Skip to main content

Advertisement

SpringerLink
Log in

MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression

  • Original Paper
  • Published:
Cellular Oncology Aims and scope Submit manuscript

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. R. A. Becker, R. Montesano, Repair of O 4-methyldeoxythymidine residues in DNA by mammalian liver extracts. Carcinogenesis 6, 313–317 (1985)

    Article  CAS  PubMed  Google Scholar 

  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)

  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)

  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)

  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)

  6. A. E. Pegg, Repair of O(6)-alkylguanine by alkyltransferases. Mutat. Res. 462, 83–100 (2000)

  7. S. L. Gerson, Clinical relevance of MGMT in the treatment of cancer. J. Clin. Oncol. 20, 2388–2399 (2002)

    Article  CAS  PubMed  Google Scholar 

  8. S. L. Gerson, MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev. Cancer 4, 296–307 (2004)

    Article  CAS  PubMed  Google Scholar 

  9. G. P. Margison, M. F. Santibáñez Koref, A. C. Povey, Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine. Mutagenesis 17, 483–487 (2002)

    Article  CAS  PubMed  Google Scholar 

  10. G. P. Margison, A. C. Povey, B. Kaina, M. F. Santibáñez Koref, Variability and regulation of O 6-alkylguanine-DNA alkyltransferase. Carcinogenesis 24, 625–635 (2003)

    Article  CAS  PubMed  Google Scholar 

  11. J. R. Silber, M. S. Bobola, A. Blank, M. C. Chamberlain, O(6)-methylguanine-DNA methyltransferase in glioma therapy: promise and problems. Biochim. Biophys. Acta 1826, 71–82 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. W. Wick, M. Weller, M. van den Bent, M. Sanson, M. Weiler, A. von Deimling, C. Plass, M. Hegi, M. Platten, G. Reifenberger, MGMT testing–the challenges for biomarker-based glioma treatment. Nat. Rev. Neurol. 10(7), 372–385 (2014)

    Article  CAS  PubMed  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)

  14. A. E. Pegg, Q. Fang, N. A. Loktionova, Human variants of O6-alkylguanine-DNA alkyltransferase. DNA Repair 6, 1071–1078 (2007)

  15. K. K. Bhakat, S. Mitra, CpG methylation-dependent repression of the human O6-methylguanine-DNA methyltransferase gene linked to chromatin structure alteration. Carcinogenesis 24, 1337–1345 (2003)

    Article  CAS  PubMed  Google Scholar 

  16. R. P. Danam, S. R. Howell, T. P. Brent, L. C. Harris, Epigenetic regulation of O 6-methylguanine-DNA methyltransferase gene expression by histone acetylation and methyl-CpG binding proteins. Mol. Cancer Ther. 4, 61–69 (2005)

    CAS  PubMed  Google Scholar 

  17. M. Krześniak, D. Butkiewicz, A. Samojedny, M. Chorazy, M. Rusin, Polymorphisms in TDG and MGMT genes - epidemiological and functional study in lung cancer patients from Poland. Ann. Hum. Genet. 68, 300–312 (2004)

    Article  PubMed  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)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  PubMed Central  Google 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)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  Google Scholar 

  22. S. E. Johnatty, M. Abdellatif, L. Shimmin, R. B. Clark, E. Boerwinkle, Beta 2 adrenergic receptor 5′ haplotypes influence promoter activity. Br. J. Pharmacol. 137, 1213–1216 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. H. Takane, D. Kobayashi, T. Hirota, J. Kigawa, N. Terakawa, K. Otsubo, I. Ieiri, Haplotype-oriented genetic analysis and functional assessment of promoter variants in the MDR1 (ABCB1) gene. J. Pharmacol. Exp. Ther. 311, 1179–1187 (2004)

    Article  CAS  PubMed  Google Scholar 

  24. N. J. Hawkins, J. H. Lee, J. J. Wong, C. T. Kwok, R. L. Ward, M. P. Hitchins, MGMT methylation is associated primarily with the germline C > T SNP (rs16906252) in colourectal cancer and normal colonic mucosa. Mod. Pathol. 22, 1588–1599 (2009)

    Article  CAS  PubMed  Google Scholar 

  25. I. L. Candiloro, A. Dobrovic, Detection of MGMT promoter methylation in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter single nucleotide polymorphism. Cancer Prev. Res. 2, 862–867 (2009)

