Isolation and characterization of the human CLC-5 chloride channel gene promoter.
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.
Hayama
A
A
Second Department of Internal Medicine, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima Bunkyo-ku, 113-8519, Tokyo, Japan.
Uchida
S
S
Sasaki
S
S
Marumo
F
F
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics*; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010281:Parathyroid Hormone / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic
C097503:CLC-5 chloride channel; D018118:Chloride Channels; D018076:DNA, Complementary; D010281:Parathyroid Hormone; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D018808:Transcription Factor AP-1; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate