title: Transcriptional regulation of the rat NHE3 gene. Functional interactions between GATA-5 and Sp family transcription factors.
abstract: Expression of sodium-hydrogen exchanger isoform 3 (NHE3) in the intestinal and renal epithelium plays a critical role in sodium absorption and acid/base homeostasis. To decipher rat NHE3 gene regulation, its cis-acting regulatory elements and associated transcription factors were characterized by transient transfection of Caco-2, IEC-6, Qt6, and Drosophila SL2 cells. Deletion and mutational analyses demonstrated that the atypical TATA box located at bp -26/-31 was not necessary for promoter activity, and that a -20/+8-bp fragment represents a functional initiator. Within the 81-bp upstream region, three Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrate that rat NHE3 promoter is initiator-driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction represents a novel regulatory mechanism, which is likely to participate in a gradient of intestinal gene expression along the crypt-villus axis.
authors.affiliation: Departmentf Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA.
authors.forename: Pawel R
authors.initials: PR
authors.lastname: Kiela
authors.forename: Jeffrey
authors.initials: J
authors.lastname: LeSueur
authors.forename: James F
authors.initials: JF
authors.lastname: Collins
authors.forename: Fayez K
authors.initials: FK
authors.lastname: Ghishan
mesh_terms: D000230:Adenocarcinoma; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology / Q000235:genetics; D003110:Colonic Neoplasms; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D050992:GATA5 Transcription Factor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D019459:Urothelium / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D050992:GATA5 Transcription Factor; C494755:GATA5 protein, human; C494756:Gata5 protein, mouse; C494757:Gata5 protein, rat; D020033:Protein Isoforms; D011994:Recombinant Proteins; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D014157:Transcription Factors; D008156:Luciferases