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A Combined Bisulfite Restriction Analysis Bioinformatics Tool: Methyl-Typing

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Epigenetics Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 791))

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Abstract

In this chapter, we introduce our developed freeware tool Methyl-Typing. It provides methylation-related bioinformatics with a special focus on combined bisulfite restriction analysis (COBRA). We give an overview of the implementation and program modules for Methyl-Typing. Various databases and methylation-related functions are integrated into Methyl-Typing and a helpful example is illustrated in detail. Several input protocols and their outputs for COBRA-related information are demonstrated, such as the inputs of multiple gene names in official gene symbols, multiple accession numbers for nucleotide sequence retrieval, multiple template sequences in a free format, primer sequences, and file uploads. The program goal of Methyl-Typing is to provide computation and visualization of the essential information for COBRA assay so that methylation can easily be analyzed by COBRA. It is a fast and efficient tool for providing all possible methylation sites of restriction enzymes.

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References

  1. Phillips, T. (2008) The role of methylation in gene expression. Nature Education 1.

    Google Scholar 

  2. Vucic, E. A., Brown, C. J., and Lam, W. L. (2008) Epigenetics of cancer progression. Pharmacogenomics 9, 215–34.

    Article  PubMed  CAS  Google Scholar 

  3. Herman, J. G., and Baylin, S. B. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349, 2042–54.

    Article  PubMed  CAS  Google Scholar 

  4. Chu, D. C., Chuang, C. K., Fu, J. B., Huang, H. S., Tseng, C. P., and Sun, C. F. (2002) The use of real-time quantitative polymerase chain reaction to detect hypermethylation of the CpG islands in the promoter region flanking the GSTP1 gene to diagnose prostate carcinoma. J Urol 167, 1854–8.

    Article  PubMed  CAS  Google Scholar 

  5. Weber, M., Davies, J. J., Wittig, D., Oakeley, E. J., Haase, M., Lam, W. L., and Schubeler, D. (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37, 853–62.

    Article  PubMed  CAS  Google Scholar 

  6. Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89, 1827–31.

    Article  PubMed  CAS  Google Scholar 

  7. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93, 9821–6.

    Article  PubMed  CAS  Google Scholar 

  8. Eads, C. A., Danenberg, K. D., Kawakami, K., Saltz, L. B., Blake, C., Shibata, D., Danenberg, P. V., and Laird, P. W. (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28, E32.

    Article  PubMed  CAS  Google Scholar 

  9. Tost, J., and Gut, I. G. (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2, 2265–75.

    Article  PubMed  CAS  Google Scholar 

  10. Xiong, Z., and Laird, P. W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25, 2532–4.

    Article  PubMed  CAS  Google Scholar 

  11. Rohde, C., Zhang, Y., Jurkowski, T. P., Stamerjohanns, H., Reinhardt, R., and Jeltsch, A. (2008) Bisulfite sequencing Data Presentation and Compilation (BDPC) web server--a useful tool for DNA methylation analysis. Nucleic Acids Res 36, e34.

    Article  PubMed  Google Scholar 

  12. Leakey, T. I., Zielinski, J., Siegfried, R. N., Siegel, E. R., Fan, C. Y., and Cooney, C. A. (2008) A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms. Nucleic Acids Res 36, e64.

    Article  PubMed  Google Scholar 

  13. Rahner, N., Friedrichs, N., Steinke, V., Aretz, S., Friedl, W., Buettner, R., Mangold, E., Propping, P., and Walldorf, C. (2008) Coexisting somatic promoter hypermethylation and pathogenic MLH1 germline mutation in Lynch syndrome. J Pathol 214, 10–6.

    Article  PubMed  CAS  Google Scholar 

  14. Nagasaka, T., Goel, A., Notohara, K., Takahata, T., Sasamoto, H., Uchida, T., Nishida, N., Tanaka, N., Boland, C. R., and Matsubara, N. (2008) Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis. Int J Cancer 122, 2429–36.

    Article  PubMed  CAS  Google Scholar 

  15. Zhang, F., Pomerantz, J. H., Sen, G., Palermo, A. T., and Blau, H. M. (2007) Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons. Proc Natl Acad Sci USA 104, 4395–400.

    Article  PubMed  CAS  Google Scholar 

  16. Brena, R. M., Auer, H., Kornacker, K., Hackanson, B., Raval, A., Byrd, J. C., and Plass, C. (2006) Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform. Nucleic Acids Res 34, e17.

