MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression
- 338 Downloads
The O6-methylguanine-DNA methyltransferase (MGMT) protein removes O6-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes are located within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentially affect transcription factor binding and, subsequently, alter MGMT promoter activity.
In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively.
Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p ≤ 0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system.
The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.
KeywordMGMT P/E haplotypes Transcription factor binding siRNA regulation Promoter activity
This work was supported by grants from the National Institutes of Health (R03 NS065392-01 to S.A.R.; T-32-ES007254 to C.C. and J.S.) and the John Sealy Memorial Endowment fund for Biomedical Research (to S.A.R.). Additional partial support was provided by the NIEHS Center in Environmental Toxicology at the University of Texas Medical Branch funded through P30 ES006676, The Molecular Genomics Core (GMC) at the University of Texas Medical Branch, the Institute for Translational Sciences at the University of Texas Medical Branch, supported in part by a Clinical and Translational Science Award (8UL1TR000071) from the National Center for Research Resources, now the National Center for Advancing Translational Sciences, as well as the National Institutes of Health, R01 DA 030998-01 (to GDH/TN) and 2 U54 HD047891 (to GDH).
Compliance with ethical standards
Conflict of interest
- 2.M. Christmann, B. Verbeek, W. P. Roos, B. Kaina, O(6)-methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: enzyme activity, promoter methylation and immunohistochemistry. Biochim. Biophys. Acta 1816, 179–190 (2011)Google Scholar
- 3.D. S. Daniels, T. T. Woo, K. X. Luu, D. M. Noll, N. D. Clarke, A. E. Pegg, J. A. Tainer, DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nat. Struct. Mol. Biol. 11, 714–720 (2004)Google Scholar
- 4.A. E. Pegg, Mammalian O 6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 50, 6119–6129 (1990)Google Scholar
- 5.P. E. Jackson, C. N. Hall, A. F. Badawi, P. J. O’Connor, D. P. Cooper, A. C. Povey, Frequency of Ki-ras mutations and DNA alkylation in colourectal tissue from individuals living in Manchester. Mol. Carcinog. 16, 12–19 (1996)Google Scholar
- 6.A. E. Pegg, Repair of O(6)-alkylguanine by alkyltransferases. Mutat. Res. 462, 83–100 (2000)Google Scholar
- 13.B. Kaina, M. Christmann, S. Naumann, W. P. Roos, MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair 6, 1079–1099 (2007)Google Scholar
- 14.A. E. Pegg, Q. Fang, N. A. Loktionova, Human variants of O6-alkylguanine-DNA alkyltransferase. DNA Repair 6, 1071–1078 (2007)Google Scholar
- 18.S. Ogino, A. Hazra, G. J. Tranah, G. J. Kirkner, T. Kawasaki, K. Nosho, M. Ohnishi, Y. Suemoto, J. A. Meyerhardt, D. J. Hunter, et al., MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colourectal cancer. Carcinogenesis 28, 1985–1990 (2007)CrossRefPubMedGoogle Scholar
- 19.S. Leng, A. M. Bernauer, C. Hong, K. C. Do, C. M. Yingling, K. G. Flores, M. Tessema, C. S. Tellez, R. P. Willink, E. A. Burki, et al., The a/G allele of rs16906252 predicts for MGMT methylation and is selectively silenced in premalignant lesions from smokers and in lung adenocarcinomas. Clin. Cancer Res. 17, 2014–2023 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
- 20.K. L. McDonald, R. W. Rapkins, J. Olivier, L. Zhao, K. Nozue, D. Lu, S. Tiwari, J. Kuroiwa-Trzmielina, J. Brewer, H. R. Wheeler, et al., The T genotype of the MGMT C > T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is associated with promoter methylation and longer survival in glioblastoma patients. Eur. J. Cancer 49, 360–368 (2013)CrossRefPubMedGoogle Scholar
- 21.M. Xu, I. Nekhayeva, C. E. Cross, C. M. Rondelli, J. K. Wickliffe, S. Z. Abdel-Rahman, Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents. Carcinogenesis 35(3), 564–571 (2014)CrossRefPubMedGoogle Scholar
