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.

Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq

Abstract

A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6–8 d.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Schematic diagram of BS-seq, MAB-seq and caMAB-seq.
Figure 2
Figure 3: Schematic diagram of base-resolution mapping methods for oxidized methylcytosines.
Figure 4: Bioanalyzer electropherogram of sequencing libraries of MAB-seq/caMAB-seq.
Figure 5: Base-resolution 5fC/5caC maps and affinity-enrichment-based 5fC/5caC maps at the TchpGit2 locus in mouse ESCs.

References

  1. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Smith, Z.D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Wu, H. & Zhang, Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 156, 45–68 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Pastor, W.A., Aravind, L. & Rao, A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat. Rev. Mol. Cell Biol. 14, 341–356 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kohli, R.M. & Zhang, Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502, 472–479 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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 

  7. 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 

  8. 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 

  9. 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 

  10. 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 

  11. 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 

  12. 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 

  13. Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 

  15. 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 

  16. 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 

  17. 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 

  18. Wu, H. & Zhang, Y. Tet1 and 5-hydroxymethylation: a genome-wide view in mouse embryonic stem cells. Cell Cycle 10 2428–2436 ( 2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Dawlaty, M.M. et al. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kang, J. et al. Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis. Proc. Natl. Acad. Sci. USA 112, E4236–4245 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

  22. Dawlaty, M.M. et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166–175 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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 

  24. Yamaguchi, S. et al. Tet1 controls meiosis by regulating meiotic gene expression. Nature 492, 443–447 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yamaguchi, S., Shen, L., Liu, Y., Sendler, D. & Zhang, Y. Role of Tet1 in erasure of genomic imprinting. Nature 504, 460–464 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Zhang, R.R. et al. Tet1 regulates adult hippocampal neurogenesis and cognition. Cell Stem Cell 13, 237–245 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Rudenko, A. et al. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kaas, G.A. et al. TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79, 1086–1093 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Cimmino, L., Abdel-Wahab, O., Levine, R.L. & Aifantis, I. TET family proteins and their role in stem cell differentiation and transformation. Cell Stem Cell 9, 193–204 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ko, M. et al. TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol. Rev. 263, 6–21 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Song, C.X. & He, C. Potential functional roles of DNA demethylation intermediates. Trends Biochem. Sci. 38, 480–484 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wu, H. & Zhang, Y. Charting oxidized methylcytosines at base resolution. Nat. Struct. Mol. Biol. 22, 656–661 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Spruijt, C.G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Iurlaro, M. et al. A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 14, R119 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Mellen, M., Ayata, P., Dewell, S., Kriaucionis, S. & Heintz, N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 151, 1417–1430 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kellinger, M.W. et al. 5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 19, 831–833 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang, L. et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex. Nature 523, 621–625 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Raiber, E.A. et al. 5-Formylcytosine alters the structure of the DNA double helix. Nat. Struct. Mol. Biol. 22, 44–49 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. Szulik, M.W. et al. Differential stabilities and sequence-dependent base pair opening dynamics of Watson-Crick base pairs with 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxylcytosine. Biochemistry 54, 1294–1305 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Wu, H., Wu, X., Shen, L. & Zhang, Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat. Biotechnol. 32, 1231–1240 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Song, C.-X. et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153, 678–691 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Shen, L. et al. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153, 692–706 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Neri, F. et al. Single-base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals promoter DNA methylation dynamics. Cell Rep. 10, 674–683 (2015).

    Article  CAS  PubMed  Google Scholar 

  44. Guo, F. et al. Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15, 447–458 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Lister, R. et al. Global epigenomic reconfiguration during Mammalian brain development. Science 341, 1237905 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. 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 

  47. Hu, L. et al. Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation. Cell 155, 1545–1555 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Hashimoto, H. et al. Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA. Nature 506, 391–395 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Sun, Z. et al. A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol. Cell 57, 750–761 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Xia, B. et al. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat. Methods 12, 1047–1050 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Booth, M.J., Marsico, G., Bachman, M., Beraldi, D. & Balasubramanian, S. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat. Chem. 6, 435–440 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lu, X. et al. Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics. Cell Res. 25, 386–389 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454, 766–770 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Lu, F., Liu, Y., Jiang, L., Yamaguchi, S. & Zhang, Y. Role of Tet proteins in enhancer activity and telomere elongation. Genes Dev. 28, 2103–2119 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Tsumura, A. et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Krueger, F. & Andrews, S.R. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Thorvaldsdottir, H., Robinson, J.T. & Mesirov, J.P. Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Quinlan, A.R. & Hall, I.M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Liu, Y., Siegmund, K.D., Laird, P.W. & Berman, B.P. Bis-SNP: combined DNA methylation and SNP calling for Bisulfite-seq data. Genome Biol. 13, R61 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Sotiriou, S. et al. Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival. Nat. Med. 8, 514–517 (2002).

    Article  CAS  PubMed  Google Scholar 

  63. Blaschke, K. et al. Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Yin, R. et al. Ascorbic Acid Enhances Tet-Mediated 5-Methylcytosine Oxidation and Promotes DNA Demethylation in Mammals. J. Am. Chem. Soc. 135, 10396–10403 (2013).

    Article  CAS  PubMed  Google Scholar 

  65. Keane, T.M. et al. Mouse genomic variation and its effect on phenotypes and gene regulation. Nature 477, 289–294 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang, K. et al. Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses. Epigenetics 10, 775–783 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  67. 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 

Download references

Acknowledgements

H.W. was supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research and is currently supported by the National Human Genome Research Institute (R00HG007982). X.W. was supported by the China Scholarship Council. Y.Z. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

Authors

Contributions

H.W. and X.W. performed experiments and carried out data analysis. H.W., X.W. and Y.Z. wrote the manuscript.

Corresponding author

Correspondence to Yi Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wu, H., Wu, X. & Zhang, Y. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc 11, 1081–1100 (2016). https://doi.org/10.1038/nprot.2016.069

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.069

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing