Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

DNA methylation reactivates GAD1 expression in cancer by preventing CTCF-mediated polycomb repressive complex 2 recruitment

Oncogene volume 35, pages 3995–4008 (2016)Cite this article

Subjects

A Corrigendum to this article was published on 27 June 2016

This article has been updated

Abstract

Levels of γ-aminobutyric acid (GABA) and glutamic acid decarboxylase 1 (GAD1), the enzyme that synthesizes GABA, are significantly increased in neoplastic tissues. However, the mechanism underlying this increase remains elusive. Instead of silencing gene transcription, we showed that the GAD1 promoter was hypermethylated in both colon and liver cancer cells, leading to the production of high levels of GAD1. GAD1 is a target gene that is silenced by H3K27me3. The key locus responsible for GAD1 reactivation was mapped to a DNA methylation-sensitive CTCF-binding site (CTCF-BS3) within the third intron of GAD1. Chromosome configuration capture (3C) analysis indicated that an intrachromosomal loop was formed by CTCF self-dimerisation in normal cells (CTCF binds to both unmethylated CTCF-BS3 and CTCF-BS2). The CTCF dimer then interacted with suppressor of zeste 12 homologue (SUZ12), which is a domain of Polycomb repressive complex 2 (PRC2), promoting the methylation of H3K27 and the silencing of GAD1 expression. This silencing was shown to be inhibited by DNA methylation in cancer cells. These findings strongly suggest that GAD1 is reactivated by DNA methylation, which provided a model for DNA methylation and the active orchestration of oncogenic gene expression by CTCF in cancer cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Change history

  • 28 July 2016

    This article has been corrected since Advance Online Publication and a corrigendum is also printed in this issue

References

  1. Joh TH, Baetge EE, Ross ME, Lai CY, Docherty M, Bradford H et al. Genes for neurotransmitter synthesis, storage, and uptake. Fed Proc 1985; 44: 2773–2779.

    CAS  PubMed  Google Scholar 

  2. Matuszek M, Jesipowicz M, Kleinrok Z . GABA content and GAD activity in gastric cancer. Med Sci Monit 2001; 7: 377–381.

    CAS  PubMed  Google Scholar 

  3. Kimura R, Kasamatsu A, Koyama T, Fukumoto C, Kouzu Y, Higo M et al. Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by beta-catenin translocation and MMP7 activation. BMC Cancer 2013; 13: 555.

    Article  Google Scholar 

  4. Maemura K, Yamauchi H, Hayasaki H, Kanbara K, Tamayama T, Hirata I et al. Gamma-amino-butyric acid immunoreactivity in intramucosal colonic tumors. J Gastroenterol Hepatol 2003; 18: 1089–1094.

    Article  CAS  Google Scholar 

  5. Azuma H, Inamoto T, Sakamoto T, Kiyama S, Ubai T, Shinohara Y et al. Gamma-aminobutyric acid as a promoting factor of cancer metastasis; induction of matrix metalloproteinase production is potentially its underlying mechanism. Cancer Res 2003; 63: 8090–8096.

    CAS  PubMed  Google Scholar 

  6. Jaraj SJ, Augsten M, Haggarth L, Wester K, Ponten F, Ostman A et al. GAD1 is a biomarker for benign and malignant prostatic tissue. Scand J Urol Nephrol 2011; 45: 39–45.

    Article  CAS  Google Scholar 

  7. Li CM, Kim CE, Margolin AA, Guo M, Zhu J, Mason JM et al. CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms' tumors. Am J Pathol 2004; 165: 1943–1953.

    Article  CAS  Google Scholar 

  8. Jorissen RN, Lipton L, Gibbs P, Chapman M, Desai J, Jones IT et al. DNA copy-number alterations underlie gene expression differences between microsatellite stable and unstable colorectal cancers. Clin Cancer Res 2008; 14: 8061–8069.

    Article  CAS  Google Scholar 

  9. Andang M, Hjerling-Leffler J, Moliner A, Lundgren TK, Castelo-Branco G, Nanou E et al. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature 2008; 451: 460–464.

    Article  Google Scholar 

  10. Huerta S, Harris DM, Jazirehi A, Bonavida B, Elashoff D, Livingston EH et al. Gene expression profile of metastatic colon cancer cells resistant to cisplatin-induced apoptosis. Int J Oncol 2003; 22: 663–670.

    CAS  PubMed  Google Scholar 

  11. Xu Y, Hu B, Choi AJ, Gopalan B, Lee BH, Kalady MF et al. Unique DNA methylome profiles in CpG island methylator phenotype colon cancers. Genome Res 2012; 22: 283–291.

    Article  CAS  Google Scholar 

  12. Razin A, Riggs AD . DNA methylation and gene function. Science 1980; 210: 604–610.

    Article  CAS  Google Scholar 

  13. Holliday R, Pugh JE . DNA modification mechanisms and gene activity during development. Science 1975; 187: 226–232.

    Article  CAS  Google Scholar 

  14. Bird A . Perceptions of epigenetics. Nature 2007; 447: 396–398.

    Article  CAS  Google Scholar 

  15. Eckhardt F, Lewin J, Cortese R, Rakyan VK, Attwood J, Burger M et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat Genet 2006; 38: 1378–1385.

