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 transcri ption factor that regulates the expression of several drug-metabolizin g enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxi cities, The mouse Ahr gene 5' proximal promoter region, which contains four potential Spl 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 Spl sites w ere protected from DNase I digestion using nuclear extracts from MLE-1 2 (lung), F9 (embryonal carcinoma), Hepa-1 (hepatoma), and 41-5a (epid ermal) cells. The Hepa-1 and F9 cell lines were shown by reverse trans criptase-polymerase chain reaction and Western blot to contain mRNA an d protein for Sp1 and Sp3, but not Sp2 and Sp4. In electrophoretic mob ility shift assays using oligonucleotide probes corresponding to the f our Ahr Sp1 sites, nuclear extracts from Hepa-1 and F9 cells formed co mplexes that were determined immunologically to contain both Sp1 and S p3 protein. The two Ahr proximal Sp1 sites (A and B) were shown to bin d both Sp1 and Sp3 proteins, whereas the more distal sites (C and D) b ound only Sp1. Competition gel shift experiments showed that sites A a nd 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 S p1 sites, we fused the Ahr promoter to a luciferase (LUC) reporter gen e and transfected the construct into the Drosophila cell line Schneide r-2, which contains no Sp1 or Sp1-like factors. Cotransfection of this construct with expression plasmids for each of the Sp factors reveale d that Sp3 was approximately 1.6-fold more efficient than Sp1 in Ahr t ransactivation. Mutation of the four Sp1 sites individually and in com bination 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 exp ression and distribution of Sp1-like factors.