FUNCTIONAL-ANALYSIS OF THE PROMOTER FOR THE HUMAN CYP1B1 GENE

Our laboratory has cloned the cDNA (Sutter, T. R., Tang Y, M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Gr eenlee, W. P. (1994) J. Biol. Chem. 269, 13092-13099) and gene (Tang, Y. M., Wo, Y.-Y.P., Jabs, E. pi,, Stewart, J. C., Sutter, T. R. and Gr eenlee, W. F. (1996) J. Biol. Chem. 271, 28324-28350) for human CYP1B1 , a new member of the cytochrome P450 superfamily, Here, we report on the mapping and function of the CYP1B1 promoter, The CYP1B1 promoter i s fully functional, when if: is uncoupled from upstream enhancer eleme nts. Deletion analysis and site-directed mutagenesis identified four r egulatory elements required for maximum promoter activity: two antisen se Spl sites (-84 to -89 and -68 to -73), a TATA-like box (-84 to -29) , and an initiator motif (-5 to +3). The initiator and the TATA-like e lements are both required for basal promoter activity, with enhanced a ctivity mediated by the two antisense Sp1 elements. The GYP1B1 initiat or was demonstrated by in vitro transcription analysis to be a positio ning element that maintained fidelity of transcription from a single s ite. Specific binding to a CYP1B1 initiator probe by human nuclear ext ract proteins was competed either by the highly homologous murine term inal deoxynucleotidyl transferase initiator or, to a lesser extent, by the adenovirus major late initiator. Taken together, these results in dicate that the structure and function of the CYP1B1 promoter confers constitutive expression of the gene and assures fidelity of transcript ion initiation from a single site, The CYP1B1 promoter is distinct fro m the promoters of the closely related cytochrome P450s CYP1A1 and CYP 1A2 and is structurally and functionally similar to the promoters of c onstitutively expressed genes and at least two viruses.