A natural CYP2B6 TATA box polymorphism (-82T--> C) leading to enhanced transcription and relocation of the transcriptional start site.
We investigated the impact of promoter polymorphisms on transcription of the human CYP2B6 gene. In total, 98 DNA samples from white persons from a previously characterized liver bank were sequenced throughout 2.3 kilobases of upstream sequence and haplotype structures were determined using additional coding sequence information. HepG2 cells and primary rat and human hepatocytes were transfected with luciferase reporter gene constructs driven by 2033 base pairs (bp) of the most frequent promoter variants. The novel haplotype *22 (-1848C--> A, -801G--> T, -750T--> C, and -82T--> C) showed 3- to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed -82T--> C, predicted to disrupt a putative TATA box, to be alone responsible for this effect. In silico analysis and electrophoretic mobility shift assay demonstrated conversion of the putative TATA box into a functional CCAAT/enhancer-binding protein binding site. Analysis of transcriptional start sites showed the mutant promoter to be transcribed from a start site located approximately 30 bp downstream of the wild-type start site, consistent with the use of a noncanonical TATA box at -55 bp. Median CYP2B6 mRNA expression and bupropion hydroxylase activity as a selective marker of CYP2B6 catalytic activity were approximately 2-fold higher in livers genotyped -82TC as in those genotyped -82TT (20.4 versus 9.8 arbitrary units, p = 0.007, and 201.8 versus 106.7 pmol/mg/min, p = 0.042, respectively). This promoter polymorphism thus contributes to CYP2B6 functional variability and represents a novel mechanism by which mutations can enhance transcription. Furthermore, a detailed interspecies comparison of CYP2B promoters and transcriptional start sites provided novel insights into evolutionary relationships.
Zukunft
Jörg
J
Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
Lang
Thomas
T
Richter
Tanja
T
Hirsch-Ernst
Karen I
KI
Nussler
Andreas K
AK
Klein
Kathrin
K
Schwab
Matthias
M
Eichelbaum
Michel
M
Zanger
Ulrich M
UM
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D065702:Cytochrome P-450 CYP2B6; D003596:Cytosine; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D016385:TATA Box / Q000235:genetics*; D013941:Thymine; D024363:Transcription Initiation Site
D003596:Cytosine; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D013941:Thymine