Skip to main content
SpringerLink
Log in

Isolation of CpG Islands Using a Methyl-CpG Binding Column

  • Protocol
DNA Methylation Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 200))

  • 1014 Accesses

Abstract

Vertebrate genomes are globally heavily methylated at the sequence CpG, with the exception of short patches of GC-rich DNA of between 1–2 kb in size that are free of methylation, and these are known as CpG islands (see refs. 1 and 2 for reviews). In addition to distinctive DNA characteristics, CpG islands also have an open chromatin structure in that they are hyperacetylated, lack histone H1, and have a nucleosome-free region (3). The major reason for interest in CpG islands is that they co-localize with the 5′ end of genes. Both promoter sequences and the 5′ parts of transcription units are found within CpG islands. It has been estimated that 45,000 (56%) of human genes and 37,000 (47%) of mouse genes are associated with a CpG island (4) and these include all genes that are ubiquitously expressed as well as many genes with a tissue-restricted pattern of expression (5,6).

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Antequera, F. and Bird, A. (1993) CpG islands, in DNA Methylation: Molecular Biology and Biological Significance (Jost, J. P. and Saluz, H. P., eds.), Birkhauser Verlag, Basel, Switzerland, pp. 169–185.

    Google Scholar 

  2. Cross, S. H. and Bird, A. P. (1995) CpG islands and genes. Curr. Opin. Genet. Dev. 5, 309–314.

    Article  PubMed  CAS  Google Scholar 

  3. Tazi, J. and Bird, A. (1990) Alternative chromatin structure at CpG islands. Cell 60, 909–920.

    Article  PubMed  CAS  Google Scholar 

  4. Antequera, F. and Bird, A. (1993) Number of CpG islands and genes in human and mouse. Proc. Natl. Acad. Sci. USA 90, 11,995–11,999.

    Article  PubMed  CAS  Google Scholar 

  5. Gardiner-Garden, M. and Mer, M. (1987) CpG islands in vertebrate genomes.J Mol. Biol. 196, 261–282.

    Article  PubMed  CAS  Google Scholar 

  6. Larsen, F., Gunderson, G., Lopez, R., and Prydz, H. (1992) CpG islands as gene markers in the human genome. Genomics 13, 1095–1107.

    Article  PubMed  CAS  Google Scholar 

  7. Riggs, A. D. and Pfeifer, G. P. (1992) X-chromosome inactivation and cell memory. Trends Genet. 8,169–174.

    PubMed  CAS  Google Scholar 

  8. Antequera, F., Boyes, J., and Bird, A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell-lines. Cell 62, 503–514.

    Article  PubMed  CAS  Google Scholar 

  9. Tilghman, S. M. (1999) The sins of the fathers and mothers: genomic imprinting in mamMalian development. Cell 96,185–193.

    Article  PubMed  CAS  Google Scholar 

  10. Greger, V., Passarge, E., Höpping, W., Messmer, E., and Horsthemke, B. (1989) Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum. Genet. 83, 155–158.

    Article  PubMed  CAS  Google Scholar 

  11. Issa, J-P. J., Ottaviano, Y. L., Celano, P., Hamilton, S. R., Davidson, N. E., and Baylin, S. B. (1994) Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nature Genet. 7, 536–540.

    Article  PubMed  CAS  Google Scholar 

  12. Schmutte, C. and Jones, P. A. (1998) Involvement of DNA methylation in human carcinogenesis. Biol. Chem. 379, 377–388.

    Article  PubMed  CAS  Google Scholar 

  13. Macleod, D., Charlton, J., Mullins, J., and Bird, A. (1994) Sp1 sites in the mouse Aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282–2292.

    Article  PubMed  CAS  Google Scholar 

  14. Brandeis, M., Frank, D., Keshet, I., Siegfreid, Z., Mendelsohn, A., Nemes, A., et al. (1994) Sp1 elements protect a CpG island from de novo methylation. Nature 371,435–438.

    Article  PubMed  CAS  Google Scholar 

  15. Tykocinski, M. L. and Max, E. E. (1984) CG dinucleotide clusters in MHC genes and in 5′ demethylated genes. Nucleic Acids Res. 12, 4385–4396.

