A Natural CYP2B6 TATA Box Polymorphism (–82T→ C) Leading to Enhanced Transcription and Relocation of the Transcriptional Start Site

Abstract

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 (Ϫ1848C3 A, Ϫ801G3 T, Ϫ750T3 C, and Ϫ82T3 C) showed 3-to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed Ϫ82T3 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 Ϫ55 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 Ϫ82TC as in those genotyped Ϫ82TT (20.4 versus 9.8 arbitrary units, p ϭ 0.007, and 201.8 versus 106.7 pmol/mg/min, p ϭ 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.The human cytochrome P450 gene superfamily currently consists of 57 functional genes and 58 pseudogenes (Nelson et al., 2004). The members of families CYP1, CYP2, and CYP3 are localized in the endoplasmic reticulum of both liver and extrahepatic tissues where they catalyze a variety of biotransformations of numerous endogenous and exogenous substrates, including many drugs currently in use. The family member CYP2B6 was first described in 1989 (Yamano et al., 1989) as the human ortholog to the phenobarbital-inducible CYP2B genes in rodents. Initially underestimated, the number of drugs recognized as CYP2B6 substrates has been constantly increasing, and several clinically important substances are now known to be preferred substrates of this enzyme. These include the anticancer prodrug cyclophosphamide (Roy et al., 1999); the narcotic propofol (Court et al., 2001); the antidepressant bupropion, which is now the most commonly used probe drug for CYP2B6 (Faucette et al., 2000); the antimalarial drug artemisinin (Svensson and Ashton, 1999); and the reverse transcriptase inhibitor efavirenz (Ward et al., 2003).

Allele and Genotype Frequencies ofCytochrome P450 2B6Gene in a Mongolian Population

“…reaction assay with predesigned primers and TaqMan MGB probes (TaqMan SNP Genotyping Assay; Applied Biosystems, Foster City, CA) or previously published primers and MGB probes (Tsuchiya et al, 2004;Klein et al, 2005;Zukunft et al, 2005;Gatanaga et al, 2007) using the ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems). All PCR reactions were performed in a 25-l volume containing 20 ng of genomic DNA, 2ϫ TaqMan Universal master mix (Applied Biosystems), and 20ϫ drug metabolism genotyping assay mix (Applied Biosystems) or 40ϫ assay mix (Applied Biosystems).…”

“…Lower expression was also found in cell lines transfected with *6, *11, *12, *13, *14, *15, *16, *18, *19, *20, and *21 alleles (Lang et al, 2004;Klein et al, 2005;Wang et al, 2006;Hofmann et al, 2008). Higher expression was detected *22-transfected cells (Zukunft et al, 2005). The *27 allele was reported to result in decreased in vitro enzymatic activity of bupropion hydroxylase, and *28 was reported to result in truncated protein (Rotger et al, 2007).…”

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

“…The thermal cycling conditions for the assay were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Previously published TaqMan assays were used for the mRNA quantification of CYP2B6 (Zukunft et al, 2005), CYP2C19 (Burk et al, 2005), CYP2D6 (Toscano et al, 2006), and CYP3A4 Wolbold et al, 2003). All primers and probes used are presented in Table 2.…”

MALDI-TOF Mass Spectrometry for Multiplex Genotyping of CYP2B6 Single-Nucleotide Polymorphisms

“…In contrast to the above-mentioned CYP genes, variations in CYP2B6 have been discovered through reverse genetics studies, i.e., by initial sequencing and variation scanning studies and subsequent analysis of the functional consequences of the identified variations (17)(18)(19)(20)(21)(22)(23)(24)(25). More than 100 DNA variations, including numerous nonsynonymous variations, as well as silent, promoter, and intronic changes, were found within the CYP2B6 gene, many of them showing extensive linkage disequilibrium giving rise to distinct haplotypes (26 ) (17,19,21,23 ), and alleles with increased expression [CYP2B6*22; (24 )]. Some of these variations are rare, but many are common, with allele frequencies between 10% and almost 50%, depending on the population studied (22,23 ).…”