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

Pharmacogenetics. 2004 Jun;14(6):369-79. doi: 10.1097/00008571-200406000-00006.


Objectives: 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.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 5' Flanking Region / genetics
  • Aryl Hydrocarbon Hydroxylases / genetics*
  • CCAAT-Binding Factor / genetics*
  • CCAAT-Binding Factor / metabolism
  • Carcinoma, Hepatocellular / enzymology
  • Carcinoma, Hepatocellular / pathology
  • Chromatin Immunoprecipitation
  • Cytochrome P-450 CYP2A6
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Enzymologic*
  • Gene Frequency
  • Haplotypes / genetics
  • Humans
  • Liver Neoplasms / enzymology
  • Liver Neoplasms / pathology
  • Luciferases / metabolism
  • Mixed Function Oxygenases / genetics*
  • Polymorphism, Genetic / genetics*
  • Promoter Regions, Genetic / genetics
  • Regulatory Sequences, Nucleic Acid / genetics*
  • Tumor Cells, Cultured


  • CCAAT-Binding Factor
  • Mixed Function Oxygenases
  • Luciferases
  • Aryl Hydrocarbon Hydroxylases
  • CYP2A6 protein, human
  • Cytochrome P-450 CYP2A6