Chuang S S, Das H K
Department of Pharmacology, University of North Texas Health Science Center at Fort Worth 76107, USA.
Biochim Biophys Acta. 1999 Jan 4;1436(3):600-5. doi: 10.1016/s0005-2760(98)00117-9.
Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located within the -128 to +122 promoter region (S.S. Chuang, H.K. Das, Identification of trans-acting factors that interact with cis-acting elements present in the first non-translated exon of the human apolipoprotein B gene, Biochem. Biophys. Res. Commun. 220 (1996) 553-562). Two cis-acting positive elements (-104 to -85; -84 to -60) are located upstream from the start of transcription. A negative element (+20 to +40) and a strong positive element (+43 to +53) are located in the first non-translated exon of the human apolipoprotein B gene. Trans-acting factors BRF-2, BRF-1, BRF-3, and BRF-4 interact with the above four cis-acting elements respectively. In this study, we examine the roles of the upstream positive elements -104 to -85 and -84 to -60 in modulating transcriptional regulation of the apoB gene by downstream elements +20 to +40 and +43 to +53. Using in vitro mutagenesis and transient transfection experiments in HepG2 cells, the cis-acting element -84 to -60 has been found to be absolutely necessary for the function of the upstream element -104 to -85 and downstream elements +20 to +40 and +43 to +53. In vitro mutagenesis of the downstream positive element +43 to +53 and transfection of the mutant promoter constructs in HepG2 cells reveal that nucleotide G at position +51 is essential for the strong positive activity of the element +43 to +53. A single substitution point mutation of nucleotide G to either A or T at position +51 reduces apolipoprotein B gene transcription substantially in HepG2 cells. These results suggest that a single substitution mutation in vivo, of nucleotide G to either A or T at position +51 in the downstream positive promoter element +43 to +53 may potentially cause hypobetalipoproteinemia, a heterozygous from of an autosomal-dominant disorder.
人载脂蛋白B（apoB）基因的肝细胞特异性表达受位于-128至+122启动子区域内的至少四个顺式作用元件调控（S.S.庄，H.K.达斯，鉴定与人载脂蛋白B基因第一个非翻译外显子中存在的顺式作用元件相互作用的反式作用因子，《生物化学与生物物理研究通讯》220（1996）553 - 562）。两个顺式作用正元件（-104至-85；-84至-60）位于转录起始点上游。一个负元件（+20至+40）和一个强正元件（+43至+53）位于人载脂蛋白B基因的第一个非翻译外显子中。反式作用因子BRF - 2、BRF - 1、BRF - 3和BRF - 4分别与上述四个顺式作用元件相互作用。在本研究中，我们研究了上游正元件-104至-85和-84至-60在调节apoB基因转录调控中对下游元件+20至+40和+43至+53的作用。通过在HepG2细胞中进行体外诱变和瞬时转染实验，发现顺式作用元件-84至-60对于上游元件-104至-85以及下游元件+20至+40和+43至+53的功能绝对必要。对下游正元件+43至+53进行体外诱变并将突变的启动子构建体转染到HepG2细胞中发现，+51位的核苷酸G对于元件+43至+53的强正活性至关重要。在HepG2细胞中，+51位的核苷酸G单一位点替换突变为A或T会显著降低载脂蛋白B基因的转录。这些结果表明，在体内，下游正启动子元件+43至+53中+51位的核苷酸G单一位点替换突变为A或T可能潜在地导致低β脂蛋白血症，这是一种常染色体显性疾病的杂合形式。
