Regulation of the constitutive expression of the human CYP1A2 gene: cis elements and their interactions with proteins.
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.
Chung
I
I
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA.
Bresnick
E
E
D001483:Base Sequence; D001665:Binding Sites; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D005809:Genes, Regulator; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D014407:Tumor Cells, Cultured
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D003850:Deoxyribonuclease I