Greenberg D, Miao C H, Ho W T, Chung D W, Davie E W
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12347-51. doi: 10.1073/pnas.92.26.12347.
Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.
对编码人凝血因子VII的基因5'侧翼区域的启动子和沉默子元件进行了鉴定和表征。通过对人肝脏cDNA的RACE（cDNA末端快速扩增）分析确定了主要转录起始位点，命名为+1，发现其位于翻译起始位点上游50bp处。在+32bp和+37bp处发现了两个次要转录起始位点。将5'侧翼区域的逐步缺失片段与氯霉素乙酰转移酶报告基因融合，并测定其在HepG2和HeLa细胞中的瞬时表达。鉴定出了两个对肝细胞特异性转录至关重要的启动子元件。第一个位点FVIIP1位于-19bp至+1bp，其功能不依赖于方向或位置，约占凝血因子VII基因启动子活性的三分之一。电泳迁移率变动、竞争和抗肝细胞细胞核因子4（HNF4）抗体超迁移实验表明，该位点含有一个与编码因子IX和因子X的基因启动子同源的HNF-4结合元件。第二个位点FVIIP2位于-50bp至-26bp，其功能也不依赖于方向或位置，约占凝血因子VII基因启动子活性的三分之二。对突变序列的功能分析表明，一个与凝血酶原基因增强子具有90%序列同一性的富含10bp G+C的核心序列对第二个位点的功能至关重要。迁移率变动和竞争分析表明，该位点还结合肝脏特异性因子以及转录因子Sp1。通过报告基因分析还鉴定出了位于启动子区域上游-130bp至-103bp（FVIIS1位点）和-202bp至-130bp（FVIIS2）的两个沉默子元件。
