Expression-Based Discovery of Variation in the Human Glutathione

Research Article

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

Xuemei Liu, Michelle R. Campbell, Gary S. Pittman, Eric C. Faulkner, Mary A. Watson, and Douglas A. Bell

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

Abstract on the efficacy of therapeutic treatments (16), characterization of Discovery and functional evaluation of biologically significant genotypic and phenotypic variation in the GST gene family is regulatory single nucleotide polymorphisms (SNP) in carcin- important. ogen metabolism genes is a difficult challenge because the GSTM3 is expressed in testis, brain, lung, and lymphocytes as phenotypic consequences may be both transient and subtle. well as in many tumor types (NCI-60; refs. 17–19). A recently We have used a gene expression screening approach to identify described GSTM3 3-bp deletion polymorphism (GSTM3*A/*B) has a functional regulatory SNP in glutathione S-transferase M3 been shown to have a frequency of 0.16 in the Caucasian population (GSTM3). Anttila et al. proposed that variation in GSTM3 (20, 21). This polymorphism was associated with an increased risk expression was affected by exposure to cigarette smoke and of cutaneous basal cell carcinomas and laryngeal carcinomas and inheritance of the GSTM1-null genotype. To investigate the interacted with other GST variants in modifying astrocytoma risk but had no effect on pharyngeal carcinoma risk (20–24). Anttila mechanism of GSTM3 expression variation, we measured GSTM3 expression in lymphoblast cells from a human Centre et al. (17) observed variation in GSTM3 expression in lung tissue d’Etude du Polymorphisme Humain family and observed a low and proposed a relationship among smoking exposure, GSTM3 expression phenotype. Promoter sequencing revealed two expression level, and GSTM1-null genotype. novel GSTM3 promoter SNPs: A/C and A/G SNPs, 63 and 783 To explore the mechanism for the Anttila et al. observations of bp upstream of the codon 1 start site, respectively. In this GSTM3 expression variation, we identified human cell lines with low expression GSTM3 phenotypes in a Centre d’Etude du Polymor- pedigree, the two children homozygous for the À63C/C genotype had 8-fold lower GSTM3 expression relative to the phisme Humain (CEPH) family and sequenced the GSTM3 promoters of these individuals. Using expression analyses (quanti- two children with the À63A/A genotype, with no association tative real-time PCR and Western blot), luciferase reporter assays between AÀ783G SNP and GSTM3 expression. Further evaluation using genotyped glioma cell lines and with luciferase and allele-specific chromatin immunoprecipitation (ChIP), we have shown that a GSTM3 promoter polymorphism causes a dramatic reporter constructs showed that the À63C allele was associated with lower GSTM3 expression (P < 0.0001 and P < 0.003). reduction in GSTM3 transcription and protein expression in the lymphoblast and glioma cell lines we investigated. 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 Materials and Methods binding capacity for the À63C allele. These results show that the GSTM3 À63C allele strongly affects gene expression in human Cell Culture. The human lymphoblast cell lines GM07348, GM07349, cell lines and suggests that individuals who carry the low GM07350, GM07351, GM07353, GM07354, GM07355, and GM07356 (CEPH/ UTAH Pedigree 1345) were purchased from Coriell Cell Repositories and expression allele may be deficient in glutathione transferase were maintained in RPMI 1640 supplemented with 15% heat-inactivated catalyzed biological functions. (Cancer Res 2005; 65(1): 99-104) fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin-streptomy-

