Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-Cl cotransporter gene

Peter Igarashi, Dilys A. Whyte, Kui Li, Glenn T. Nagami

Research output: Contribution to journalArticle

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Abstract

The murine Nkcc2/Slc12a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A) 28·(C-T) 28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na- K(NH 4 +)-Cl cotransport activity and endogenously expressed the 5.0- kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.

Original languageEnglish (US)
Pages (from-to)9666-9674
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number16
DOIs
StatePublished - Apr 19 1996

Fingerprint

Sodium-Potassium-Chloride Symporters
Cloning
Organism Cloning
5' Flanking Region
Genes
Kidney
Transcription Initiation Site
Extremities
Luciferases
Base Pairing
Loop of Henle
Hepatocyte Nuclear Factor 1
Binding Sites
CCAAT-Enhancer-Binding Proteins
Bumetanide
Cyclic AMP Response Element-Binding Protein
TATA Box
Transfection
Furosemide
Transcription

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-Cl cotransporter gene. / Igarashi, Peter; Whyte, Dilys A.; Li, Kui; Nagami, Glenn T.

In: Journal of Biological Chemistry, Vol. 271, No. 16, 19.04.1996, p. 9666-9674.

Research output: Contribution to journalArticle

Igarashi, Peter ; Whyte, Dilys A. ; Li, Kui ; Nagami, Glenn T. / Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-Cl cotransporter gene. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 16. pp. 9666-9674.
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abstract = "The murine Nkcc2/Slc12a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A) 28·(C-T) 28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na- K(NH 4 +)-Cl cotransport activity and endogenously expressed the 5.0- kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76{\%} decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.",
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