Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity.
Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a lambda bacteriophage clone containing 3.7 kb of the proximal 5' flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5' rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5' untranslated region. The proximal 5' flanking region was "TATA-less" but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5' flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from -31 to -113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5' flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from -113 to -31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.
Whyte
D A
DA
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Li
C
C
Thomson
R B
RB
Nix
S L
SL
Zanjani
R
R
Karp
S L
SL
Aronson
P S
PS
Igarashi
P
P
D000818:Animals; D001483:Base Sequence / Q000235:genetics; D015820:Cadherins / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004847:Epithelial Cells / Q000502:physiology; D017353:Gene Deletion; D015870:Gene Expression; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic
D015820:Cadherins; C491946:Cdh16 protein, mouse; D009687:Nuclear Proteins