    Article  CAS  Google Scholar 

  16. Gius D, Cui H, Bradbury CM, Cook J, Smart DK, Zhao S et al. Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell 2004; 6: 361–371.

    Article  CAS  Google Scholar 

  17. Lai AY, Fatemi M, Dhasarathy A, Malone C, Sobol SE, Geigerman C et al. DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas. J Exp Med 2010; 207: 1939–1950.

    Article  CAS  Google Scholar 

  18. Renaud S, Loukinov D, Abdullaev Z, Guilleret I, Bosman FT, Lobanenkov V et al. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene. Nucleic Acids Res 2007; 35: 1245–1256.

    Article  CAS  Google Scholar 

  19. Huang HS, Akbarian S . GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia. PloS One 2007; 2: e809.

    Article  Google Scholar 

  20. Ha M, Ng DW, Li WH, Chen ZJ . Coordinated histone modifications are associated with gene expression variation within and between species. Genome Res 2011; 21: 590–598.

    Article  CAS  Google Scholar 

  21. Kim BN, Yamamoto H, Ikeda K, Damdinsuren B, Sugita Y, Ngan CY et al. Methylation and expression of p16INK4 tumor suppressor gene in primary colorectal cancer tissues. Int J Oncol 2005; 26: 1217–1226.

    CAS  PubMed  Google Scholar 

  22. Zhang J, Tsoi H, Li X, Wang H, Gao J, Wang K et al. Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP-WT1-TBL1 axis. Gut 2015; e-pub ahead of print 12 June 2015; doi:10.1136/gutjnl-2014-308614.

  23. Bell AC, Felsenfeld G . Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 2000; 405: 482–485.

    Article  CAS  Google Scholar 

  24. Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilghman SM . CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 2000; 405: 486–489.

    Article  CAS  Google Scholar 

  25. Euskirchen GM, Rozowsky JS, Wei CL, Lee WH, Zhang ZD, Hartman S et al. Mapping of transcription factor binding regions in mammalian cells by ChIP: comparison of array- and sequencing-based technologies. Genome Res 2007; 17: 898–909.

    Article  CAS  Google Scholar 

  26. Rago C, Vogelstein B, Bunz F . Genetic knockouts and knockins in human somatic cells. Nat Protoc 2007; 2: 2734–2746.

    Article  CAS  Google Scholar 

  27. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G . Genome regulation by polycomb and trithorax proteins. Cell 2007; 128: 735–745.

    Article  CAS  Google Scholar 

  28. Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K . Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J 2004; 23: 4061–4071.

    Article  CAS  Google Scholar 

  29. Li T, Hu JF, Qiu X, Ling J, Chen H, Wang S et al. CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop. Mol Cell Biol 2008; 28: 6473–6482.

    Article  CAS  Google Scholar 

  30. Kobayashi T, Ebihara S, Ishii K, Kobayashi T, Nishijima M, Endo S et al. Structural and functional characterization of mouse glutamate decarboxylase 67 gene promoter. Biochim Biophys Acta 2003; 1628: 156–168.

    Article  CAS  Google Scholar 

  31. Catalano V, Loupakis F, Graziano F, Torresi U, Bisonni R, Mari D et al. Mucinous histology predicts for poor response rate and overall survival of patients with colorectal cancer and treated with first-line oxaliplatin- and/or irinotecan-based chemotherapy. Br J Cancer 2009; 100: 881–887.

    Article  CAS  Google Scholar 

  32. Cotter PD, Gahan CG, Hill C . A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol Microbiol 2001; 40: 465–475.

    Article  CAS  Google Scholar 

  33. Labrador M, Corces VG . Setting the boundaries of chromatin domains and nuclear organization. Cell 2002; 111: 151–154.

    Article  CAS  Google Scholar 

  34. West AG, Huang S, Gaszner M, Litt MD, Felsenfeld G . Recruitment of histone modifications by USF proteins at a vertebrate barrier element. Mol Cell 2004; 16: 453–463.

    Article  CAS  Google Scholar 

  35. Phillips JE, Corces VG . CTCF: master weaver of the genome. Cell 2009; 137: 1194–1211.

    Article  Google Scholar 

  36. Ren L, Wang Y, Shi M, Wang X, Yang Z, Zhao Z . CTCF mediates the cell-type specific spatial organization of the Kcnq5 locus and the local gene regulation. PloS One 2012; 7: e31416.

    Article  CAS  Google Scholar 

  37. Rodriguez C, Borgel J, Court F, Cathala G, Forne T, Piette J . CTCF is a DNA methylation-sensitive positive regulator of the INK/ARF locus. Biochem Biophys Res Commun 2010; 392: 129–134.

    Article  CAS  Google Scholar 

  38. Nozoe T, Anai H, Nasu S, Sugimachi K . Clinicopathological characteristics of mucinous carcinoma of the colon and rectum. J Surg Oncol 2000; 75: 103–107.