cin (Invitrogen) at 5% CO2 at 37jC. The human glioma cell lines SNB-19, SF-539, and SF-268 were provided by National Cancer Institute and cultured Introduction under the same conditions listed above. All cultures were sampled during Human glutathione S-transferases (GST) protect cells against logarithmic growth phase. toxic insults by catalyzing nucleophilic addition or substitution DNA Extraction, Genotyping, and Sequencing. For genotyping, we reactions between glutathione and reactive electrophilic com- used a community-based sample of healthy, unrelated (19-53 years old) pounds (1–6). The GST gene superfamily has numerous families volunteers of African and European descent from Durham and Chapel Hill, (7–12), and many have notable polymorphisms that have been NC (protocol 86E-0037), and placental DNA samples from anonymous full- term, normal, unrelated maternity patients with Asian ancestry were shown to alter disease risk (13–15). Because GSTs are important for provided by Dr. L.L. Hsieh (Chang Gung University, Taiwan, China). DNA both their protection against environmental insults and their impact was isolated from all samples with standard proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation. Genotyping of the promoter single nucleotide polymorphism (SNP; rs1332018) was carried out using a Taqman allelic discrimination assay Request for reprints: Douglas A. Bell, Environmental Genomics Section, C3-03, (Applied Biosystems, Foster City, CA). The primers used were forward: 5V- P.O. Box 12233, National Institute of Environmental Health Sciences, NIH, Research GCGGAGGCGGAGTCA-3V and reverse: 5V-TCGAGGACTAGGGAAACTGTG- Triangle Park, NC 27709. Phone: 919-541-7686; Fax: 919-541-4634; E-mail: [email protected]. 3V. The probes used were VIC: 5V-CCCCCTTATGTAGGGTATA-3V and FAM: I2005 American Association for Cancer Research. 5V-CCCTTATGTCGGGTATA-3V. The reaction mix included 20 ng genomic www.aacrjournals.org 99 Cancer Res 2005; 65: (1). January 1, 2005

