Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6)

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Abstract
In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2A6 gene variants are known which contribute to the highly variable expression of this enzyme among individuals. In this study we report a novel polymorphism located in the 5' flanking region (-745A > G) of the CYP2A6 gene disrupting a CCAAT box. Electrophoretic mobility shift assays (EMSA) indicated that NF-YA is part of this nuclear protein complex. Chromatin immunoprecipitation revealed that NF-Y recognizes a region of the CYP2A6 5' flanking region located between -932 and -606. EMSA showed that out of the three CCAAT boxes in the CYP2A6 promoter, with CCAAT core sequences located between -839/-835, -748/-744, and -689/-685, only the one at -748/-744 was able to compete with the nuclear protein complex binding to the -748/-744 CCAAT box. Cotransfection experiments indicated that NF-Y acts as a positive regulatory element on CYP2A6 gene regulation. EMSA demonstrated that an NF-Y consensus oligonucleotide but not the -745A > G oligonucleotide competed efficiently with binding of the protein complex to the -748/-744 CCAAT box. Promoter activity of the -745A > G variant was significantly reduced to 78% relative to the wild-type allele in HepG2 cells transfected with luciferase reporter plasmids. Finally, haplotype analysis was carried out comprising the -745A > G variant in combination with all known CYP2A6 3' and 5' flanking single nucleotide polymorphisms: -1013A > G, -48T > G, and the CYP2A6/CYP2A7 3' flank conversion. A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.
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