Background: Accurate risk assessment for carcinogens requires understanding how genetic variation modulates exposure. Following discovery of new polymorphic genes in environmental response pathways, it is necessary to characterize their impact on function (genotype/phenotype relationship).
Aims : 1) Characterize functional role of promoter and coding region polymorphisms of metabolism genes using standard biochemical, molecular and cellular bioassay techniques (e.g. CYPs, GSTs, PGEs). 2) Develop model approaches for characterizing functional effects of polymorphisms in transcription factors, transcription factor binding sites, and other regulatory sequence elements using expression profiling. This project focuses on the functional characterization of variation in environmental response genes. These projects tend to be labor intensive and time consuming. We have recently developed methods which will improve productivity in this area (see future plans). Accomplishments: 1. 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. Discovery and functional evaluation of biologically significant regulatory polymorphisms (rSNPs) 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 rSNP in glutathione S-transferase M3 (GSTM3). Antilla et al. (1) 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 variation, we measured GSTM3 expression in lymphoblast cells from a human CEPH family and observed a low expression phenotype. Promoter sequencing revealed two novel GSTM3 promoter SNPs: an A/C and A/G SNP, 63-bp and 783-bp upstream of the codon 1 start site, respectively. In this pedigree, the two children homozygous for the -63C/C genotype had eight-fold lower GSTM3 expression relative to the two children with the -63A/A genotype, with no association between the A-783G SNP and GSTM3 expression. Further evaluation using genotyped glioma cell lines and with luciferase reporter constructs showed the -63C allele was associated with lower GSTM3 expression (p < 0.0001; p < 0.003). RNA polymerase II chromatin immunoprecipitation3 (ChIP) was combined with quantitative probed-based allelic-discrimination genotyping to provide direct evidence of a nine-fold reduced RNA polymerase II binding capacity for the -63C allele. These results demonstrate 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. 2.A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 gene (UGT1A1). The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert?s syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K) positive or micronuclei K negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7-TA and 8-TA displayed marginally lower GPA_NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7-TA and 8-TA) were associated with modestly increased HPRT mutation frequency (p<0.05) while the same low expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high expression genotypes (5-TA and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7-TA and 8-TA were associated with increased GPA_N? mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and genetic damage will be needed.