DNA, 1Â primer (0.2 Amol/L)/probe (0.9 Amol/L) mix, and 1 Â Taqman conditions. Cells were washed with PBS twice and harvested after Universal Master Mix (Applied Biosystems) in a final volume of 10 AL. incubation (2 hours, room temperature) in 500 AL passive lysis buffer Thermocycling consisted of a 95jC, 10-minute hold for AmpliTaq Gold (Promega) with agitation. Luciferase activity was determined using 20 AL activation followed by 40 cycles of 95jC, 15 seconds and 60jC, 60 seconds. cell lysate aliquots. The lysates were mixed with 100 AL luciferase firefly VIC and FAM probe signals were recorded post-PCR using an ABI PRISM assay reagent and read on a model TD-20/20 luminometer (Turner Designs, 7700 instrument (Applied Biosystems). GSTM3 promoter sequencing in Sunnyvale, CA) every 0.5 second for 25 seconds in a 96-well plate. Luciferase lymphoblasts was done using an ABI PRISM BigDye Terminator Cycle activity was normalized to the intensity of pRL-TK (Promega) and expressed Sequencing kit and an ABI PRISM 377 DNA sequencer (Applied Biosystems). as relative light units. All luciferase assays were carried out in triplicate. Sequences were compared with the GSTM3 consensus (Genbank accession ChIP Assay. We have used an approach similar to Knight et al. (25), no. AF043105) using the PileUp program of GCG Wisconsin package version combining a ChIP protocol in heterozygous À63AC (SNB-19) cells with 10.3 (Accelrys, San Diego, CA). Taqman quantitative genotyping. SNB-19 cells (3 Â 107) were used in 10 Measurement of GSTM3 Expression Using Real-time PCR. Total RNA independent experiments carried out with a ChIP procedure modified from was isolated from cells using a RNeasy Mini kit (Qiagen, Inc., Valencia, CA) Upstate Biotechnology (Lake Placid, NY). Cells were fixed using 1% j treated with DNase and measured for A260/A280 nm ratios using a NanoDrop formaldehyde and incubated (37 C, 10 minutes). Fixation was stopped with spectrometer (NanoDrop, Rockland, ME). cDNA was generated from 1 Ag 125 mmol/L glycine (37jC, 5 minutes). Cells were washed, pelleted (4jC, total RNA using the SuperScript First-Strand Synthesis System (Invitrogen). 2,000 rpm, 4 minutes), and lysed with 200 AL SDS lysis buffer (Upstate The cDNA reaction solution was diluted to a final concentration of 1.33 ng/ Biotechnology) containing the protease inhibitors. The lysate was sonicated AL. Real-time PCR constituents were 22.5 AL DNA, 1Â Taqman Universal (20 seconds, six times) using a Sonicator 3000 (Misonix, Farmingdale, NY) Master Mix, and 1Â primer/probe mix (Hs00356079, Applied Biosystems) in at 90% of output level, resulting in an average of 500-bp DNA length, a50AL final volume. Real-time PCR amplification was done using the same determined by 1% agarose Tris-borate EDTA gel electrophoresis. Samples conditions as for genotyping, except for the addition of an initial 50jC, 2- were centrifuged (13,000 rpm, 10 minutes) and the supernatant was diluted minute hold for optimal AmpErase UNG activity. Signals were recorded 10-fold in ChIP dilution buffer (Upstate Biotechnology) to a 2 mL final during PCR using an ABI PRISM 7700 instrument. All gene expression volume. For each sample, 20 AL supernatant was kept as input/starting results were normalized to HPRT expression. Gene expression was material. Immunoprecipitations were done by incubating (4jC, 30 minutes) calculated using the DCt method (Applied Biosystems). 2 mL diluted supernatant in 80 AL salmon sperm DNA/protein A agarose Measurement of GSTM3 Protein Level Using Western Blot. Cells were 50% slurry with agitation and then by centrifugation (2,000 rpm, 3 minutes). harvested and incubated (30 minutes on ice) in lysis buffer. After The supernatant was incubated (4jC, overnight) in 3 Ag RNA pol II antibody centrifugation (13,000 rpm, 15 minutes), proteins in the supernatants were (Santa Cruz Biotechnology, Santa Cruz, CA). A no-antibody immunopre- quantified (Bio-Rad Laboratories, Hercules, CA). Cell lysates (10 Ag) were cipitation served as a negative control. Following precipitation, protein- denatured (80jC, 15 minutes), loaded onto an 18% Tris-glycine gel DNA complexes were washed and eluted from the antibody by adding (Invitrogen), run on a XCell Surelock Mini-Cell (Invitrogen), and transferred elution buffer (Upstate Biotechnology). Protein-DNA cross-links were onto a 0.2 Am nitrocellulose membrane (Invitrogen). The membrane was rinsed (PBS with 0.05% Tween), blocked (1% bovine serum albumin in PBS), rinsed again, and incubated in primary rabbit anti-human GSTM3 antibody (1:2,000 dilution) generously provided by Dr. John D. Hayes (Biomedical Research Centre, University of Dundee, Dundee, United Kingdom). Protein was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) autoradiography. The membrane was stripped and similarly blotted with h-actin antibody. Preparation of pGL3 Vector Constructs. A fragment of the GSTM3 5V untranslated region, À948 to +3 nucleotides (relative to codon 1 start, Genbank accession no. AF043105), was generated by PCR amplification using genomic DNA from GM07354 cells (À783AA/À63AA). PCR was done using Expand High-Fidelity PCR System (Roche Molecular Diagnostics Corp., Indianapolis, IN) according to the manufacturer’s instructions. The primers used were forward: 5V-AAGAAGCTCGAGGTTGGACAGATGAATGAGTG-3V and reverse: 5V-AAGAAGAGATCTCATGGTGACGGGCTTCCGAG-3V.The PCR product was purified and inserted into the firefly luciferase reporter plasmid, pGL3-enhancer vector (Promega, Madison, WI), resulting in the reporter plasmid. In pGL3, the four possible haplotypes for the AÀ783G and AÀ63C sites were generated using the QuickChange Site-Directed Mutagen- esis kit (Stratagene, La Jolla, CA). The primers used were forward: 5V- CCGCCCCCTTATGTgGGGTATAAAGCCCCTC-3V and reverse: 5V- GGAGGGGCTTTATACCCcACATAAGGGGGCG-3V for AÀ783G site and forward: 5V-GATTAATTTCCCTAAATGCcTGAGCATTAGG-3V and reverse: 5V-CCTAATGCTCAgGCATTTAGGGAAATTAATC-3Vfor AÀ63C site. Sequen- ces were verified using the dye terminator cycle sequencing method on an ABI PRISM. Transient Transfection. The human glioma cell line SF-268 was plated in six-well plates at a density of 2 Â 105 cells per well and incubated at 37jC in RPMI 1640 containing 15% fetal bovine serum and 1% penicillin- streptomycin (Invitrogen) for 24 hours. After washing the cells with PBS, the cells were cotransfected (37jC, 4 hours) with 3 Ag pGL3-enhancer construct, Figure 1. Real-time PCR analysis of GSTM3 mRNA expression in CEPH1345 0.3 Ag pRL-TK (for normalization of luciferase expression), and 6 AL family. A, pedigree of CEPH1345 family. B, GSTM3 mRNA expression. The A amount of GSTM3 mRNA was calculated relative to a standard curve of Transfectam (Promega) in 500 L RPMI 1640 without fetal bovine serum reference RNA and the HPRT gene in the same cDNA sample. Columns, and then incubated (37jC, 48 hours) with fresh medium. The pRL-TK, pGL- mean from triplicate measurements; bars, SD. Each lymphoblast cell line was 3 control, and pGL-3 empty vectors were used to establish transfection evaluated with three independent experiments.

