Regulation of mouse Ah receptor (Ahr) gene basal expression by members of the Sp family of transcription factors.
The aromatic hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of several drug-metabolizing enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxicities. The mouse Ahr gene 5' proximal promoter region, which contains four potential Sp1 motifs, is required for efficient basal expression. Using a fragment spanning the region from nt -174 to +70 of the Ahr promoter, we found that four regions corresponding to four Sp1 sites were protected from DNase I digestion using nuclear extracts from MLE-12 (lung), F9 (embryonal carcinoma), Hepa-1 (hepatoma), and 41-5a (epidermal) cells. The Hepa-1 and F9 cell lines were shown by reverse transcriptase-polymerase chain reaction and Western blot to contain mRNA and protein for Sp1 and Sp3, but not Sp2 and Sp4. In electrophoretic mobility shift assays using oligonucleotide probes corresponding to the four Ahr Sp1 sites, nuclear extracts from Hepa-1 and F9 cells formed complexes that were determined immunologically to contain both Sp1 and Sp3 protein. The two Ahr proximal Sp1 sites (A and B) were shown to bind both Sp1 and Sp3 proteins, whereas the more distal sites (C and D) bound only Sp1. Competition gel shift experiments showed that sites A and B had 10-fold higher affinity for Sp factors than did sites C and D. To determine the transactivation potential of each of the four Ahr Sp1 sites, we fused the Ahr promoter to a luciferase (LUC) reporter gene and transfected the construct into the Drosophila cell line Schneider-2, which contains no Sp1 or Sp1-like factors. Cotransfection of this construct with expression plasmids for each of the Sp factors revealed that Sp3 was approximately 1.6-fold more efficient than Sp1 in Ahr transactivation. Mutation of the four Sp1 sites individually and in combination demonstrated that each site contributes to the overall level of expression of the reporter gene and that interactions between these sites play a minor role in regulation of the Ahr-LUC construct. These results suggest that basal Ahr expression may be regulated by the expression and distribution of Sp1-like factors.
Center for Environmental Genetics and Department of Environmental Health, University of Cincinnati Medical Center, OH 45267-0925, USA.
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Fitzgerald
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Nebert
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Puga
D000818:Animals; D001667:Binding, Competitive; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D004330:Drosophila; D051379:Mice; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection
D002457:Cell Extracts; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004247:DNA