    Article  CAS  Google Scholar 

  26. L. S. Kristensen, H. M. Nielsen, H. Hager, L. L. Hansen, Methylation of MGMT in malignant pleural mesothelioma occurs in a subset of patients and is associated with the T allele of the rs16906252 MGMT promoter SNP. Lung Cancer 71, 130–136 (2011)

    Article  PubMed  Google Scholar 

  27. M. H. Chae, J. S. Jang, H. G. Kang, J. H. Park, J. M. Park, W. K. Lee, S. Kam, E. B. Lee, J. W. Son, J. Y. Park, O6-alkylguanine-DNA alkyltransferase gene polymorphisms and the risk of primary lung cancer. Mol. Carcinog. 45, 239–249 (2006)

    Article  CAS  PubMed  Google Scholar 

  28. Z. Hu, H. Wang, M. Shao, G. Jin, W. Sun, Y. Wang, H. Liu, Y. Wang, H. Ma, J. Qian, et al., Genetic variants in MGMT and risk of lung cancer in southeastern Chinese: a haplotype-based analysis. Hum. Mutat. 28, 431–440 (2007)

    Article  CAS  PubMed  Google 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)

  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)

  31. I. Zawlik, S. Vaccarella, D. Kita, M. Mittelbronn, S. Franceschi, H. Ohgaki, Promoter methylation and polymorphisms of the MGMT gene in glioblastomas: a population-based study. Neuroepidemiology 32, 21–29 (2009)

    Article  PubMed  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)

  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)

  34. S. B. Gabriel, S. F. Schaffner, H. Nguyen, J. M. Moore, J. Roy, B. Blumenstiel, J. Higgins, M. DeFelice, A. Lochner, M. Faggart, et al., The structure of haplotype blocks in the human genome. Science 296, 2225–2229 (2002)

    Article  CAS  PubMed  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)

  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)

  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)

  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)

  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)

  40. I. Boldogh, C. V. Ramana, Z. Chen, T. Biswas, T. K. Hazra, S. Grösch, T. Grombacher, S. Mitra, B. Kaina, Regulation of expression of the DNA repair gene O6-methylguanine-DNA methyltransferase via protein kinase C-mediated signaling. Cancer Res. 58, 3950–3956 (1998)

    CAS  PubMed  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)

  42. I. Lavon, D. Fuchs, D. Zrihan, G. Efroni, B. Zelikovitch, Y. Fellig, T. Siegal, Novel mechanism whereby nuclear factor κB mediates DNA damage repair through regulation of O6-methylguanine-DNA-methyltransferase. Cancer Res. 67, 8952–8959 (2007)

    Article  CAS  PubMed  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)

  44. S. Ueda, T. Mineta, Y. Nakahara, H. Okamoto, T. Shiraishi, K. Tabuchi, Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas. J. Neurosurg. 101, 659–663 (2004)

    Article  CAS  PubMed  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)

  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)

  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)

  48. A. Reményi, H. R. Schöler, M. Wilmanns, Combinatorial control of gene expression. Nat. Struct. Mol. Biol. 11, 812–815 (2004)

  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)

  50. C. J. Fry, P. J. Farnham, Context-dependent transcriptional regulation. J. Biol. Chem. 274, 29583–29586 (1999)

  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)

Download references

Acknowledgments

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).

Author information

Author notes

  1. Courtney E. Cross

    Present address: A.T. Still University School of Osteopathic Medicine, 5850 E. Still Circle, Mesa, AZ, 85206, USA

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Maternal-Fetal Pharmacology and Biodevelopment Laboratories, University of Texas Medical Branch, Galveston, TX, 77555-1066, USA

    Meixiang Xu, Courtney E. Cross, Jordan T. Speidel & Sherif Z. Abdel-Rahman

Authors
  1. Meixiang Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Courtney E. Cross
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jordan T. Speidel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sherif Z. Abdel-Rahman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sherif Z. Abdel-Rahman.

Ethics declarations

Conflict of interest

None declared.

Electronic Supplementary Material

Supplementary tables 1 and 2 was also submitted for details of TFBS in-silico prediction.

ESM 1

(PDF 189 kb)

ESM 2

(PDF 300 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, M., Cross, C.E., Speidel, J.T. et al. MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression. Cell Oncol. 39, 435–447 (2016). https://doi.org/10.1007/s13402-016-0286-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13402-016-0286-4

Keyword

Search

Navigation