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.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA

Nature Chemical Biology volume 10, pages 574–581 (2014)Cite this article

Subjects

Abstract

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Metabolism of cytosine derivatives, their detection by LC-UV-ESI-MS/MS and levels in mESCs and mouse cortex.
Figure 2: hmU is present at elevated levels in mESCs compared to tissue.
Figure 3: hmU and fU are thymine oxidation products in WT mESCs with no detectable contribution from hmC or fC deamination.
Figure 4: Tet1 and Tet2 generate hmU in mESCs.
Figure 5: hmU is produced during mESC differentiation.
Figure 6: Identification of hmU:A readers.

References

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

    Article  CAS  Google Scholar 

  2. Franchini, D.M., Schmitz, K.M. & Petersen-Mahrt, S.K. 5-Methylcytosine DNA demethylation: more than losing a methyl group. Annu. Rev. Genet. 46, 419–441 (2012).

    Article  CAS  Google Scholar 

  3. 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  Google Scholar 

  4. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. 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  Google Scholar 

  7. Tan, L. & Shi, Y.G. Tet family proteins and 5-hydroxymethylcytosine in development and disease. Development 139, 1895–1902 (2012).

    Article  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. Bjelland, S. et al. Oxidation of thymine to 5-formyluracil in DNA: mechanisms of formation, structural implications, and base excision by human cell free extracts. Biochemistry 34, 14758–14764 (1995).

    Article  CAS  Google Scholar 

  10. Mouret, J.F., Polverelli, M., Sarrazini, F. & Cadet, J. Ionic and radical oxidations of DNA by hydrogen peroxide. Chem. Biol. Interact. 77, 187–201 (1991).

    Article  CAS  Google Scholar 

  11. Guo, J.U., Su, Y., Zhong, C., Ming, G.L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).

    Article  CAS  Google Scholar 

  12. Cortellino, S. et al. Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell 146, 67–79 (2011).

    Article  CAS  Google Scholar 

  13. Nabel, C.S. et al. AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation. Nat. Chem. Biol. 8, 751–758 (2012).

    Article  CAS  Google Scholar 

  14. Rangam, G., Schmitz, K.M., Cobb, A.J. & Petersen-Mahrt, S.K. AID enzymatic activity is inversely proportional to the size of cytosine C5 orbital cloud. PLoS ONE 7, e43279 (2012).

    Article  CAS  Google Scholar 

  15. Jacobs, A.L. & Schär, P. DNA glycosylases: in DNA repair and beyond. Chromosoma 121, 1–20 (2012).

    Article  CAS  Google Scholar 

  16. Zhang, Q.M. et al. DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the hNEIL1 and hNTH1 enzymes in human cells. DNA Repair (Amst.) 4, 71–79 (2005).

    Article  CAS  Google Scholar 

  17. Burrows, C.J. Surviving an oxygen atmosphere: DNA damage and repair. ACS Symp. Ser. Am. Chem. Soc. 2009, 147–156 (2009).

    PubMed  PubMed Central  Google Scholar 

  18. Taghizadeh, K. et al. Quantification of DNA damage products resulting from deamination, oxidation and reaction with products of lipid peroxidation by liquid chromatography isotope dilution tandem mass spectrometry. Nat. Protoc. 3, 1287–1298 (2008).

    Article  CAS  Google Scholar 

  19. Globisch, D. et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010).

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

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

    Article  Google Scholar 

  22. Liu, S. et al. Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA. Nucleic Acids Res. 41, 6421–6429 (2013).

    Article  CAS  Google Scholar 

  23. Ficz, G. et al. FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13, 351–359 (2013).

    Article  CAS  Google Scholar 

  24. Habibi, E. et al. Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13, 360–369 (2013).

    Article  CAS  Google Scholar 

  25. Leitch, H.G. et al. Naive pluripotency is associated with global DNA hypomethylation. Nat. Struct. Mol. Biol. 20, 311–316 (2013).

    Article  CAS  Google Scholar 

  26. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).