- 29.L. Wang, H. Liu, Z. Zhang, M. R. Spitz, Q. Wei, Association of genetic variants of O 6-methylguanine-DNA methyltransferase with risk of lung cancer in non-Hispanic whites. Cancer Epidemiol. Biomark. Prev. 15, 2364–2369 (2006)Google Scholar
- 30.J. H. Park, N. S. Kim, J. Y. Park, Y. S. Chae, J. G. Kim, S. K. Sohn, J. H. Moon, B. W. Kang, H. M. Ryoo, S. H. Bae, et al., MGMT -535G > T polymorphism is associated with prognosis for patients with metastatic colourectal cancer treated with oxaliplatin-based chemotherapy. J. Cancer Res. Clin. Oncol. 136, 1135–1142 (2010)Google Scholar
- 32.J. Felsberg, M. Rapp, S. Loeser, R. Fimmers, W. Stummer, M. Goeppert, H. J. Steiger, B. Friedensdorf, G. Reifenberger, M. C. Sabel, Prognostic significance of molecular markers and extent of resection in primary glioblastoma patients. Clin. Cancer Res. 15, 6683–6693 (2009)Google Scholar
- 33.Z. Zhang, L. Wang, S. Wei, Z. Liu, L. E. Wang, E. M. Sturgis, Q. Wei, Polymorphisms of the DNA repair gene MGMT and risk and progression of head and neck cancer. DNA Repair 9, 558–566 (2010)Google Scholar
- 35.L. C. Harris, P. M. Potter, K. Tano, S. Shiota, S. Mitra, T. P. Brent, Characterization of the promoter region of the human O6-methylguanine-DNA methyltransferase gene. Nucleic Acids Res. 19, 6163–6167 (1991)Google Scholar
- 36.M. Christmann, B. Kaina, Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation. Nucleic Acids Res. 41, 8403–8420 (2013)Google Scholar
- 37.X. Messeguer, R. Escudero, D. Farré, O. Nuñez, J. Martínez, M. M. Albà, PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics 18, 333–334 (2002)Google Scholar
- 38.D. Farré, R. Roset, M. Huerta, J. E. Adsuara, L. Roselló, M. M. Albà, X. Messeguer, Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res. 31, 3651–3653 (2003)Google Scholar
- 39.A. Yousuf, M. Y. Bhat Arshad, A. Pandith, D. Afroze, N. P. Khan, K. Alam, P. Shah, M. A. Shah, S. Mudassar, MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area. Cell. Oncol. 37, 245–252 (2014)Google Scholar
- 41.J. F. Costello, B. W. Futscher, R. A. Kroes, R. O. Pieper, Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines. Mol. Cell. Biol. 14, 6515–6521 (1994)Google Scholar
- 43.L. Persano, F. Pistollato, E. Rampazzo, A. Della Puppa, S. Abbadi, C. Frasson, F. Volpin, S. Indraccolo, R. Scienza, G. Basso, BMP2 sensitizes glioblastoma stem-like cells to temozolomide by affecting HIF-1α stability and MGMT expression. Cell Death Dis. 3, e412 (2012)Google Scholar
- 45.K. K. Bhakat, S. Mitra, Regulation of the human O6-methylguanine-DNA methyltransferase gene by transcriptional coactivators cAMP response element-binding protein-binding protein and p300. J. Biol. Chem. 275, 34197–34204 (2000)Google Scholar
- 46.K. S. Srivenugopal, J. Shou, S. R. Mullapudi, F. F. Lang Jr., J. S. Rao, F. Ali-Osman, Enforced expression of wild-type p53 curtails the transcription of the O(6)-methylguanine-DNA methyltransferase gene in human tumor cells and enhances their sensitivity to alkylating agents. Clin. Cancer Res. 7, 1398–1409 (2001)Google Scholar
- 47.A. Natsume, D. Ishii, T. Wakabayashi, T. Tsuno, H. Hatano, M. Mizuno, J. Yoshida, IFN-beta down-regulates the expression of DNA repair gene MGMT and sensitizes resistant glioma cells to temozolomide. Cancer Res. 65, 7573–7579 (2005)Google Scholar
- 48.A. Reményi, H. R. Schöler, M. Wilmanns, Combinatorial control of gene expression. Nat. Struct. Mol. Biol. 11, 812–815 (2004)Google Scholar
- 49.R. Pique-Regi, J. F. Degner, A. A. Pai, D. J. Gaffney, Y. Gila, J. K. Pritchard, Accurate inference of transcription factor binding from DNA sequence and chromatin accessibility data. Genome Res. 21(3), 447–455 (2011)Google Scholar
- 50.C. J. Fry, P. J. Farnham, Context-dependent transcriptional regulation. J. Biol. Chem. 274, 29583–29586 (1999)Google Scholar
- 51.K. Vazquez-Santillan, J. Melendez-Zajgla, L. Jimenez-Hernandez, G. Martínez-Ruiz, V. Maldonado, NF-κB signaling in cancer stem cells: a promising therapeutic target? Cell. Oncol. 38, 327–339 (2015)Google Scholar