Isolation and characterization of the human CLC-5 chloride channel gene promoter
Isolation and characterization of the human CLC-5 chloride channel gene promoter
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DOI：
10.1016/s0378-1119(00)00493-5
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发表时间：
2000-12-31
期刊：
影响因子：
3.5
通讯作者：
Marumo, F
中科院分区：
文献类型：
--
作者：
Hayama, A;Uchida, S;Marumo, F
人类ClC-5氯通道主要在肾脏中表达，其突变导致登特氏病(一种家族性肾小管综合征，伴有高钙尿、管状蛋白尿、软骨病、肾钙质沉着症和最终的肾功能衰竭)。为了深入了解ClC-5基因表达的调控机制，我们分离并鉴定了一个包含人ClC-5基因5‘侧翼区的基因组克隆。用5‘-末端快速扩增法分离到两种类型的cDNA5’端，其中一种类似于含有ATG的外显子II上游的2.1kbp，在人类中首次被发现。主要启动子活性位于这个新发现的外显子Ia的5‘侧翼区。近端5‘侧翼区序列含有激活蛋白(AP)-1样位点和cAMP反应元件，但缺少TATA盒、富含GC元件和SP-1位点。5‘-侧翼区的缺失分析表明，位于-38位的含AP-L样元件的片段在表达ClC-5的LLC-PK1细胞中具有较高的启动子活性，但进一步缺失不含该AP-L样元件的片段会导致荧光素酶活性的丧失。凝胶滞留分析表明，在LLC-PK1细胞中存在一种与这种AP-L样元件结合的特异性蛋白，这似乎与真正的AP-1不同。这项研究阐明了人类CLCN5启动子的关键元件，该区域的突变可能是Dent病的原因。(C)2000 Elsevier Science B.V.保留所有权利。
The human CLC-5 chloride channel is expressed mainly in the kidney and its mutations cause Dent's disease (a familial renal tubular syndrome with hypercalciuria, tubular proteinuria, rickets, nephrocalcinosis, and eventual renal failure). To gain insight into the regulatory mechanism of CLC-5 expression, a genomic clone that contains the 5'-flanking region of the human CLC-5 gene was isolated and characterized. Two types of 5'-ends of cDNA were isolated by 5'-rapid amplification of cDNA ends, and one of them, similar to2.1 kbp upstream of ATG-containing exon II, was first identified in human. The major promoter activity was detected in the 5'-flanking region of this newly identified exon Ia. The sequence of the proximal 5'-flanking region contained an activator protein (AP)-1-like site and cAMP-responsive element, but it lacked a TATA box, a GC-rich element, and an SP-1 site. Deletion analysis of the 5'-flanking region showed that the fragments containing the AP-l-like element (TGACTCC) positioned at -38 exhibited high promoter activities in CLC-5 expressing LLC-PK1 cells, but that further deletions not containing this AP-l-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-l-like element in LLC-PK1 cells, which seemed to differ from an authentic AP-1. This study clarified the key element of the human CLCN5 promoter, and the mutation in this region could be the cause of Dent's disease. (C) 2000 Elsevier Science B.V. All rights reserved.