title: Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.
abstract: Discovery and functional evaluation of biologically significant regulatory single nucleotide polymorphisms (SNP) in carcinogen metabolism genes is a difficult challenge because the phenotypic consequences may be both transient and subtle. We have used a gene expression screening approach to identify a functional regulatory SNP in glutathione S-transferase M3 (GSTM3). Anttila et al. proposed that variation in GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression was affected by exposure to cigarette smoke and inheritance of the GSTM1-null genotype. To investigate the mechanism of GSTM3 expression variation, we measured GSTM3 expression in lymphoblast cells from a human Centre d'Etude du Polymorphisme Humain family and observed a low expression phenotype. Promoter sequencing revealed two novel GSTM3 promoter SNPs: A/C and A/G SNPs, 63 and 783 bp upstream of the codon 1 start site, respectively. In this pedigree, the two children homozygous for the -63C/C genotype had 8-fold lower GSTM3 expression relative to the two children with the -63A/A genotype, with no association between A-783G SNP and GSTM3 expression. Further evaluation using genotyped glioma cell lines and with luciferase reporter constructs showed that the -63C allele was associated with lower GSTM3 expression (P < 0.0001 and P < 0.003). RNA pol II chromatin immunoprecipitation was combined with quantitative probed-based allelic discrimination genotyping to provide direct evidence of a 9-fold reduced RNA pol II binding capacity for the -63C allele. These results show that the GSTM3 -63C allele strongly affects gene expression in human cell lines and suggests that individuals who carry the low expression allele may be deficient in glutathione transferase catalyzed biological functions.
authors.affiliation: Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Campbell
authors.forename: Gary S
authors.initials: GS
authors.lastname: Pittman
authors.forename: Eric C
authors.initials: EC
authors.lastname: Faulkner
authors.forename: Mary A
authors.initials: MA
authors.lastname: Watson
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Bell
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D005910:Glioma; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008297:Male; D010375:Pedigree; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D002843:Chromatin; D017931:DNA Primers; D004273:DNA, Neoplasm; C483009:GSTM3 protein, human; D005982:Glutathione Transferase