MULTIPLICITY AND EXPRESSIN OF RAT CYP3A

Abstract

We have isolated 5 different CYP3A genes (P450/6βA, P450/6βB, P450/6βD, P450/6βE and P450/6βF) containing 5'-flanking region and the first exon. Several clones covering the whole structure of two genes (P450/6βA and P450/6βB) were isolated and analyzed nucleotide sequences around exon-intron boundary, resulting that these gene consisted in 13 exons. P450/6βA and P450/6βB may correspond to CYP3A2 and CYP3A1, respectively, based on their nucleotide sequences and expression profiles of the two mRNA. The 5'-flanking region of P450/6βA and P450/βB genes were further analyzed. A high conserved region was observed in proximal promoter region (-180bp to -1bp) of both genes. Using DNase I footprinting analysis and gel shift assay, three sites (6βA-A, 6βA-B and 6βA-C) interacting with nuclear proteins were found in the proximal promoter region of P450/6βA gene. Thus, we carried out to analyze the role of these sites on transcriptional activation of P450/6βA gene using CAT assay. A site (60A-A: -89bp to -106bp) was mainly responsible for the transcriptional activation of P450/6βA gene in HepG2 cells, but not in Y1 cells. The nuclear protein binding to 6βA-A site was competitively inhibited by the addition of APF 1 oligonucleotide which bound hepatocyte nuclear factor-4 (HNF-4) in gel shift assay. These results suggest that HNF-4 plays an essential role of the transcriptional activation of P450/6βA gene in rat liver.