Shi Q, Black T A, Gross K W, Sigmund C D
Departments of Internal Medicine and Physiology & Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Circ Res. 1999 Sep 17;85(6):479-88. doi: 10.1161/01.res.85.6.479.
A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4. 1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.
先前有报道称，在小鼠肾素基因上游存在一个远端转录增强子。在人类肾素基因上游也存在一个同源序列，但小鼠和人类肾素增强子在激活肾素启动子转录的能力上存在显著差异。尽管这两种增强子在其202bp的启动子远端部分具有高度同源性，但其40bp的近端部分是异质的。通过在表达小鼠肾脏肾素的As4.1细胞中进行瞬时转染分析，利用嵌合增强子来测试增强子40bp片段（m40）的作用。从小鼠肾素增强子中删除m40或将其添加到人类肾素增强子中，当靠近小鼠或人类肾素启动子时，转录活性没有显著变化。然而，当置于肾素启动子更上游时，相同的缺失和替换显著改变了增强子活性。电泳凝胶迁移率变动分析在m40中鉴定出两个元件，a和b，它们特异性结合As4.1细胞的核蛋白。诱变和瞬时转染分析表明，元件b负责m40的功能，而元件a拮抗元件b的正向影响。凝胶竞争和超迁移分析表明，核因子-Y，一种普遍存在的CAAT盒结合蛋白，与元件a结合。序列分析表明，人类肾素增强子在元件b中存在一个天然的功能丧失突变，这影响了其在启动子上游远处时的反式激活能力。将人类肾素元件b恢复为与小鼠序列匹配，可恢复增强子在小鼠As4.1细胞中的反式激活。这些数据表明，元件b与增强子的其余部分协同作用以维持完整的增强子活性，而元件a可能调节增强子活性。这些元件的序列差异可能解释了小鼠和人类肾素增强子序列的功能差异。
