Abstract
Cytochrome P4501A2 (CYP1A2) is a member of the cytochrome P450 family that is involved in phase I drug metabolism in vertebrates. To understand how the constitutive expression of the human CYP1A2 gene is regulated, its 5' flanking region was analyzed. The promoter activity of a human CYP1A2 gene sequence [base pairs (bp) -3203 to +58 bp] was measured in transiently transfected HepG2 cells using fusion constructs containing the luciferase reporter gene. Using 5'-end deletion analysis, two functionally important cis elements, i.e., a proximal 42-bp DNA from bp -72 to bp -31 and a distal 259-bp DNA from bp -2352 to bp -2094, were identified. The proximal sequence (bp -72 to -31) contained CCAAT and GC boxes, with which well characterized transcription factors such as nuclear factor-1/CCAT transcription factor and simian virus 40 promoter factor-1 could interact. With regard to the 259-bp fragment (bp -2352 to bp -2094), gel mobility shift analyses with HepG2 nuclear lysates indicated high affinity, specific interactions of several trans-acting factors. Three protein binding sites within the 259-bp fragment were identified by DNase 1 footprinting analysis; these sites contained activator protein-1, nuclear factor-E1.7, and one-half hepatic nuclear factor-1 (HNF-1) binding consensus sequences. Only the region from bp -2124 to bp -2098, in which the HNF-1 binding site was located, was markedly protected by a HepG2 nuclear extract, compared with a MCF7 human breast cancer nuclear extract. These results suggested that the 259-bp DNA fragment contained positive regulator binding sites and HNF-1 could contribute to the liver-specific expression of human CYP1A2.
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|