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5-Formylcytosine to cytosine conversion by C–C bond cleavage in vivo

Abstract

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C–C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2′-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC–MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C–C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

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Figure 1: Isotope tracing studies.
Figure 2: Conversion of isotopically labeled fdC into dC in mESCs.
Figure 3: F-fdC is converted into F-dC within the genome.
Figure 4: Demodification of 2′-fluorinated fdC is a rapid process, does not require Dnmt or Tet enzymes and occurs also in somatic cell types.

References

  1. 1

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

    CAS  PubMed  Google Scholar 

  2. 2

    Schübeler, D. Function and information content of DNA methylation. Nature 517, 321–326 (2015).

    PubMed  Google Scholar 

  3. 3

    Jeltsch, A. & Jurkowska, R.Z. Allosteric control of mammalian DNA methyltransferases – a new regulatory paradigm Nucleic Acids Res. 44, 8556–8575 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

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

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

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

    CAS  PubMed  Google Scholar 

  7. 7

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

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

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

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Münzel, M., Globisch, D. & Carell, T. 5-Hydroxymethylcytosine, the sixth base of the genome. Angew. Chem. Int. Ed. Engl. 50, 6460–6468 (2011).

    PubMed  Google Scholar 

  11. 11

    Pfaffeneder, T. et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat. Chem. Biol. 10, 574–581 (2014).

    CAS  PubMed  Google Scholar 

  12. 12

    Wagner, M. et al. Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues. Angew. Chem. Int. Edn Engl. 54, 12511–12514 (2015).

    CAS  Google Scholar 

  13. 13

    Branco, M.R., Ficz, G. & Reik, W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 13, 7–13 (2011).

    PubMed  Google Scholar 

  14. 14

    Wu, H. & Zhang, Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev. 25, 2436–2452 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Bachman, M. et al. 5-Formylcytosine can be a stable DNA modification in mammals. Nat. Chem. Biol. 11, 555–557 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Su, M. et al. 5-Formylcytosine could be a semipermanent base in specific genome sites. Angew. Chem. Int. Ed. Engl. 55, 11797–11800 (2016).

    CAS  PubMed  Google Scholar 

  17. 17

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

    CAS  PubMed  Google Scholar 

  18. 18

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

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Zhu, C. et al. Single-cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution. Cell Stem Cell 20, 720–731.e5 (2017).

    CAS  PubMed  Google Scholar 

  21. 21

    Hill, P.W., Amouroux, R. & Hajkova, P. DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story. Genomics 104, 324–333 (2014).

    CAS  PubMed  Google Scholar 

  22. 22

    Wu, X., Inoue, A., Suzuki, T. & Zhang, Y. Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells. Genes Dev. 31, 511–523 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Guo, F. et al. Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15, 447–459 (2014).

    CAS  PubMed  Google Scholar 

  25. 25

    Wu, S.C. & Zhang, Y. Active DNA demethylation: many roads lead to Rome. Nat. Rev. Mol. Cell Biol. 11, 607–620 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    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).

    CAS  PubMed  Google Scholar 

  27. 27

    Liutkevičiūtė, Z. et al. Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases. J. Am. Chem. Soc. 136, 5884–5887 (2014).

    PubMed  Google Scholar 

  28. 28

    Schiesser, S. et al. Mechanism and stem-cell activity of 5-carboxycytosine decarboxylation determined by isotope tracing. Angew. Chem. Int. Ed. Engl. 51, 6516–6520 (2012).

    CAS  PubMed  Google Scholar 

  29. 29

    Jekunen, A. & Vilpo, J.A. 5-Methyl-2′-deoxycytidine. Metabolism and effects on cell lethality studied with human leukemic cells in vitro. Mol. Pharmacol. 25, 431–435 (1984).

    CAS  PubMed  Google Scholar 

  30. 30

    Vilpo, J.A. & Vilpo, L.M. Biochemical mechanisms by which reutilization of DNA 5-methylcytosine is prevented in human cells. Mutat. Res. 256, 29–35 (1991).

    CAS  PubMed  Google Scholar 

  31. 31

    Schröder, A.S. et al. Synthesis of (R)-configured 2′-fluorinated mC, hmC, fC, and caC phosphoramidites and oligonucleotides. Org. Lett. 18, 4368–4371 (2016).

    PubMed  Google Scholar 

  32. 32

    Schröder, A.S. et al. 2′-(R)-Fluorinated mC, hmC, fC and caC triphosphates are substrates for DNA polymerases and TET-enzymes. Chem. Commun. (Camb.) 52, 14361–14364 (2016).