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Table 1. Genotyping and allele frequencies among Taiwanese, African Americans, and European Americans

n A/A, n (%) A/C, n (%) C/C, n (%) % Allele frequency (C)

Taiwanese 118 90 (76.3) 24 (20.3) 4 (3.4) 14 African Americans 107 72 (67.3) 33 (30.8) 2 (1.9) 17 European Americans 175 57 (32.6) 82 (46.9) 36 (20.6) 44

NOTE: Hardy-Weinberg equilibrium test indicates that all populations were in equilibrium.

reversed in 0.2 mol/L NaCl by heating (65jC, 4 hours). The protein was three heterozygous cell lines, possibly due to the presence of the digested in 40 Ag/mL proteinase K (45jC, 1 hour, Qiagen), 0.01 EDTA, and À783 G allele. However, this was not consistent across all cell lines. 0.40 mol/L Tris-HCl (pH 6.5). DNA was extracted with phenol-chloroform, Genotyping of GSTM3. To estimate the frequencies of the precipitated with ice-cold 100% ethanol, and resuspended in 50 AL Tris- GSTM3 AÀ63C genotypes in human populations, allele discrimina- EDTA. DNA concentrations were estimated using a NanoDrop method tion genotyping was carried out on samples from European according to the manufacturer’s directions. Quantitative Allelic Discrimination Assay. Quantitative allelic dis- American, African American, and Taiwanese individuals. Table 1 crimination of the AÀ63C SNP was done using an ABI PRISM 7700 with the shows the genotype and allele frequencies in these groups. Among same primer and probe set used for genotyping. A standard curve was created those of European descent, the C allele was common (0.44) and had a using À63AA and À63CC genomic DNA mixtures at varying percentages (0%, high frequency, relative to individuals of African (0.17) or Asian (0.14) 10%, 20%, ..., 100%). PCR constituents and thermocycling were the same as descent. Of note, the GSTM1*0 null allele is also common and occurs for AÀ63C genotyping, except that 11.25 AL immunoprecipitated DNA at similar frequencies in these groups (European, 0.49; African, 0.38; f (estimated to be 10 ng) in a 25 AL final volume and 36 cycles were used. VIC ref. 14). However, despite the relative close proximity (f50 kb) of and FAM signals were recorded post-PCR using an ABI PRISM 7700. GSTM3 to the GSTM1*0/*1 deletion polymorphism, the GSTM3 Statistical Analysis. The results for gene expression, transient transfec- À63C allele can occur on the same chromosome with either tion, and allele-specific ChIP assays are given as mean F SD of at least triplicate measurements. The significance of the results was determined GSTM1*1 or GSTM1*0 allele (all four haplotypes are present), using the Student’s t test at a = 0.05. suggesting that these polymorphisms are not strongly in linkage. GSTM3 Expression in Glioma Cell Lines. We also examined the NCI-60 tumor cell line gene expression database (http://genome- Results and Discussion www.stanford.edu/nci60/) and observed GSTM3 expression vari- Phenotype-Directed Discovery of GSTM3 Promoter SNPs. ability across the various tumor types. This finding suggested that SNPs can alter protein function (coding SNP) or affect gene the À63C SNP might also affect GSTM3 expression in cancer cell expression regulation (rSNP), and these effects may lead to lines. Because GSTM3 is highly expressed in brain tissue, we interindividual differences in drug metabolism or disease suscepti- obtained three glioma cell lines, SNB-19, SF-268, and SF-539, from bility (26–31). Although numerous projects have focused on coding the National Cancer Institute Developmental Therapeutics Program. SNPs, few biologically directed approaches for identifying and Protein levels determined by Western blotting with GSTM3 antibody evaluating function of rSNPs are known (32). In this work, we used a (Fig. 2A) were consistent with values determined by the Develop- gene expression screening approach to discover a functional rSNP in mental Therapeutics Program. Both SNB-19 and