Active turnover of genomic methylcytosine in pluripotent cells

Abstract

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2′-deoxycytidine (mdC), 5-hydroxymethyl-2′-deoxycytidine (hmdC) and 5-formyl-2′-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3–6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

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Fig. 1: Drastic reduction of global genomic mdC oxidation and increase of mdC to mdU conversion upon transition to primed pluripotency.
Fig. 2: Turnover of genomic mdC and its derivatives under a2i/LIF and priming conditions.
Fig. 3: The active component of mdC turnover in primed pluripotent cells largely depends on Tet proteins.
Fig. 4: All detectable mdC deamination takes place in the soluble pool.
Fig. 5: Oxidation-independent mdC-to-dT turnover is developmentally regulated.

Data availability

The raw sequencing data, matrix table and targeted bisulfite amplicon sequencing data for Extended Data Fig. 2b are available at the Gene Expression Omnibus (GEO) under accession code GSE152174. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files.

References

  1. 1.

    Greenberg, M. V. C. & Bourc’his, D. The diverse roles of DNA methylation in mammalian development and disease. Nat. Rev. Mol. Cell Biol. 20; 590–607 (2019).

  2. 2.

    He, Y. & Ecker, J. R. Non-CG methylation in the human genome. Annu. Rev. Genomics Hum. Genet. 16, 55–77 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner Tet1. Science 324, 930–935 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    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 

  5. 5.

    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 

  6. 6.

    Schuermann, D., Weber, A. R. & Schär, P. Active DNA demethylation by DNA repair: facts and uncertainties. DNA Repair 44, 92–102 (2016).

    CAS  PubMed  Google Scholar 

  7. 7.

    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 

  8. 8.

    Slyvka, A., Mierzejewska, K. & Bochtler, M. Nei-like 1 (NEIL1) excises 5-carboxylcytosine directly and stimulates TDG-mediated 5-formyl and 5-carboxylcytosine excision. Sci. Rep. 7, 9001 (2017).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Schomacher, L. et al. NEIL DNA glycosylases promote substrate turnover by Tdg during DNA demethylation. Nat. Struct. Mol. Biol. 23, 116–124 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Chen, C.-C., Wang, K.-Y. & Shen, C.-K. J. The mammalian de novo DNA methyltransferases Dnmt3a and Dnmt3b Are Also DNA 5-hydroxymethyl cytosine dehydroxymethylases. J. Biol. Chem. 287, 33116–33121 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Liutkevičiūtė, Z., Lukinavičius, G., Masevičius, V., Daujotytė, D. & Klimašauskas, S. Cytosine-5-methyltransferases add aldehydes to DNA. Nat. Chem. Biol. 5, 400–402 (2009).

    PubMed  Google Scholar 

  12. 12.

    Iwan, K. et al. 5-formylcytosine to cytosine conversion by C–C bond cleavage in vivo. Nat. Chem. Biol. 14, 72–78 (2017).

    PubMed  Google Scholar 

  13. 13.

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

    PubMed  Google Scholar 

  14. 14.

    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 

  15. 15.

    Rai, K. et al. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase and Gadd45. Cell 135, 1201–1212 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

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

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

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

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).

    CAS  PubMed  Google Scholar 

  19. 19.

    Grin, I. & Ishchenko, A. A. An interplay of the base excision repair and mismatch repair pathways in active DNA demethylation. Nucleic Acids Res. 44, 3713–3727 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Franchini, D.-M. et al. Processive DNA demethylation via DNA deaminase-induced lesion resolution. PLoS ONE 9, e97754 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Atlasi, Y. & Stunnenberg, H. G. The interplay of epigenetic marks during stem cell differentiation and development. Nat. Rev. Genet. 18, 643–658 (2017).

    CAS  PubMed  Google Scholar 

  22. 22.

    von Meyenn, F. et al. Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells. Mol. Cell 62, 848–861 (2016).

    Google Scholar 

  23. 23.

    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 

  24. 24.

    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 

  25. 25.

    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 

  26. 26.

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

    CAS  PubMed  Google Scholar 

  27. 27.

    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 

  28. 28.

    van Mierlo, G., Wester, R. A. & Marks, H. A mass spectrometry survey of chromatin-associated proteins in pluripotency and early lineage commitment. Proteomics 19, e1900047 (2019).

