Activation of an SP binding site is crucial for the expression of Claudin 1 in rat epididymal principal cells
Activation of an SP binding site is crucial for the expression of Claudin 1 in rat epididymal principal cells
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DOI：
10.1095/biolreprod.106.057430
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发表时间：
2007-05-01
影响因子：
3.6
通讯作者：
Cyr, Daniel G.
中科院分区：
文献类型：
--
作者：
Dufresne, Julie;Cyr, Daniel G.
Claudin 1（CLDN 1）是存在于附睾中的紧密连接蛋白。关于Cldn 1转录调控的信息有限。在附睾中，编码紧密连接蛋白的基因的5'侧翼区的调节是未知的。本研究的目的是探讨Cldn 1基因在大鼠附睾中的转录调控。克隆了大鼠Cldn 1基因5'侧翼区的1.8kb序列。转录起始位点是位于相对于第一个密码子的-198位置的腺嘌呤，并且在推定的TATA盒下游26 bp处。它是大鼠附睾中Cldn 1基因转录的唯一起始位点。将Cldn 1启动子插入到荧光素酶基因表达载体中，并转染到大鼠附睾头细胞系（RCE-1）中。序列缺失分析显示，含有-61至+164 bp启动子的构建体实现了最小的启动子活性。该序列含有一个TATA盒和两个共有的SP1结合位点。电泳迁移率改变和超迁移率测定证实，SP1和SP3存在于RCE-1细胞和附睾核提取物中，并且它们结合到启动子的5' SP1结合基序。5' SP1结合位点的定点诱变导致最小启动子序列的反式激活降低4倍。这些发现表明，SP1和SP3结合到Cidn 1启动子区域，并且这种相互作用影响Cidn 1在大鼠附睾中的表达。
Claudin 1 (CLDN1) is a tight junctional protein present in the epididymis. Limited information exists regarding the regulation of Cldn1 transcription. In the epididymis, the regulation of the 5' flanking region of genes coding for tight junctional proteins is unknown. The present objectives were to investigate the transcriptional regulation of the Cldn1 gene in the rat epididymis. A 1.8-kb sequence of the 5' flanking region of the rat Cldn1 gene was cloned. The transcriptional start site is an adenine located at the -198 position relative to the first codon, and 26 bp downstream of the putative TATA box. It is the only start site for the Cldn1 gene transcription in the rat epididymis. The Cldn1 promoter was inserted into a luciferase gene expression vector and transfected into a rat caput epididymal cell line (RCE-1). Sequential deletion analysis revealed that minimal promoter activity was achieved with the construct containing -61 to +164 bp of the promoter. This sequence contained a TATA box and two consensus SP1 binding sites. Electrophoretic mobility shift and supershift assays confirmed that SP1 and SP3 were present in RCE-1 cells and epididymal nuclear extracts, and that they bind to the 5' SP1 binding motif of the promoter. Site-directed mutagenesis of the 5' SP1 binding site resulted in a 4-fold decrease in transactivation of the minimal promoter sequence. These findings indicate that SP1 and SP3 bind to the Cidn1 promoter region, and that this interaction influences the expression of Cidn1 in the rat epididymis.