Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity

Dilys A. Whyte, Congyi Li, R. Brent Thomson, Stagey L. Nix, Reza Zanjani, Sharon Karp, Peter S. Aronson, Peter Igarashi

Research output: Contribution to journalArticle

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Abstract

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 λ 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.

Original languageEnglish
JournalAmerican Journal of Physiology - Renal Physiology
Volume277
Issue number4 46-4
StatePublished - Oct 1999

Fingerprint

Cadherins
5' Flanking Region
Epithelial Cells
Kidney
Hepatocyte Nuclear Factors
Genes
Nuclear Proteins
Luciferases
Genetic Promoter Regions
Binding Sites
GATA Transcription Factors
CCAAT-Enhancer-Binding Proteins
NIH 3T3 Cells
5' Untranslated Regions
Transcription Initiation Site
Electrophoretic Mobility Shift Assay
Ribonucleases
Protein Binding
Bacteriophages
Introns

Keywords

  • Cell adhesion
  • Gene regulation
  • Kidney specific
  • Transcription factor

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

Whyte, D. A., Li, C., Thomson, R. B., Nix, S. L., Zanjani, R., Karp, S., ... Igarashi, P. (1999). Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity. American Journal of Physiology - Renal Physiology, 277(4 46-4).

Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity. / Whyte, Dilys A.; Li, Congyi; Thomson, R. Brent; Nix, Stagey L.; Zanjani, Reza; Karp, Sharon; Aronson, Peter S.; Igarashi, Peter.

In: American Journal of Physiology - Renal Physiology, Vol. 277, No. 4 46-4, 10.1999.

Research output: Contribution to journalArticle

Whyte, DA, Li, C, Thomson, RB, Nix, SL, Zanjani, R, Karp, S, Aronson, PS & Igarashi, P 1999, 'Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity', American Journal of Physiology - Renal Physiology, vol. 277, no. 4 46-4.
Whyte, Dilys A. ; Li, Congyi ; Thomson, R. Brent ; Nix, Stagey L. ; Zanjani, Reza ; Karp, Sharon ; Aronson, Peter S. ; Igarashi, Peter. / Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity. In: American Journal of Physiology - Renal Physiology. 1999 ; Vol. 277, No. 4 46-4.
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AU - Zanjani, Reza

AU - Karp, Sharon

AU - Aronson, Peter S.

AU - Igarashi, Peter

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AB - 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 λ 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.

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