SF-268 cells showed GSTM3. Anttila et al. (17) reported that the GSTM1-null (GSTM1*0) higher GSTM3 protein levels than those of SF-539, which was genotype was associated with GSTM3 expression. undetectable. Real-time PCR was used to determine GSTM3 mRNA We screened GSTM1 genotype and GSTM3 expression in expression levels (Fig. 2B) and these results were consistent with the lymphoblast cell lines from a CEPH family (CEPH1345) containing Western blot data. SF-539, homozygous À63C/C, had 9-fold lower parents heterozygous for the GSTM1*1/*0 null genotype and expression relative to SF-268, homozygous À63A/A (P < 0.0001; children with various combinations of the GSTM1 alleles. These Fig. 2B). SNB-19, heterozygous À63A/C, expressed similar GSTM3 individuals were all homozygous for the GSTM3*A allele (the mRNA levels (1.3-fold lower) compared with SF-268 cells (Fig. 2B). GSTM3*Balleleisa3-nucleotidedeletioninintron6).No These genotype/phenotype results mirrored those observed in our relationship between GSTM1 genotype and GSTM3 expression was lymphoblast lines. Taken together, these data suggest that the observed1; however, several children displayed GSTM3 low expres- absence of GSTM3 mRNA and protein is the consequence of the sion phenotypes (Fig. 1). GSTM3 promoter sequencing in these À63C SNP in the proximal GSTM3 promoter and is not due to other children revealed two SNPs, AÀ783G and AÀ63C, relative to the start post transcriptional processes. of codon 1. The individuals homozygous for the À63CC genotype Functional Analysis of GSTM3 Promoter Haplotypes. To had 8-fold lower expression compared with those with the À63AA evaluate relative promoter activity of the ÀA783G and ÀA63C f genotype (P < 0.003). Mean GSTM3 expression in the two parents and alleles, we amplified 900 bp of the GSTM3 promoter (containing two other children, all À63AC heterozygotes, was 1.6-fold lower than the À783A/À63A haplotype) and cloned this fragment into a pGL3- the À63AA children, suggesting a recessive mode of action for this enhancer vector upstream of the luciferase reporter. After transient SNP. GM07350 displays somewhat lower expression than the other transfection into SF-268 cells, strong luciferase expression was observed for the À783A/À63A vector compared with an empty pGL3-enhancer vector (Fig. 3). Using in vitro mutagenesis, we gen- 1 M.A. Watson and D.A. Bell, unpublished data. erated luciferase reporter constructs containing the four possible www.aacrjournals.org 101 Cancer Res 2005; 65: (1). January 1, 2005

Allele-Specific ChIP Assay. ChIP can be used to precipitate DNA sequences directly bound to cellular proteins in vivo (33). Immunoprecipitation of the cross-linked transcription complex has been used to study specific molecular events occurring in transcription, and Knight et al. (25) showed that ChIP could be used in an allele-specific format. We modified this approach and quantified the relative abundance of the AÀ63C GSTM3 alleles in immunoprecipitated DNA from a AÀ63C heterozygous cell line (SNB-19) using Taqman-based allelic discrimination genotyping. We validated the quantitative genotyping component by measur- ing the VIC (À63A) and FAM (À63C) probe signals in cell line DNA with the three possible AÀ63C genotypes (À63A/A, À63C/C, and À63A/C) and in genomic DNA mixtures of varying À63A/A and À63C/C DNA percentages (0%, 10%, 20%, ..., 100%). The DNA mixture experiment showed a linear relationship (r2 = 0.93) between allele composition and VIC/FAM ratios (Fig. 4A). VIC/FAM ratios for the homozygous À63A/A and À63C/C DNA samples were 2.82 F 0.41 and 0.28 F 0.03 (Fig. 4B), respectively, and the heterozygous À63A/C cell line was 0.93 F 0.02. Furthermore, the VIC/FAM ratio observed from a heterozygous À63A/C cell line, GM01032A (0.93 F 0.02), was not significantly different from the 50%:50% allele mixture experiment (1.17 F 0.04). VIC and FAM signals were linear down to