    PubMed  Google Scholar 

  29. 29.

    Yagi, M. et al. Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 548, 224–227 (2017).

    CAS  PubMed  Google Scholar 

  30. 30.

    Choi, J. et al. Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells. Nature 548, 219–223 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Kalkan, T. et al. Tracking the embryonic stem cell transition from ground state pluripotency. Development 144, 1221–1234 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Wang, L. et al. Programming and inheritance of parental DNA methylomes in mammals. Cell 157, 979–991 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    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 

  35. 35.

    Jekunen, A., Puukka, M. & Vilpo, J. Exclusion of exogenous 5-methyl-2'-deoxycytidine from DNA in human leukemic cells: A study with [2-14C]- and [methyl-14C]5-methyl-2’-deoxycytidine. Biochem. Pharmacol. 32, 1165–1168 (1983).

  36. 36.

    Maley, G. F., Lobo, A. P. & Maley, F. Properties of an affinity-column-purified human deoxycytidylate deaminase. Biochim. Biophys. Acta 1162, 161–170 (1993).

    CAS  PubMed  Google Scholar 

  37. 37.

    de Saint Vincent, B. R., Déchamps, M. & Buttin, G. The modulation of the thymidine triphosphate pool of Chinese hamster cells by dCMP deaminase and UDP reductase. Thymidine auxotrophy induced by CTP in dCMP deaminase-deficient lines. J. Biol. Chem. 255, 162–167 (1980).

    Google Scholar 

  38. 38.

    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 

  39. 39.

    Bilyard, M. K., Becker, S. & Balasubramanian, S. Natural, modified DNA bases. Curr. Opin. Chem. Biol. 57, 1–7 (2020).

    CAS  PubMed  Google Scholar 

  40. 40.

    Shen, J.-C., Rideout, W. M. & Jones, P. A. The rate of hydrolytic deamination of 5-methylcytosine in double-stranded DNA. Nucleic Acids Res. 22, 972–976 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Ladstätter, S. & Tachibana-Konwalski, K. A surveillance mechanism ensures repair of DNA lesions during zygotic reprogramming. Cell 167, 1774–1787 (2016).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Jiang, D., Wei, S., Chen, F., Zhang, Y. & Li, J. Tet3‐mediated DNA oxidation promotes ATR‐dependent DNA damage response. EMBO Rep. 18, 781–796 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Kweon, S.-M. et al. Erasure of Tet-oxidized 5-methylcytosine by a SRAP nuclease. Cell Rep. 21, 482–494 (2017).

    CAS  PubMed  Google Scholar 

  44. 44.

    Robertson, A. B., Robertson, J., Fusser, M. & Klungland, A. Endonuclease G preferentially cleaves 5-hydroxymethylcytosine-modified DNA creating a substrate for recombination. Nucleic Acids Res. 42, 13280–13293 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    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 

  46. 46.

    Shipony, Z. et al. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells. Nature 513, 115–119 (2014).

    CAS  PubMed  Google Scholar 

  47. 47.

    Ooi, S. K. et al. Dynamic instability of genomic methylation patterns in pluripotent stem cells. Epigenetics Chromatin 3, 17 (2010).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Bachman, M. et al. 5-hydroxymethylcytosine is a predominantly stable DNA modification. Nat. Chem. 6, 1049–1055 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Rulands, S. et al. Genome-scale oscillations in DNA methylation during exit from pluripotency. Cell Syst. 7, 63–76 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Zhou, X., Chadarevian, J. P., Ruiz, B. & Ying, Q.-L. Cytoplasmic and nuclear TAZ exert distinct functions in regulating primed pluripotency. Stem Cell Rep. 9, 732–741 (2017).

    CAS  Google Scholar 

  52. 52.

    Moser, M., Nieswandt, B., Ussar, S., Pozgajova, M. & Fässler, R. Kindlin-3 is essential for integrin activation and platelet aggregation. Nat. Med. 14, 325–330 (2008).

    CAS  PubMed  Google Scholar 

  53. 53.

