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Transcriptional Elements Directing a Liver-Specific Expression of P450/6βA (CYP3A2) Gene-Encoding Testosterone 6β-Hydroxylase

https://doi.org/10.1006/abbi.1995.1206Get rights and content

Abstract

The P450/6βA (CYP3A2) gene encoding a testosterone 6β-hydroxylase is expressed predominantly in liver and induced by the treatment of rats with various compounds. To understand the mechanism of the basal transcriptional activation of the CYP3A2 gene, the cis-acting elements in the proximal promoter region (−165 to −73) of the CYP3A2 gene were identified in this study. Nuclear extract from rat livers interacted with three sites, 6βA-A (−106 to −87), 6βA-B (−140 to −119) and 6βA-C (−163 to −145). These sites were detectable by DNase I footprinting and gel mobility shift assays and found to share nucleotide sequence similarity with each other (T(A/C)(A/C)N(A/G)AAG(G/ T)(C/T)CA). Direct repeats of AGTTCA (−134 to −120) and AG(G/C)TCA (−162 to −148) are also detected in 6βA-B and 6βA-C sites, respectively. To elucidate the relationship of these sites with basal transcriptional activation of the CYP3A2 gene, varying lengths of the proximal promoter region (−164 to +41) fused to a CAT reporter gene were transfected in human hepatoma (HepG2) and mouse adrenal tumor (Y-1) cells. The relative level of CAT activity in HepG2 cells was slightly increased by the deletion of the 5′-portion from −164 to −111 bp, but was reduced to 14% of the control (the construct including from -110 to +41) by the deletion from −110 to −81 including the 6βA-A site. On the other hand, these deletions have no clear effect on the level of the activity in Y-1 cells. Substitution mutations at two nucleotides in the 6βA-A site resulted in the reduction of CAT activity in HepG2 cells to 12% of the activity in the wild-type construct, The interaction of an oligonucleotide corresponding to the 6βA-A site (−106 to −87) with liver nuclear factors was completely inhibited by the addition of a typical oligonucleotide for hepatocyte nuclear factor-4 (HNF-4) binding site (F, M. Sladek, W. Zhong, E. Lai, and J. E. Darnell, Jr,, 1990, Genes Dev. 4, 2353-2365) but not of oligonucleotides corresponding to 6βA-B or 6βA-C sites. These results suggest an essential role of the binding of HNF-4 and/or HNF-4-related nuclear factors to the 6βA-A site on the basal transcriptional activation of the CYP3A2 gene in liver cells.

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