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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

Laboratory of Computational Biology and Risk Analysis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina

Request for reprints: Douglas A. Bell, Environmental Genomics Section, C3-03, P.O. Box 12233, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709. Phone: 919-541-7686; Fax: 919-541-4634. E-mail: bell1{at}niehs.nih.gov.

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.

Key Words: SNPs • gene expression • glioma • ChIP • GSTM3 • promoter

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