    Nichols, J. & Ying, Q.-L. Derivation and propagation of embryonic stem cells in serum- and feeder-free culture. in Embryonic Stem Cell Protocols (ed. Turksen, K.) Vol. 329, 91–98 (Humana Press, 2006).

  54. 54.

    Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Rahimoff, R. et al. 5-formyl- and 5-carboxydeoxycytidines do not cause accumulation of harmful repair intermediates in stem cells. J. Am. Chem. Soc. 139, 10359–10364 (2017).

    CAS  PubMed  Google Scholar 

  56. 56.

    Weiner, K. X., Weiner, R. S., Maley, F. & Maley, G. F. Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli. J. Biol. Chem. 268, 12983–12989 (1993).

    CAS  PubMed  Google Scholar 

  57. 57.

    Traube, F. R. et al. Isotope-dilution mass spectrometry for exact quantification of non-canonical DNA nucleosides. Nat. Protoc. 14, 283–312 (2019).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are very thankful to the following colleagues: M. Möser (Max Planck Institute for Biochemistry) for K3 mPSCs (kindlin3+/+); H. Niwa and M. Okano (both at Kumamoto University) for Oct4-YFP-Puro E14tg2a and Dnmt TKO J1 mPSCs, respectively; Y. Zhang (Boston Children’s Hospital) for parental and Tet TKO E14tg2a cells; and S. Bultmann and C. Mulholland (both at Ludwig Maximilian University) for guidance on high-depth bisulfite amplicon sequencing. Anti-DCTD antibody and purified recombinant DCTD protein were generous gifts from F. Maley (New York State University). Funding was provided by the Deutsche Forschungsgemeinschaft via the programs SFB1309 (TP: A4), SFB1361 (TP: 2), GRK 2338 and SPP1784. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. EPiR 741912). Y.Z. is supported by the China Scholarship Council (CSC no. 201806200069).

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F.S. and T.C. conceived the study and designed the experiments. F.S., S.S., A.K., Y.Z., G.A. and O.K. performed experiments and analyzed data. F.S., T.C. and S.S. interpreted data. R.R., C.E. and E.K. synthesized isotopically labeled nucleosides used as standards for LC–MS2. F.S., T.C. and S.S. wrote the manuscript.

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Correspondence to Fabio Spada or Thomas Carell.

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Extended data

Extended Data Fig. 1 Expression of pluripotency and early lineage specification factors in mPSCs under different culture conditions.

a, YFP fluorescence and bright field images of an Oct4 YFP reporter mPSC line after adaptation to naïve conditions in chemically defined medium (CDM/2i/LIF; left), followed by six days of priming under serum/CHIR/IWR-1 conditions (serum/CR; middle) or long term priming as mEpiSCs in chemically defined medium containing CHIR/IWR-1/FGF 2/activin A (CDM/CRFA; right). Scale bars = 100 µm. b, Transcript levels of pluripotency and early lineage specification factors assayed by reverse transcription-qPCR from cultures of the Oct4 YFP mPSC line shown in a under CDM/2i/LIF (naïve), serum/a2i/LIF as well as both serum/CR and CDM/CRFA primed conditions. Note that under serum/a2i/LIF conditions relatively high and low levels of naïve and primed markers are expressed, respectively, while the levels of Oct4 and Nanog transcripts are similar to those in naïve conditions. Also, transcript levels of naïve and primed pluripotency factors are similar under short term serum/CR priming conditions and long term primed EpiSCs in CDM/CRFA. Mean (bars) and individual values of fold change Log (diamonds) from three technical replicates are shown. ND = not detectable. Gapdh was used as housekeeping gene reference to calculate ΔCt values.

Extended Data Fig. 2 Global and local transitions of genomic dC modification levels upon priming of mPSCs.

