5'-侧翼序列中跨度为-78至-52，区域二跨度为-125至-100。通过用合成寡核苷酸进行凝胶阻滞试验，鉴定出至少两种不同的肝核因子，即HNF-1和HNF-2（肝细胞核因子），它们分别与第一和第二区域特异性结合。我们提供的证据表明，HNF-1和HNF-2是正向作用的、组织特异性转录因子，可调节人类AAT基因的肝脏表达。
