Yan Y, Jones C A, Sigmund C D, Gross K W, Catanzaro D F
Cardiovascular Center, Cornell University Medical College, New York, NY 10021, USA.
Circ Res. 1997 Oct;81(4):558-66. doi: 10.1161/01.res.81.4.558.
Despite the strong conservation of proximal 5'-flanking DNA sequences, cell transfection and transgenic animal studies have failed to provide a unifying hypothesis to explain the expression of both mouse and human renin genes. Recently, sequences contained in the mouse Ren-1c gene 5'-flanking DNA (-2866 to -2625) were shown to contain an enhancer-like element that stimulates Ren-1c promoter activity in renin-expressing As4.1 cells approximately 80-fold. Earlier studies using transgenic mice had suggested that this same region is required for the cell-specific expression of mouse renin genes. Since existing human renin genomic clones lack sequences homologous to the mouse renin enhancer, we isolated several human P1 and P1 artificial chromosome genomic clones that contain > 80 kb spanning the human renin gene. Analysis of these clones by Southern blot hybridization and long-rang polymerase chain reaction showed that they contain sequences homologous to the mouse enhancer at approximately 12 kb upstream of the transcription start site. Mouse and human sequences were 59% identical over a 650-bp region that contained the minimal enhancer from the mouse Ren-1c gene. However, a 1-kb fragment containing the entire human enhancer homology failed to stimulate human renin promoter activity in transiently transfected As4.1 cells. Further deletional analysis showed that a 220-bp region of the human sequence highly conserved in the mouse Ren-1c gene exhibited up to 47-fold transcriptional stimulation, although this was lower than the maximal effect exhibited by the minimal mouse enhancer (223-fold). Taken together, these observations suggest that sequences surrounding the conserved enhancer core stimulate enhancer activity in the mouse gene but suppress activity in the human gene. The high transcriptional activity of the mouse enhancer may have evolved to support the exceptionally high plasma renin concentrations found in mice. However, the enhancer core and surrounding conserved sequences may play an additional role in directing cell specificity.
尽管5'-侧翼DNA序列近端具有很强的保守性，但细胞转染和转基因动物研究未能提供一个统一的假说来解释小鼠和人类肾素基因的表达。最近，小鼠Ren-1c基因5'-侧翼DNA（-2866至-2625）中包含的序列被证明含有一个增强子样元件，该元件可刺激肾素表达的As4.1细胞中Ren-1c启动子活性约80倍。早期使用转基因小鼠的研究表明，同一区域是小鼠肾素基因细胞特异性表达所必需的。由于现有的人类肾素基因组克隆缺乏与小鼠肾素增强子同源的序列，我们分离了几个包含跨越人类肾素基因> 80 kb的人类P1和P1人工染色体基因组克隆。通过Southern印迹杂交和长距离聚合酶链反应对这些克隆进行分析表明，它们在转录起始位点上游约12 kb处含有与小鼠增强子同源的序列。在包含小鼠Ren-1c基因最小增强子的650 bp区域内，小鼠和人类序列的同一性为59%。然而，一个包含整个人类增强子同源性的1 kb片段在瞬时转染的As4.1细胞中未能刺激人类肾素启动子活性。进一步的缺失分析表明，人类序列中在小鼠Ren-1c基因中高度保守的220 bp区域表现出高达47倍的转录刺激，尽管这低于最小小鼠增强子所表现出的最大效应（223倍）。综上所述，这些观察结果表明，保守增强子核心周围的序列在小鼠基因中刺激增强子活性，但在人类基因中抑制活性。小鼠增强子的高转录活性可能已经进化以支持小鼠中发现的异常高的血浆肾素浓度。然而，增强子核心和周围保守序列可能在指导细胞特异性方面发挥额外作用。