a, Global levels of genomic mdC (left panel) and hmdC (right panel) assayed by UHPLC-MS2 in wt J1 mPSCs transitioning from naïve state (serum/2i/LIF; day 0) to primed state over five days in serum/CHIR/IWR-1 (serum/CR) and in the Oct4-YFP reporter mEpiSC line derived and permanently cultured in serum free CDM containing CHIR/IWR-1/FGF 2/activin A (CRFA). Mean (bars) and individual values (black squares) from three technical replicates are shown. b, High depth targeted amplicon bisulfite sequencing analysis of selected secondary imprints in mPSCs upon transition from a2i/LIF (a2iL) to serum/CR (FCR) conditions. Primary mouse embryonic fibroblasts (mEFs) are shown as reference for differentiated primary somatic cells. CpG methylation levels at individual positions were averaged over each amplicon. Size, numbers of CpG sites and genomic coordinates are reported in Supplementary Table 2. Boxes display the inter-quartile range (IQR), where lower and upper edges represent the first and third quartile, respectively, and the thicker horizontal line is the median. Whiskers mark 1.5xIQR and dots represent outliers beyond 1.5xIQR. Magenta and green box plots represent distinct amplicons within the H19 promoter region and Nespas. Results from individual culture samples are shown. Numbers of aligned reads used for quantifications (range depends on sample and CpG position): Dlk1: 5.5k-23k; H19: green 13k-35K, purple 22k-39k; Nespas: green 26k-67k; purple 32k-80k; Magel-Mrkn3: 14k-36.7k; Cdkn1c: 10k-26.7k.

Extended Data Fig. 3 Drastic reduction of global genomic mdC oxidation and increase of mdC-to-mdU conversion upon transition to primed pluripotency (related to Fig. 1).

Time course analysis of genomic dC derivatives upon metabolic labelling with m+4Met under naïve (a; a2i/LIF) and priming (b; serum/CR) conditions. Independent biological replicate of the experiment shown in Fig. 1c,d. The labelling/time course schedules are shown at the top. Global levels of unlabeled mC (pale yellow) and m+4dC (dark yellow), hm+3dC (blue) and f+2dC (green) are shown as mean (bars) and individual values (black squares) of three technical replicates. LOD = limt of detection.

Extended Data Fig. 4 Turnover of genomic mdC and its derivatives under priming conditions (related to Fig. 2).

a, b, Independent biological replicate of the experiment reported in Fig. 2c,e. a, Global profiles of the indicated labelled cytosine derivatives and dT+12 in the genome of mPSCs under priming conditions (serum/CR) upon chasing of m+4Met. Each panel shows fold change values relative to the highest level for each modification from an individual experiment. Dashes represent single technical replicate measurements (n = 3) from single samples. Full lines lines represent first order decay curves fitted to mean values (in the case of f+2dC t = 0 h was excluded from curve fitting). b, Absolute levels of genomic m+4Met from the same samples as in a. Black squares represent single technical replicate measurements (n = 3) from single samples and bars show their mean values.

Extended Data Fig. 5 Genomic m+4dU is generated exclusively from genomic m+4dC.

Global levels of m+4dC (a; yellow) and m+4dU (b; red) in the genome of wt and Dnmt TKO J1 mPSCs after priming for five days in the presence m+4Met. In Dnmt TKO cells neither m+4dC nor m+4dU were above background levels (LOD = limit of detection). The two panels in b show results from two independent replicate cultures (n = 2). Mean (bars) and individual values (black squares) of three technical replicates are shown.

Extended Data Fig. 6 Double labelling of primed wt and CD DKO J1 with m+4Met and dC[D9] (related to Fig. 4).

a, Conversion of dC[D9] into m+4dC[D8] and potentially m + 4dU[D8] within gDNA. SAH = S adenosylhomocysteine. b, UHPLC chromatogram (UV trace) showing the retention delay of dT[D8] relative to natural dT. c, Quantification of m+4dU[D8] accumulated in wt and CD DKO PSCs upon priming in the presence of m+4Met and dC[D8]. Mean (bars) and individual values (black squares) of three technical replicates are shown.

Extended Data Fig. 7 Tet proteins do not trigger DNA repair events that contribute to turnover of mdC into dT (related to Fig. 3).

Global levels of mdC, m+4dC and m+4dU (red) in the genome of parental (Tet1-3 proficient) and Tet TKO E14tg2a mPSCs after priming for five days in the presence m+4Met. a) and b) show independent biological replicates. Mean (bars) and individual values (black squares) of three technical replicates are shown.

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Spada, F., Schiffers, S., Kirchner, A. et al. Active turnover of genomic methylcytosine in pluripotent cells. Nat Chem Biol 16, 1411–1419 (2020). https://doi.org/10.1038/s41589-020-0621-y

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