Igarashi P, Whyte D A, Li K, Nagami G T
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520.
J Biol Chem. 1996 Apr 19;271(16):9666-74. doi: 10.1074/jbc.271.16.9666.
The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.
小鼠Nkcc2/Slcl2a1基因编码一种布美他尼敏感的钠-钾-氯共转运体，该共转运体仅在肾脏的髓袢升支粗段表达。核转录分析表明，Nkcc2的肾脏特异性表达至少部分归因于肾脏特异性基因转录。为了开始对该基因启动子的研究，分离出了一个包含Nkcc2 5'侧翼区13.5千碱基的基因组克隆。在起始密码子上游1330个碱基对（bp）处定位到一个单一的转录起始位点。近端5'侧翼区的序列包含典型的真核启动子元件，包括一个TATA盒、两个CCAAT盒和一个起始子。还鉴定出一个（G-A）28·（C-T）28微卫星以及肝细胞核因子1、cAMP反应元件结合蛋白、CCAAT/增强子结合蛋白和碱性螺旋-环-螺旋蛋白的共有结合位点。为了在功能上表达该启动子，将近端5'侧翼区的2255 bp连接到荧光素酶报告基因上，并转染到髓袢升支粗段（TAL）细胞中，TAL细胞是一种从Tg（SV40E）Bri7小鼠显微切割的髓袢中获得的稳定细胞系。TAL细胞表现出对呋塞米敏感的钠-钾-（铵离子）-氯共转运活性，并内源性表达5.0千碱基的Nkcc2转录本。与转染到不表达Nkcc2的细胞（NIH 3T3成纤维细胞）相比，转染到TAL细胞后的荧光素酶活性高130倍。缺失分析表明，从转录起始位点延伸至-1529至-469的构建体在TAL细胞中的启动子活性相似，而进一步缺失至-190导致活性降低76%。我们得出结论，Nkcc2启动子表现出肾细胞特异性活性。最大启动子活性所需的调控元件位于一个280 bp的DNA片段中，该片段包含在肾脏中表达的几种转录因子의共有结合位点。