    Google Scholar 

  33. 33

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

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Minor, E.A., Court, B.L., Young, J.I. & Wang, G. Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine. J. Biol. Chem. 288, 13669–13674 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    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).

    CAS  PubMed  Google Scholar 

  36. 36

    Wu, X. & Zhang, Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 18, 517–534 (2017).

    CAS  PubMed  Google Scholar 

  37. 37

    Hargrove, T.Y. et al. Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum. J. Biol. Chem. 286, 26838–26848 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Lepesheva, G.I. et al. CYP51: A major drug target in the cytochrome P450 superfamily. Lipids 43, 1117–1125 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Aukema, K.G. et al. Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes. ACS Catal. 3, 2228–2238 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Jia, C. et al. Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases. Protein Cell 6, 55–67 (2015).

    CAS  PubMed  Google Scholar 

  41. 41

    Fujihashi, M., Mnpotra, J.S., Mishra, R.K., Pai, E.F. & Kotra, L.P. Orotidine monophosphate decarboxylase--a fascinating workhorse enzyme with therapeutic potential. J. Genet. Genomics 42, 221–234 (2015).

    CAS  PubMed  Google Scholar 

  42. 42

    Smiley, J.A., Angelot, J.M., Cannon, R.C., Marshall, E.M. & Asch, D.K. Radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes. Anal. Biochem. 266, 85–92 (1999).

    CAS  PubMed  Google Scholar 

  43. 43

    Xu, S. et al. Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase. Cell Res. 23, 1296–1309 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Kim, H. et al. Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nat. Commun. 4, 2403 (2013).

    PubMed  PubMed Central  Google Scholar 

  45. 45

    Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K. & Niwa, H. Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135, 909–918 (2008).

    CAS  PubMed  Google Scholar 

  46. 46

    Shirane, K. et al. Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells. Dev. Cell 39, 87–103 (2016).

    CAS  PubMed  Google Scholar 

  47. 47

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    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).

    CAS  PubMed  Google Scholar 

  49. 49

    Hu, X. et al. Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Cell Stem Cell 14, 512–522 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Dawlaty, M.M. et al. Loss of Tet enzymes compromises proper differentiation of embryonic stem cells. Dev. Cell 29, 102–111 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Liu, N. et al. Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules. PLoS One 8, e62755 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    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).

    CAS  PubMed  Google Scholar 

  53. 53

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

    CAS  PubMed  Google Scholar 

  54. 54

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Dietmair, S., Timmins, N.E., Gray, P.P., Nielsen, L.K. & Krömer, J.O. Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. Anal. Biochem. 404, 155–164 (2010).

    CAS  PubMed  Google Scholar 

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Acknowledgements

Tet TKO mESC lines were kindly provided by G.-L. Xu (Shanghai Institutes for Biological Sciences) and R. Jaenisch (Whitehead Institute, MIT, Cambridge). We are grateful to M. Okano and H. Niwa (both at Kumamoto University, Japan) for providing the Dnmt TKO mESC line and the Oct4-YFP reporter cell line, respectively. A.S.S. is supported by a fellowship from the Fonds der Chemischen Industrie. We thank the Deutsche Forschungsgemeinschaft for financial support through the programs: SFB749 (TP A4), SFB1032 (TP A5), SPP1784 and CA275-11/1. We thank the European Union Horizon 2020 program for funding the ERC Advanced project EPiR (741912). Further support is acknowledged from the Excellence Cluster CiPSM (Center for Integrated Protein Science).

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K.I. developed and performed the UHPLC–MS/MS studies. R.R. and A.S.S. synthesized the fluorinated and isotopically labeled nucleosides. A.K. designed and performed cell culture work. F.S. designed, supervised and performed cell culture work. O.K. and J.S. analyzed feeding studies of isotopically labeled dC. S.F. contributed to experiments for the analysis of soluble nucleoside pools. M.M. supervised the biochemical work, interpreted and discussed results. T.C. designed and supervised the study. All members discussed results, interpreted data and wrote the manuscript.

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Correspondence to Thomas Carell.

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Iwan, K., Rahimoff, R., Kirchner, A. et al. 5-Formylcytosine to cytosine conversion by C–C bond cleavage in vivo. Nat Chem Biol 14, 72–78 (2018). https://doi.org/10.1038/nchembio.2531

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