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


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.


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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).

Author information




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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Supplementary Results, Supplementary Tables 1–4, Supplementary Figures 1–13 (PDF 1111 kb)

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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).

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