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The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine

Nature Protocols volume 7, pages 1897–1908 (2012)Cite this article

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Abstract

5-hydroxymethylcytosine (5hmC) is a recently discovered base in the mammalian genome, produced upon oxidation of 5-methylcytosine (5mC) in a process catalyzed by TET proteins. The biological functions of 5hmC and further oxidation products of 5mC are under intense investigation, as they are likely intermediates in DNA demethylation pathways. Here we describe a novel protocol to profile 5hmC at a genome-wide scale. This approach is based on sodium bisulfite–mediated conversion of 5hmC to cytosine-5-methylenesulfonate (CMS); CMS-containing DNA fragments are then immunoprecipitated using a CMS-specific antiserum. The anti-CMS technique is highly specific with a low background, and is much less dependent on 5hmC density than anti-5hmC immunoprecipitation (IP). Moreover, it does not enrich for CA and CT repeats, as noted for 5hmC DNA IP using antibodies to 5hmC. The anti-CMS protocol takes 3 d to complete.

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Figure 1: Effect of sodium bisulfite treatment on C, 5mC and 5hmC.
Figure 2: Testing antibody specificity and comparing the density dependence of anti-5hmC and anti-CMS methods.
Figure 3: Precipitation of genomic DNA using the anti-CMS method.
Figure 4: Optimization of the anti-CMS IP method.
Figure 5: Sequence of hmC-containing oligos for optimization of pull-down.
Figure 6

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References

  1. Li, Y. & Sasaki, H. Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Res. 21, 466–473 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jackson-Grusby, L. et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nat. Genet. 27, 31–39 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Walsh, C.P., Chaillet, J.R. & Bestor, T.H. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet 20, 116–117 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Bourc'his, D. & Bestor, T.H. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431, 96–99 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Munzel, M. et al. Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew. Chem. Int. Ed. Engl. 49, 5375–5377 (2010).

    Article  PubMed  CAS  Google Scholar 

  9. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Valinluck, V. & Sowers, L.C. Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1. Cancer Res. 67, 946–950 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Valinluck, V. et al. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 32, 4100–4108 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jin, S.G., Kadam, S. & Pfeifer, G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Ed. Engl. 50, 7008–7012 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. He, Y.F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maiti, A. & Drohat, A.C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Koh, K.P. et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bacher, U. et al. Mutations of the TET2 and CBL genes: novel molecular markers in myeloid malignancies. Ann. Hematol. 89, 643–652 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Quivoron, C. et al. TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 20, 25–38 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li, Z. et al. Deletion of Tet2 in mice leads to dysregulated hematopoietic stem cells and subsequent development of myeloid malignancies. Blood 118, 4509–4518 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ko, M. et al. Ten-Eleven-Translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc. Natl. Acad. Sci. USA 108, 14566–14571 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ko, M. et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839–843 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Haffner, M.C. et al. Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget. 2, 627–637 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Wossidlo, M. et al. 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming. Nat. Commun. 2, 241 (2011).

    Article  PubMed  CAS  Google Scholar 

  25. Gu, T.P. et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Pastor, W.A., Huang, Y., Henderson, H.R., Agarwal, S. & Rao, A. The GLIB technique for mapping 5-hydroxymethylcytosine. Nat. Protoc. 7, 1909–1917 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pastor, W.A. et al. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature 473, 394–397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hayatsu, H., Wataya, Y., Kai, K. & Iida, S. Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 9, 2858–2865 (1970).

    Article  CAS  PubMed  Google Scholar 

  29. Hayatsu, H. & Shiragami, M. Reaction of bisulfite with the 5-hydroxymethyl group in pyrimidines and in phage DNAs. Biochemistry 18, 632–637 (1979).

    Article  CAS  PubMed  Google Scholar 

  30. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Weber, M. et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853–862 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Zemach, A., McDaniel, I.E., Silva, P. & Zilberman, D. Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 328, 916–919 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Wu, H. et al. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Xu, Y. et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol. Cell 42, 451–464 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Matarese, F., Carrillo-de Santa Pau, E. & Stunnenberg, H.G. 5-Hydroxymethylcytosine: a new kid on the epigenetic block? Mol. Syst. Biol. 7, 562 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Booth, M.J. et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934–937 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Song, C.X. et al. Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat. Methods 9, 75–77 (2012).

    Article  CAS  Google Scholar 

  41. Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the Mammalian genome. Cell 149, 1368–1380 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kornberg, S.R., Zimmerman, S.B. & Kornberg, A. Glucosylation of deoxyribonucleic acid by enzymes from bacteriophage-infected Escherichia coli. J. Biol. Chem. 236, 1487–1493 (1961).

    Article  CAS  PubMed  Google Scholar 

  43. Lister, R. et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462, 315–322 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Y.H. is supported by a postdoctoral fellowship from the Leukemia and Lymphoma Society. This study was supported by US National Institutes of Health grants AI44432 and HD065812, grant RM1-01729 from the California Institute for Regenerative Medicine and Translational Research grant 6187-12 from the Leukemia and Lymphoma Society (to A.R.).

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Authors and Affiliations

  1. La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Yun Huang, William A Pastor & Anjana Rao

  2. Licenciatura en Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico

    Jorge A Zepeda-Martínez

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Contributions

A.R. conceptualized and directed the project. Y.H. and W.A.P. developed the CMS-IP method. J.A.Z.-M. contributed to optimizing the CMS-IP method.

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Correspondence to Anjana Rao.

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Huang, Y., Pastor, W., Zepeda-Martínez, J. et al. The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7, 1897–1908 (2012). https://doi.org/10.1038/nprot.2012.103

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