Petrovic N, Black T A, Fabian J R, Kane C, Jones C A, Loudon J A, Abonia J P, Sigmund C D, Gross K W
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
J Biol Chem. 1996 Sep 13;271(37):22499-505. doi: 10.1074/jbc.271.37.22499.
Mouse As4.1 cells, obtained after transgene-targeted oncogenesis to induce neoplasia in renal renin-expressing cells, express high levels of renin mRNA from the endogenous Ren-1(c) gene. We have used these cells to characterize the role of the Ren-1(c) proximal promoter (+6 to -117) in the regulation of renin gene transcription. It was found that 4.1 kilobases (kb) of Ren-1(c) 5'-flanking sequence, in combination with the proximal promoter, are required for strong activation (approximately 2 orders of magnitude over the basal level of the promoter alone) of the chloramphenicol acetyltransferase reporter in transfection assays. Within the 4.1-kb fragment, a 241-base pair region was identified that retains full activity in an orientation-independent manner in combination with the promoter. The resulting transcripts initiate at the normal renin start site. Electrophoretic mobility shift assays identified a sequence at approximately position -60 in the promoter region that binds nuclear proteins specific for renin-expressing As4.1 cells. Mutations in this sequence, which disrupt binding of nuclear protein(s), completely abolish activation of transcription by the 4. 1-kb fragment. Activation of transcription by the 241-base pair enhancer was still observed, although it was diminished in magnitude (60-fold over the mutated promoter alone). We present a model derived from the current data that suggests that regulation of renin expression is achieved through cooperation of transcription factors binding at the proximal promoter element and a distal enhancer element to abrogate or override the effects of an intervening negative regulatory region.
通过转基因靶向肿瘤发生在表达肾素的肾细胞中诱导肿瘤形成后获得的小鼠As4.1细胞，从内源性Ren-1(c)基因表达高水平的肾素mRNA。我们利用这些细胞来表征Ren-1(c)近端启动子（+6至-117）在肾素基因转录调控中的作用。发现在转染实验中，4.1千碱基（kb）的Ren-1(c) 5'侧翼序列与近端启动子结合，是氯霉素乙酰转移酶报告基因强烈激活（比单独启动子的基础水平高约2个数量级）所必需的。在4.1-kb片段内，鉴定出一个241碱基对的区域，该区域与启动子结合时以方向独立的方式保留全部活性。产生的转录本在正常肾素起始位点起始。电泳迁移率变动分析确定了启动子区域中大约-60位置的一个序列，该序列与表达肾素的As4.1细胞特异性的核蛋白结合。该序列中的突变破坏了核蛋白的结合，完全消除了4.1-kb片段对转录的激活。尽管241碱基对增强子对转录的激活程度有所降低（比单独的突变启动子高60倍），但仍可观察到。我们根据当前数据提出了一个模型，表明肾素表达的调控是通过结合在近端启动子元件和远端增强子元件上的转录因子协同作用来消除或克服中间负调控区域的影响而实现的。