    Article  PubMed  CAS  Google Scholar 

  16. Stöger, R., Kubicka, P, Liu, C. G., Kafri, T., Razin, A., Cedar, H., and Barlow, D. P. (1993) Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73, 61–71.

    Article  PubMed  Google Scholar 

  17. Macleod, D., Ali, R. R., and Bird, A. (1998) An alternative promoter in the mouse major histocompatibility complex class II I-Aβ gene: implications for the origin of CpG islands. Mol. Cell Biol. 18, 4433–4443.

    PubMed  CAS  Google Scholar 

  18. Wutz, A., Smrzka, O. W., Schweifer, N., Schellander, K., Wagner, E. F., and Barlow, D. P. (1998) Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature 389, 745–749.

    Article  Google Scholar 

  19. Delgado, S., Gómez, M., Bird, A., and Antequera, F. (1998) Initiation of DNA replication at CpG islands in mamMalian chromosomes. EMBO J. 17, 2426–2435.

    Article  PubMed  CAS  Google Scholar 

  20. Bickmore, W. A. and Bird A. P. (1992) Use of restriction endonucleases to detect and isolate genes from mamMalian cells. Methods Enzymol. 216, 224–245

    Article  PubMed  CAS  Google Scholar 

  21. Cross, S. H., Charlton, J. A., Nan, X., and Bird, A. P. (1994) Purification of CpG islands using a methylated DNA binding column. Nature Genet. 6, 236–244.

    Article  PubMed  CAS  Google Scholar 

  22. Lewis, J. D., Meehan, R. R., Henzel, W. J., Maurer-Fogy, I., Jeppesen, P., Klein, H., and Bird, A. (1992) Purification, sequence and cellular localisation of a novel chromosomal protein that binds to methylated DNA. Cell 69, 905–914.

    Article  PubMed  CAS  Google Scholar 

  23. Nan, X., Meehan, R. R., and Bird, A. (1993) Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Res. 21, 4886–4892.

    Article  PubMed  CAS  Google Scholar 

  24. Nan, X. Campoy, J., and Bird, A. (1997) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88,471–481.

    Article  PubMed  CAS  Google Scholar 

  25. Nan, X., Ng, H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N., and Bird, A. (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386–389.

    Article  PubMed  CAS  Google Scholar 

  26. Jones, P. L., Veenstra, G. J. C., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., et al. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genet. 19,187–191.

    Article  PubMed  CAS  Google Scholar 

  27. Cross, S. H., Lee, M., Clark, V. H., Craig, J. M., Bird, A. P., and Bickmore, W. A. (1997) The chromosomal distribution of CpG islands in the mouse: evidence for genome scrambling in the rodent lineage. Genomics 40,454–461.

    Article  PubMed  CAS  Google Scholar 

  28. McQueen, H. A., Fantes, J., Cross, S. H., Clark, V. H., Archibald, A. L., and Bird, A. P. (1996) CpG islands of chicken are concentrated on microchromosomes. Nature Genet. 12, 321–324.

    Article  PubMed  CAS  Google Scholar 

  29. McQueen, H. A., Clark, V. H., Bird, A. P., Yerle, M., and Archibald, A. L. (1997) CpG islands of the pig. Genome Res. 7, 924–931.

    PubMed  CAS  Google Scholar 

  30. McQueen, H. A., Siriaco, G., and Bird, A. P. (1998) Chicken microchromo-somes are hyperacetylated, early replicating, and gene rich. Genome Res. 8, 621–630.

    PubMed  CAS  Google Scholar 

  31. Craig, J. M. and Bickmore, W. A. (1994) The distribution of CpG islands in mamMalian chromosomes. Nature Genet. 7, 376–382.

    Article  PubMed  CAS  Google Scholar 

  32. Watanabe, T., Inoue, S., Hiroi, H., Orimo, A., Kawashima, H., and Muramatsu, M. (1998) Isolation of estrogen-responsive genes with a CpG island library. Mol. CellBiol. 18, 442–449.

    CAS  Google Scholar 

  33. Cross, S. H., Clark, V. H., and Bird, A. P. (1999) Isolation of CpG islands from large genomic clones. Nucleic Acids Res. 27, 2099–2107.