    Article  PubMed  Google Scholar 

  17. Estecio, M. R., Youssef, E. M., Rahal, P., Fukuyama, E. E., Gois-Filho, J. F., Maniglia, J. V., Goloni-Bertollo, E. M., Issa, J. P., and Tajara, E. H. (2006) LHX6 is a sensitive methylation marker in head and neck carcinomas. Oncogene 25, 5018–26.

    Article  PubMed  CAS  Google Scholar 

  18. Yang, C. H., Chuang, L. Y., Cheng, Y. H., Gu, D. L., Chen, C. H., and Chang, H. W. (2010) Methyl-Typing: An improved and visualized COBRA software for epigenomic studies. FEBS Lett 584, 739–44.

    Article  PubMed  CAS  Google Scholar 

  19. Bao, L., Zhou, M., and Cui, Y. (2008) CTCFBSDB: a CTCF-binding site database for characterization of vertebrate genomic insulators. Nucleic Acids Res 36, D83–7.

    Article  PubMed  CAS  Google Scholar 

  20. Gaszner, M., and Felsenfeld, G. (2006) Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7, 703–13.

    Article  PubMed  CAS  Google Scholar 

  21. Wakaguri, H., Yamashita, R., Suzuki, Y., Sugano, S., and Nakai, K. (2008) DBTSS: database of transcription start sites, progress report 2008. Nucleic Acids Res 36, D97–101.

    Article  PubMed  CAS  Google Scholar 

  22. Takai, D., and Jones, P. A. (2003) The CpG island searcher: a new WWW resource. In Silico Biol 3, 235–40.

    PubMed  CAS  Google Scholar 

  23. Karolchik, D., Kuhn, R. M., Baertsch, R., Barber, G. P., Clawson, H., Diekhans, M., Giardine, B., Harte, R. A., Hinrichs, A. S., Hsu, F., Kober, K. M., Miller, W., Pedersen, J. S., Pohl, A., Raney, B. J., Rhead, B., Rosenbloom, K. R., Smith, K. E., Stanke, M., Thakkapallayil, A., Trumbower, H., Wang, T., Zweig, A. S., Haussler, D., and Kent, W. J. (2008) The UCSC Genome Browser Database: 2008 update. Nucleic Acids Res 36, D773–9.

    Article  PubMed  CAS  Google Scholar 

  24. Roberts, R. J., Vincze, T., Posfai, J., and Macelis, D. (2007) REBASE--enzymes and genes for DNA restriction and modification. Nucleic Acids Res 35, D269–70.

    Article  PubMed  CAS  Google Scholar 

  25. Chang, H. W., Cheng, Y. H., Chuang, L. Y., and Yang, C. H. (2010) SNP-RFLPing 2: an updated and integrated PCR-RFLP tool for SNP genotyping. BMC Bioinformatics 11, 173.

    Article  PubMed  Google Scholar 

  26. Zweig, A. S., Karolchik, D., Kuhn, R. M., Haussler, D., and Kent, W. J. (2008) UCSC genome browser tutorial. Genomics 92, 75–84.

    Article  PubMed  CAS  Google Scholar 

  27. Chang, H. W., Yang, C. H., Chang, P. L., Cheng, Y. H., and Chuang, L. Y. (2006) SNP-RFLPing: restriction enzyme mining for SNPs in genomes. BMC Genomics 7, 30.

    Article  PubMed  Google Scholar 

  28. Grippo, P., Iaccarino, M., Parisi, E., and Scarano, E. (1968) Methylation of DNA in developing sea urchin embryos. J Mol Biol 36, 195–208.

    Article  PubMed  CAS  Google Scholar 

  29. Doskocil, J., and Sorm, F. (1962) Distribution of 5-methylcytosine in pyrimidine sequences of deoxyribonucleic acids. Biochim Biophys Acta 55, 953–9.

    Article  PubMed  CAS  Google Scholar 

  30. Finnegan, E. J., and Kovac, K. A. (2000) Plant DNA methyltransferases. Plant Mol Biol 43, 189–201.

    Article  PubMed  CAS  Google Scholar 

  31. Bender, J. (2004) DNA methylation and epigenetics. Annu Rev Plant Biol 55, 41–68.

    Article  PubMed  CAS  Google Scholar 

  32. Chan, S. W., Henderson, I. R., and Jacobsen, S. E. (2005) Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6, 351–60.

