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Isolation and characterization of the human CLC-5 chloride channel gene promoter
1Second Department of Internal Medicine, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima Bunkyo-ku, 113-8519, Tokyo, Japan.
Abstract:
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, approximately 2.1 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-1-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-1-like element resulted in a great loss of luciferase activities. Gel-retardation analysis demonstrated the existence of a specific protein binding to this AP-1-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.
Insights
Researchers identified a key regulatory element in the human CLC-5 gene promoter. This activator protein (AP)-1-like site is crucial for CLC-5 gene expression and may be implicated in Dent's disease.
Area of Science:
- Molecular Biology
- Genetics
- Renal Physiology
Background:
- The human CLC-5 chloride channel is primarily expressed in the kidney.
- Mutations in CLC-5 cause Dent's disease, a renal tubular disorder characterized by hypercalciuria, proteinuria, rickets, nephrocalcinosis, and renal failure.
Purpose of the Study:
- To investigate the regulatory mechanisms controlling human CLC-5 gene expression.
- To identify the promoter region responsible for CLC-5 gene transcription.
Main Methods:
- Isolation and characterization of a genomic clone containing the 5'-flanking region of the human CLC-5 gene.
- 5'-rapid amplification of cDNA ends (RACE) to identify transcription start sites.
- Reporter gene assays (luciferase) with deletion analysis to map promoter activity.
- Gel-retardation assays to study protein-DNA interactions.
Main Results:
- A novel exon (exon Ia) and its upstream 5'-flanking region were identified as the major promoter site.
- The proximal 5'-flanking region contains an activator protein (AP)-1-like site and a cAMP-responsive element, but lacks TATA and SP-1 sites.
- Deletion analysis revealed that the AP-1-like element at position -38 is essential for high promoter activity in kidney cells (LLC-PK1).
- Specific protein binding to the AP-1-like element was observed, suggesting a unique regulatory interaction.
Conclusions:
- The study identified a critical AP-1-like element within the human CLCN5 promoter that drives gene expression.
- This element is essential for CLC-5 channel function in the kidney.
- Mutations affecting this regulatory region could be a cause of Dent's disease.