Objectives: In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2AS 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. Methods and results: 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. Conclusion: A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.
|Titolo:||A novel single nucleotide polymorphism in the CCAAT-box of the CYP2A6 promoter impairs binding of NF-Y|
|Parole Chiave:||CYP2A6; CYP2A6* 1H; CYP2A6* 1J; Dynamic allele-specific hybridization (DASH); Genetic polymorphism; NF-Y; Nicotine|
|Settore Scientifico Disciplinare:||Settore BIO/18 - Genetica|
|Data di pubblicazione:||2004|
|Digital Object Identifier (DOI):||10.1097/00008571-200406000-00006|
|Appare nelle tipologie:||01 - Articolo su periodico|