Studies on the mechanisms that control transcription of the cytochrome P450 Cyp1a1 gene were carried out in three areas: A. Cis-acting regulatory sequences; B. Regulatory pathways; and C. Trans-activating proteins. A. Cis-acting regulatory sequences. The effect of individual, pairwise and simultaneous mutations of the three distal aromatic hydrocarbon responsive elements (Ah REs) were studied using fusions with a reporter gene. In wild type cells, loss of each individual element affected expression to different degrees. The effect was additive in pairwise mutations, and mutations in all three elements practically abolished all expression. In a mutant cell line that lacks P450 activity, basal levels were highly elevated, and addition of inducer had little or no additional effect. These results suggest that the basal expression level is controlled by interactions occurring at the 5'-most AhRE and involving an autoregulatory effect of the cyp1a1 protein. B. Regulatory pathways. Two other regulatory pathways of Cyp1a1 expression have been uncovered. One consists of the apparent requirement for Ah receptor phosphorylation to bind to its cognate AhRE sequences. The other involves the role that inhibition of hydroxymethylglutaryl coenzyme A reductase plays in derepression of Cyp1a1 transcription. Functional receptor is required, since the effect does not occur in receptorless cells. These data suggest that the Ah receptor may function as a repressor under certain conditions. C. Trans-activating proteins. A cDNA clone coding for an AhRE-binding protein has been isolated and partially sequenced. Its deduced amino acid sequence contains a possible DNA-binding zinc-finger domain. Its localization to mouse chromosome 2 makes it unlikely that it corresponds to the Ah receptor gene, known to reside on chromosome 12. Several DNA-binding proteins that interact with the AhRE sequences have been isolated by DNA-affinity chromatography. The ligand-binding activity characteristic of the Ah receptor copurifies with these proteins, leading to the conclusion that the Ah receptor is one of them. Antibodies will be used for cloning.
