Role of AP2 consensus sites in regulation of rat Npt2 (sodium-phosphate cotransporter) promoter.
Expression of the Npt2 gene, encoding the type II sodium-dependent phosphate cotransporter, is restricted to renal proximal tubule epithelium. We have isolated a 4,740-bp fragment of the 5'-flanking sequence of the rat Npt2 gene, identified the transcription initiation site, and demonstrated that this 5'-flanking sequence drives luciferase-reporter gene expression, following transfection in the proximal tubule cell-derived opossum kidney (OK) cell line but not in unrelated cell lines. Analysis of the promoter sequence revealed the presence of 10 consensus binding motifs for the AP2 transcription factor. Transient transfection assays revealed an important effect of the number of tandemly repeated AP2 sites in enhancing promoter activity. The promoter sequence also revealed a pair of inverted repeats enclosing 1,324 bp of intervening sequence and containing 8 of the total 10 AP2 consensus sites in the promoter sequence. Deletion or reversal of orientation of the distal inverted repeat resulted in marked enhancement of promoter activity. Electrophoretic mobility shift analysis revealed a distinct pattern of transcription factor binding to oligonucleotides containing AP2 sites, using nuclear extracts from OK cells, compared with unrelated cell lines. Taken together, these results suggest an important role for AP2 consensus binding sites in regulating Npt2 gene expression and suggest a mechanism of regulation mediated by the interaction of inverted repeats enclosing these sites.
Shachaf
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Bruce Rappaport Faculty of Medicine and Research Institute, Technion, Israel Institute of Technology, Department of Molecular Medicine, Rambam Medical Center, Haifa 31096, Israel.
Skorecki
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Tzukerman
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics* / Q000494:pharmacology; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009893:Opossums; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid / Q000502:physiology; D050608:Sodium-Phosphate Cotransporter Proteins; D050609:Sodium-Phosphate Cotransporter Proteins, Type I; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D013045:Species Specificity; D027981:Symporters; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C493548:SLC17A2 protein, human; C493549:Slc17a2 protein, mouse; D050608:Sodium-Phosphate Cotransporter Proteins; D050609:Sodium-Phosphate Cotransporter Proteins, Type I; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D027981:Symporters; D050656:Transcription Factor AP-2; D014157:Transcription Factors