    Article  CAS  Google Scholar 

  27. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

    Article  CAS  Google Scholar 

  28. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Burley, G.A. et al. Directed DNA metallization. J. Am. Chem. Soc. 128, 1398–1399 (2006).

    Article  CAS  Google Scholar 

  31. Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    Article  CAS  Google Scholar 

  32. Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003); erratum 75, 4942 (2003); erratum 78, 4235 (2006).

    Article  CAS  Google Scholar 

  33. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  34. Frauer, C. et al. Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain. PLoS ONE 6, e21306 (2011).

    Article  CAS  Google Scholar 

  35. Pichler, G. et al. Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways. J. Cell. Biochem. 112, 2585–2593 (2011).

    Article  CAS  Google Scholar 

  36. Lewis, H.L., Muhleman, D.R. & Ward, J.F. Serologic assay of DNA base damage. I. 5-Hydroxymethyldeoxyuridine, a radiation product of thymidine. Radiat. Res. 75, 305–316 (1978).

    Article  CAS  Google Scholar 

  37. Boorstein, R.J. et al. Definitive identification of mammalian 5-hydroxymethyluracil DNA N-glycosylase activity as SMUG1. J. Biol. Chem. 276, 41991–41997 (2001).

    Article  CAS  Google Scholar 

  38. Kavli, B., Otterlei, M., Slupphaug, G. & Krokan, H.E. Uracil in DNA—general mutagen, but normal intermediate in acquired immunity. DNA Repair (Amst.) 6, 505–516 (2007).

    Article  CAS  Google Scholar 

  39. Santos, F. et al. Active demethylation in mouse zygotes involves cytosine deamination and base excision repair. Epigenetics Chromatin 6, 39 (2013).

    Article  Google Scholar 

  40. Silva, J. & Smith, A. Capturing pluripotency. Cell 132, 532–536 (2008).

    Article  CAS  Google Scholar 

  41. Moore, S.P.G., Toomire, K.J. & Strauss, P.R. DNA modifications repaired by base excision repair are epigenetic. DNA Repair (Amst.) 12, 1152–1158 (2013).

    Article  CAS  Google Scholar 

  42. Schiesser, S. et al. Deamination, oxidation, and C–C bond cleavage reactivity of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. J. Am. Chem. Soc. 135, 14593–14599 (2013).

    Article  CAS  Google Scholar 

  43. Gierlich, J. et al. Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA. Org. Lett. 8, 3639–3642 (2006).

    Article  CAS  Google Scholar 

  44. Cao, H. & Wang, Y. Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives. J. Am. Soc. Mass Spectrom. 17, 1335–1341 (2006).

    Article  CAS  Google Scholar 

  45. Wang, J. et al. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method. Anal. Chem. 83, 2201–2209 (2011).

    Article  CAS  Google Scholar 

  46. Chen, T., Ueda, Y., Dodge, J.E., Wang, Z. & Li, E. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605 (2003).

    Article  CAS  Google Scholar 

  47. Cortazar, D. et al. Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature 470, 419–423 (2011).

    Article  CAS  Google Scholar 

  48. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428 (1993).

    Article  CAS  Google Scholar 

  49. Li, E., Bestor, T.H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992).

    Article  CAS  Google Scholar 

  50. Montanez, E. et al. Kindlin-2 controls bidirectional signaling of integrins. Genes Dev. 22, 1325–1330 (2008).

    Article  CAS  Google Scholar 

  51. Ying, Q.L. & Smith, A.G. Defined conditions for neural commitment and differentiation. Methods Enzymol. 365, 327–341 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  53. Kittler, R., Heninger, A.K., Franke, K., Habermann, B. & Buchholz, F. Production of endoribonuclease-prepared short interfering RNAs for gene silencing in mammalian cells. Nat. Methods 2, 779–784 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Excellence Cluster Center for Integrated Protein Science Munich (CiPSM) and the collaborative research centers SFB749, SFB646 and SFB1032 as well as German Research Foundation (DFG) grant CA275/8-4, the Volkswagen foundation, NGFNplus (01GS0870) and the Netherlands Organization for Scientific Research (NWO-VIDI) for financial support. T.P. and S.S. thank the Fonds der Chemischen Industrie for predoctoral fellowships. C.B. thanks the Boehringer Ingelheim Fonds for a predoctoral fellowship. G.K. thanks the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship for research abroad. We thank M. Moser (Max Planck Institute for Biochemistry) for providing R1- and C57Bl6/129–derived mESCs, G. Höfner and K.T. Wanner for their initial help with MS as well as M. Wirsing, L. Belzner and P. Laube for providing bioinformatic tools for data processing.