    Article  CAS  Google Scholar 

  39. Du W, Mah JT, Lee J, Sankila R, Sankaranarayanan R, Chia KS . Incidence and survival of mucinous adenocarcinoma of the colorectum: a population-based study from an Asian country. Dis Colon Rectum 2004; 47: 78–85.

    Article  Google Scholar 

  40. Negri FV, Wotherspoon A, Cunningham D, Norman AR, Chong G, Ross PJ . Mucinous histology predicts for reduced fluorouracil responsiveness and survival in advanced colorectal cancer. Ann Oncol 2005; 16: 1305–1310.

    Article  CAS  Google Scholar 

  41. Maisano R, Azzarello D, Maisano M, Mafodda A, Bottari M, Egitto G et al. Mucinous histology of colon cancer predicts poor outcomes with FOLFOX regimen in metastatic colon cancer. J Chemother 2012; 24: 212–216.

    Article  CAS  Google Scholar 

  42. Xiang YY, Wang S, Liu M, Hirota JA, Li J, Ju W et al. A GABAergic system in airway epithelium is essential for mucus overproduction in asthma. Nat Med 2007; 13: 862–867.

    Article  CAS  Google Scholar 

  43. Fu XW, Wood K, Spindel ER . Prenatal nicotine exposure increases GABA signaling and mucin expression in airway epithelium. Am J Respir Cell Mol Biol 2011; 44: 222–229.

    Article  CAS  Google Scholar 

  44. Leopoldo S, Lorena B, Cinzia A, Gabriella DC, Angela Luciana B, Renato C et al. Two subtypes of mucinous adenocarcinoma of the colorectum: clinicopathological and genetic features. Ann Surg Ocol 2008; 15: 1429–1439.

    Article  Google Scholar 

  45. Gatenby RA, Gawlinski ET, Gmitro AF, Kaylor B, Gillies RJ . Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res 2006; 66: 5216–5223.

    Article  CAS  Google Scholar 

  46. Hernandez A . Proton dynamics in cancer. Curr Pharm Des 2012; 18: 1317–1318.

    Article  CAS  Google Scholar 

  47. Stuhmer T, Puelles L, Ekker M, Rubenstein JL . Expression from a Dlx gene enhancer marks adult mouse cortical GABAergic neurons. Cereb Cortex 2002; 12: 75–85.

    Article  Google Scholar 

  48. Yan HL, Xue G, Mei Q, Wang YZ, Ding FX, Liu MF et al. Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis. EMBO J 2009; 28: 2719–2732.

    Article  CAS  Google Scholar 

  49. Dekker J, Rippe K, Dekker M, Kleckner N . Capturing chromosome conformation. Science 2002; 295: 1306–1311.

    Article  CAS  Google Scholar 

  50. Wang Z . Epitope tagging of endogenous proteins for genome-wide chromatin immunoprecipitation analysis. Methods Mol Biol 2009; 567: 87–98.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (grants no. 81071680, 81272280, 81172307) Shanghai Pujiang Outstanding Young Scientist Project.

Author contributions

Conception and design: Hongli Yan, Shuhan SUN, Anmei Deng. Development of methodology: Hongli Yan, Guannan Tang, Hao Wang, Tianlin He, Xiaobo Sun. Analysis and interpretation of data (for example, statistical analysis, biostatistics, computational analysis): Hongli Yan, Hao Wang, Liqiang Hao, Anmei Deng. Writing, review of the manuscript: Hongli Yan, Shuhan SUN, Anmei Deng. Conception and design: Hongli Yan, Shuhan SUN, Anmei Deng. Development of methodology: Hongli Yan, Guannan Tang, Hao Wang, Tianlin He, Xiaobo Sun. Analysis and interpretation of data (for example, statistical analysis, biostatistics, computational analysis): Hongli Yan, Hao Wang, Liqiang Hao, Anmei Deng. Writing, review of the manuscript: Hongli Yan, Shuhan SUN, Anmei Dengonception and design: Hongli Yan, Shuhan SUN, Anmei Deng.

Author information

Author notes

  1. H Yan, G Tang, H Wang and L Hao: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Laboratory Medicine, Changhai Hospital, The Second Military Medical University, Shanghai, China

    H Yan, X Sun & A Deng

  2. Institute of Genetics, Second Military Medical University, Shanghai, China

    G Tang & S Sun

  3. Department of Colorectal Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China

    H Wang & L Hao

  4. Division of Hepatopancreatobiliary Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China

    T He

  5. Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA

    A H Ting

Authors
  1. H Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L Hao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T He
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. X Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A H Ting
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. S Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to H Yan, A Deng or S Sun.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, H., Tang, G., Wang, H. et al. DNA methylation reactivates GAD1 expression in cancer by preventing CTCF-mediated polycomb repressive complex 2 recruitment. Oncogene 35, 3995–4008 (2016). https://doi.org/10.1038/onc.2015.423

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2015.423

This article is cited by

Search

Advanced search

Quick links