Transcriptional regulation of rat Na(+)/H(+) exchanger isoform-2 (NHE-2) gene by Sp1 transcription factor.
The rat Na(+)/H(+) exchanger isoform-2 (NHE-2) gene promoter lacks a TATA box and is very GC rich. A minimal promoter extending from bp -36 to +116 directs high-level expression of NHE-2 in mouse inner medullary collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found in this region. The introduction of mutations within these Sp1 consensus elements and DNA footprinting revealed that only two of them were utilized and are critical for basal transcriptional activation in mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 100-fold, suggesting that Sp1 is critical for transcriptional regulation. However, neither Sp3 nor Sp4 was able to activate transcription in these cells. Furthermore, in mIMCD-3 cells, Sp1-mediated transcriptional activation was repressed by expression of Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may repress transcriptional activation by competing with Sp1 for binding to core cis-elements.
Bai
L
L
Department of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA.
Collins
J F
JF
Xu
H
H
Ghishan
F K
FK
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D004331:Drosophila melanogaster; D005786:Gene Expression Regulation / Q000502:physiology*; D007679:Kidney Medulla / Q000166:cytology / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D011993:Recombinant Fusion Proteins; C513111:Slc9a2 protein, mouse; C513110:Slc9a2 protein, rat; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D008156:Luciferases