Figure 2. Analysis of GSTM3 expression level in glioma cell lines. A, Western at least 1 ng genomic DNA per reaction (data not shown). This low blot of GSTM3 using 10 Ag cell lysates from SF-268, SF-539, and SNB-19. detection limit and low variability indicated that this method would h-Actin was used as a loading control. B, real-time PCR analysis of GSTM3 permit quantitative allelic discrimination in small quantities of mRNA expression in SF-268, SF-539, and SNB-19. Columns, mean from triplicate measurements; bars, SD. Expression measurements for each cell line immunoprecipitated DNA and would allow multiple analyses from were evaluated in three independent experiments. the same RNA pol II–precipitated sample. For allele-specific ChIP experiments, heterozygous À63A/C SNB- 19 cells were cross linked, lysed, sonicated, and then mixed with RNA À783A and À63A haplotypes and measured luciferase expression pol II antibody and protein A agarose beads to precipitate DNA from activity in SF-268 cells (Fig. 3). Relative to the À783A/À63A allele, actively transcribed promoters. SNB-19 input DNA (DNA extracted both À63C containing alleles reduced expression. The À783A/À63C from sonicated cells with no further treatment) produced a VIC/ allele reduced luciferase expression f4-fold from 9.4 F 1.36 to 2.36 FAM ratio of 1.04 F 0.02 (Fig. 4C), consistent with VIC/FAM ratio of F 0.3 (P < 0.0004) and the À783G/À63C allele was reduced 2.7-fold. the other À63A/C heterozygous cell line, GM 01032A (0.93 F 0.02). The À783G polymorphism also reduced luciferase expression but to These values represent the expected equal representation of the two a lesser extent (1.6-fold lower in the À783G/À63A haplotype relative alleles in the genomic DNA of heterozygous cells and are the to the À783A/À63A haplotype; P < 0.02). These observations reference value for comparison with the ChIP samples. indicate that the AÀ63C site is more important for promoter activity To determine the relative binding of RNA polymerase to the À63A than the AÀ783G site. We hypothesized that the AÀ63C site, which and À63C promoter alleles, we quantified the presence of each of the is 4-nucleotide upstream of the TATA box, could alter the binding of alleles in the DNA recovered from the ChIP procedure. Figure 4C RNA pol II and examined this possibility using ChIP. displays the mean VIC/FAM ratios obtained from quantitative

Figure 3. Functional analysis of the four GSTM3 promoter haplotypes. A, fragments of human GSTM3 promoter regions were constructed in pGL3-enhanced luciferase (Luc) reporter plasmid. pGL3 plasmids were transiently cotransfected with pRL-TK into SF-268 cells, and luciferase activities were analyzed after 48 hours of treatment. B, columns, mean from triplicate measurements of three independent experiments; bars, SD.