    Article  PubMed  CAS  Google Scholar 

  34. Cross, S. H., Clark, V. H., SimMen, M. W., Bickmore, W. A., Maroon, H., Langford, C.., et al. (1999) Preparation and characterisation of CpG islands libraries from human chromosomes 18 and 22: landmarks for novel genes. Mam M. Gen. 11, 373–383.

    Article  Google Scholar 

  35. Brock, G. J. R. and Bird, A. (1997) Mosaic methylation of the repeat unit of the human ribosomal RNA genes. Hum. Mol. Genet. 6,451–456.

    Article  PubMed  CAS  Google Scholar 

  36. Huang, T. H., Perry, M. R., and Laux, D. E. (1999) Methylation profiling of CpG islands in human breast cancer cells. Hum. Mol. Genet. 8, 459–470.

    Article  PubMed  CAS  Google Scholar 

  37. Shiraishi, M., Chuu, Y., and Sekiya, T. (1999) Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using 130 a methylated DNA binding column and denaturing gradient gel electrophoresis. Proc. Natl. Acad. Sci. USA 96, 2913–2918.

    Article  PubMed  CAS  Google Scholar 

  38. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  39. Studier, E W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89.

    Article  PubMed  CAS  Google Scholar 

  40. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein dye binding. Anal. Biochem. 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  41. John, R. M. and Cross, S. H. (1997) Gene detection by the identification of CpG islands, in Genome Analysis: A Laboratory Manual, vol. 2 Detecting Genes(Birren, B., Green, E. D., Klapholz, S., Myers, R. M., and Roskams, J., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 217–285.

    Google Scholar 

  42. Sanford, J., Chapman, V. M., and Rossant, J. (1985) DNA methylation in extraembryonic lineages of mamMals. Trends Genet. 1, 89–93.

    Article  CAS  Google Scholar 

  43. Valdes J. M., Tagle, D. A., and Collins E S. (1994) Island rescue PCR: a rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids. Proc. Nat. Acad. Sci. USA 91, 5377–5381.

    Article  PubMed  CAS  Google Scholar 

  44. Parimoo, S., Patanjali, S. R., Shukla, H., Chaplin, D. D., and Weissman, S. M. (1991) cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments. Proc. Natl. Acad. Sci. USA 88, 9623–9627.

    Article  PubMed  CAS  Google Scholar 

  45. Lovett, M., Kere, J., and Hinton, L. M. (1991) Direct selection: a method for the isolation of cDNAs encoded by large genomic regions. Proc. Natl. Acad. Sci. USA 88, 9628–9632.

    Article  PubMed  CAS  Google Scholar 

  46. Bird, A. P. and Taggart, M. H. (1980) Variable patterns of total DNA and rDNA methylation in animals. Nucleic Acids Res. 8,1485–1497.

    Article  PubMed  CAS  Google Scholar 

  47. Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D., Haber, D. A., Sharp P. A., and Housman, D. E. (1991) Exon amplification: A strategy to isolate mamMalian genes based on RNA splicing. Proc. Natl. Acad. Sci. USA 88, 4005–4009.

    Article  PubMed  CAS  Google Scholar 

  48. Ewing, B., and Green, P. (2000) Analysis of expressed sequence tags indicates 35,000 human genes. Nat. Genet. 25, 232–234.

    Article  PubMed  CAS  Google Scholar 

  49. International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK

    Sally H. Cross

Authors
  1. Sally H. Cross
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Haematology, University of Wales College of Medicine, Cardiff, UK

    Ken I. Mills PhD

  2. Department of Haematology, Western General Hospital, Edinburgh, UK

    Bernard H. Ramsahoye MD, PhD

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Cross, S.H. (2002). Isolation of CpG Islands Using a Methyl-CpG Binding Column. In: Mills, K.I., Ramsahoye, B.H. (eds) DNA Methylation Protocols. Methods in Molecular Biology™, vol 200. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-182-5:111

Download citation

  • DOI: https://doi.org/10.1385/1-59259-182-5:111

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-618-5

  • Online ISBN: 978-1-59259-182-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation