title: Identification of a novel polymorphic enhancer of the human CYP3A4 gene.
abstract: CYP3A4, the most abundant form of cytochrome P450 in the human adult liver, shows wide interindividual variation in its activity. This variability is thought to be caused largely by transcriptional and genetic factors, yet the underlying mechanisms are poorly understood. The purpose of this study was to clarify the mechanisms controlling the CYP3A4 gene transcription and to search for genetic polymorphisms in the 5'-flanking region of the CYP3A4 gene. Transient transfection of human hepatoma HepG2 cells and of normal human hepatocytes with a series of CYP3A4 promoter-luciferase reporter plasmids revealed that a region from -11.4 to -10.5 kilobases, designated the constitutive liver enhancer module of CYP3A4 (CLEM4), was important for the constitutive activation of the CYP3A4 gene. Gel shift assay using nuclear extracts prepared from HepG2 cells showed that HNF-1alpha, HNF-4alpha, USF1, and AP-1 interacted with CLEM4. Furthermore, the introduction of mutations into their binding sites demonstrated that essentially all sites were required for the maximal enhancer activity. Screening for genetic polymorphisms within CLEM4 in genomic DNA from French persons, we identified the novel variant, TGT insertion between -11,129 and -11,128 (-11,129_-11,128insTGT), whose allele frequency was 3.1%. The -11,129_-11,128insTGT resulted in the disruption of USF1 binding and a 36% reduction of the enhancer activity. These results suggest that CLEM4 is a constitutive enhancer of the CYP3A4 gene in the liver and that -11,129_-11,128insTGT may at least partly contribute to the interindividual variability of CYP3A4 expression.
authors.affiliation: Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Matsumura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Saito
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ozeki
authors.forename: Kazuma
authors.initials: K
authors.lastname: Kiyotani
authors.forename: Masaki
authors.initials: M
authors.lastname: Fujieda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamazaki
authors.forename: Hideo
authors.initials: H
authors.lastname: Kunitoh
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kamataki
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011110:Polymorphism, Genetic / Q000235:genetics*
chemical_list: D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human