Cancer Res 2005; 65: (1). January 1, 2005 102 www.aacrjournals.org

protein A agarose beads with no antibody present showed baseline VIC and FAM signal levels (data not shown), indicating an absence of nonspecifically precipitated GSTM3 promoter DNA. These allele- specific ChIP results are consistent with both gene expression (Figs. 1 and 2) and luciferase reporter assays results (Fig. 3). It is interesting to note that the magnitude of the allele difference for the allele-specific ChIP assays and the mRNA expression assays are similar (f9-fold difference), although results for the luciferase reporter assays showed a smaller difference (f4-fold). Allele-specific ChIP is a powerful technique for assessing functional differences between promoter alleles. This method provides in vivo relative determination of transcription factor loading on an endogenous gene promoter under normal regulatory control in a chromosomal environment. This approach may reflect the ‘‘true’’ biological differences between promoter alleles in vivo and as such could prove to be more biologically relevant than the usual allele comparisons using dual luciferase reporter gene constructs. A previous study by Knight et al. (25) determined relative RNA pol II binding to tumor necrosis factor a promoter by combining ChIP with primer extension/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry–based genotyping (Sequenom, San Diego, CA). In the present study, we modified the Knight et al. approach by combining RNA pol II ChIP with allele quantification using a 5V nuclease-based allelic discrimination assay (Taqman). As there are many thousands of laboratories with instruments capable of using the 5V nuclease chemistry, this approach should be more accessible than the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method. Biological Impact of the AÀ63C Polymorphism in the GSTM3 Promoter. GSTM3 is the ancestral gene in the GSTA gene family (2), and several biological functions have been shown. Like other GSTA proteins, GSTM3 catalyzes the conjugation of numerous toxic or carcinogenic compounds to glutathione, and low levels of GSTM3 expression in the cell might lead to greater amounts of cellular DNA damage. It has also been suggested that GSTM3 expression could affect sensitivity to chemotherapeutic agents. In addition, GSTM3 was identified recently as a cytosolic glutathione-dependent prostaglandin E2 synthase in human brain (34), suggesting an endo genous role for GSTM3 protein levels in regulation of prostaglandin signaling pathways. Because prostaglandin E2 plays an important role in cell proliferation signaling, GSTM3 expression variation could have an impact on growth or cell proliferation during tumorigenesis in the brain or perhaps in other GSTM3-expressing tissues. Figure 4. Allele-specific PCR analysis of immunoprecipitated DNA using RNA In summary, we have shown that the GSTM3 ÀA63C SNP has a pol II antibody. A, fluorescence intensity measurements (VIC/FAM) ratios for mixtures of genomic DNA composed of varying percentages of allele 1 (À63A) major impact on regulation of GSTM3 expression in vivo. GSTM3 has and allele 2 (À63C; 0%, 10%, 20%, ..., 100% A). B, VIC/FAM ratios of several potentially important biological functions, and the effect AÀ63C homozygous and heterozygous lymphoblast cell lines. Columns, mean from triplicate measurements; bars, SD. C, VIC/FAM ratios of unprecipitated and prevalence of a SNP in multiple ethnic populations suggests the (left) and immunoprecipitated DNA (right ) using ChIP assay in À63A/C possibility of a significant role in carcinogenesis or tumor biology. heterozygous cells (SNB-19). Columns, mean from 10 independent ChIP To test this hypothesis, we have initiated studies examining the experiments analyzed in triplicate; bars, SD. The value of f2.5 in the ChIP sample indicates that only the A allele was bound to RNA polymerase. relationship between this GSTM3 SNP and the risk of brain, lung, and testicular cancer. genotyping (in triplicate) of the unprecipitated cell lysate reference sample (left) and the DNA recovered from 10 parallel RNA pol II Acknowledgments ChIP experiments (mean value is plotted). The mean VIC/FAM ratio Received 8/9/2004; revised 10/6/2004; accepted 10/26/2004. for immunoprecipitated DNA was 2.5 F 0.3 and this difference The costs of publication of this article were defrayed in part by the payment of page F charges. This article must therefore be hereby marked advertisement in accordance (from reference value, 1.04 0.02) was highly significant (P < with 18 U.S.C. Section 1734 solely to indicate this fact. 0.0001). Using the mixed DNA samples as a standard curve, we We thank Dr. L.L. Hsieh for providing DNA samples from Taiwanese maternity estimated that the precipitated DNA contained at least 90% of A patients, Prof. Julian Knight (University of Oxford, Oxford, United Kingdom) for kindly sharing his ChIP protocol, Dr. John Hayes for sharing his GSTM3 antibody, and the allele and 10% of C allele (compared with the 90% A:10% C DNA National Cancer Institute Developmental Therapeutics Program and Susan Holbeck mixture; VIC/FAM ratio = 2.14). SNB-19 cell extracts mixed with (National Cancer Institute) for tumor cell lines and helpful advice. www.aacrjournals.org 103 Cancer Res 2005; 65: (1). January 1, 2005

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Cancer Res 2005; 65: (1). January 1, 2005 104 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. 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

Xuemei Liu, Michelle R. Campbell, Gary S. Pittman, et al.

Cancer Res 2005;65:99-104.

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