    Article  PubMed  CAS  Google Scholar 

  33. Hetzl, J., Foerster, A. M., Raidl, G., and Mittelsten Scheid, O. (2007) CyMATE: a new tool for methylation analysis of plant genomic DNA after bisulphite sequencing. Plant J 51, 526–36.

    Article  PubMed  CAS  Google Scholar 

  34. Pontecorvo, G., De Felice, B., and Carfagna, M. (2000) Novel methylation at GpC dinucleotide in the fish Sparus aurata genome. Mol Biol Rep 27, 225–30.

    Article  PubMed  CAS  Google Scholar 

  35. Kouidou, S., Malousi, A., and Maglaveras, N. (2006) Methylation and repeats in silent and nonsense mutations of p53. Mutat Res 599, 167–77.

    Article  PubMed  CAS  Google Scholar 

  36. Rodenhiser, D., Chakraborty, P., Andrews, J., Ainsworth, P., Mancini, D., Lopes, E., and Singh, S. (1996) Heterogenous point mutations in the BRCA1 breast cancer susceptibility gene occur in high frequency at the site of homonucleotide tracts, short repeats and methylatable CpG/CpNpG motifs. Oncogene 12, 2623–9.

    PubMed  CAS  Google Scholar 

  37. Vatolin, S., Abdullaev, Z., Pack, S. D., Flanagan, P. T., Custer, M., Loukinov, D. I., Pugacheva, E., Hong, J. A., Morse, H., 3rd, Schrump, D. S., Risinger, J. I., Barrett, J. C., and Lobanenkov, V. V. (2005) Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes. Cancer Res 65, 7751–62.

    PubMed  CAS  Google Scholar 

  38. Hong, J. A., Kang, Y., Abdullaev, Z., Flanagan, P. T., Pack, S. D., Fischette, M. R., Adnani, M. T., Loukinov, D. I., Vatolin, S., Risinger, J. I., Custer, M., Chen, G. A., Zhao, M., Nguyen, D. M., Barrett, J. C., Lobanenkov, V. V., and Schrump, D. S. (2005) Reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter coincides with derepression of this cancer-testis gene in lung cancer cells. Cancer Res 65, 7763–74.

    PubMed  CAS  Google Scholar 

  39. Klenova, E. M., Morse, H. C., 3rd, Ohlsson, R., and Lobanenkov, V. V. (2002) The novel BORIS + CTCF gene family is uniquely involved in the epigenetics of normal biology and cancer. Semin Cancer Biol 12, 399–414.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was partly supported by the National Science Council in Taiwan under grants NSC98-2622-E-151-024-CC3, NSC98-2622-E-151-001-CC2, NSC98-2221-E-151-040, NSC97-2311-B-037-003-MY3, and by the grants KMU-EM-99-1.4 and DOH 100-TD-C-111-002 Dr. Hsueh-Wei Chang from the Department of Health, Taiwan.

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Authors and Affiliations

  1. Department of Electronic Engineering, National Kaohsiung University of Applied Sciences, Kaohsiung, Taiwan

    Cheng-Hong Yang & Yu-Huei Cheng

  2. Department of Chemical Engineering, I-Shou University, Kaohsiung, Taiwan

    Li-Yeh Chuang

  3. Department of Biomedical Science and Environmental Biology, Center of Excellence for Environmental Medicine, Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan

    Hsueh-Wei Chang

Authors
  1. Cheng-Hong Yang
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  2. Yu-Huei Cheng
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  3. Li-Yeh Chuang
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  4. Hsueh-Wei Chang
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Corresponding author

Correspondence to Hsueh-Wei Chang .

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Editors and Affiliations

  1. Dept. Biology, University of Alabama, Birmingham, University Blvd. 1300, Birmingham, 35294-1170, Alabama, USA

    Trygve O. Tollefsbol

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Yang, CH., Cheng, YH., Chuang, LY., Chang, HW. (2011). A Combined Bisulfite Restriction Analysis Bioinformatics Tool: Methyl-Typing. In: Tollefsbol, T. (eds) Epigenetics Protocols. Methods in Molecular Biology, vol 791. Humana Press. https://doi.org/10.1007/978-1-61779-316-5_6

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  • DOI: https://doi.org/10.1007/978-1-61779-316-5_6

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-315-8

  • Online ISBN: 978-1-61779-316-5

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