Author information

Author notes

  1. Michiel Vermeulen

    Present address: Present address: Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.,

  2. Toni Pfaffeneder, Fabio Spada and Mirko Wagner: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany

    Toni Pfaffeneder, Fabio Spada, Mirko Wagner, Caterina Brandmayr, Silvia K Laube, David Eisen, Jessica Steinbacher, Benjamin Hackner, Olga Kotljarova, Olesea Kosmatchev, Stefan Schiesser, Barbara Steigenberger, Nada Raddaoui, Gengo Kashiwazaki, Markus Müller & Thomas Carell

  2. Charité Universitätsklinikum, Otto-Heubner-Centrum für Kinder und Jugendmedizin, Klinik für Allgemeine Pädiatrie, Labor für Pädiatrische Molekularbiologie, Berlin, Germany

    Matthias Truss

  3. Department of Biomedicine, University of Basel, Basel, Switzerland

    David Schuermann & Primo Schär

  4. Center for Integrated Protein Science at the Department of Pharmacy—Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany

    Stylianos Michalakis

  5. Center for Integrated Protein Science at the Department of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Udo Müller & Heinrich Leonhardt

  6. Department of Molecular Cancer Research, Cancer Genomics Netherlands, Utrecht, The Netherlands

    Cornelia G Spruijt & Michiel Vermeulen

Authors
  1. Toni Pfaffeneder
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fabio Spada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mirko Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Caterina Brandmayr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Silvia K Laube
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David Eisen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matthias Truss
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jessica Steinbacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Benjamin Hackner
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Olga Kotljarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. David Schuermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Stylianos Michalakis
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Olesea Kosmatchev
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Stefan Schiesser
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Barbara Steigenberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Nada Raddaoui
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Gengo Kashiwazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Udo Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Cornelia G Spruijt
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Michiel Vermeulen
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Heinrich Leonhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Primo Schär
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Markus Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Thomas Carell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.P. synthesized MS standards, performed the sample preparation and ultra high-performance LC/MS/MS method development, did LC/MS analysis, interpreted data and performed statistical analysis. F.S. and N.R. performed mESC differentiation and isotope tracing experiments. M.W. and C.B. performed the HEK-293T experiments, did LC/MS analysis and interpreted data. C.B. performed qPCR and analyzed the data. S.K.L. and D.E. performed the protein pulldown studies and interpreted data. M.T. performed mESC knockdown and knockout experiments. J.S. and O. Kosmatchev did sample preparation and LC/MS analysis. B.H., S.S. and J.S. prepared MS standards. O. Kotljarova performed in vitro assays. B.S. synthesized oligonucleotides for protein capture. G.K. synthesized tandem mass tags, and S.M. provided mouse tissue samples. U.M. and H.L. constructed Tet expression plasmids. C.G.S. and M.V. performed Uhrf1/2 overexpression in HEK-293T cells. P.S. and D.S. provided plasmids and cell lines. M.M. and T.C. conceived and supervised the project, interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Markus Müller or Thomas Carell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–12, Supplementary Tables 1–22 and Supplementary Notes 1–5. (PDF 2618 kb)

Supplementary Data Set 1

5hmU reader proteins identified from 4 independent pulldown experiments. (XLSX 126 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pfaffeneder, T., Spada, F., Wagner, M. et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol 10, 574–581 (2014). https://doi.org/10.1038/nchembio.1532

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1532

This article is cited by

Search

Advanced 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