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The origin of genomic N6-methyl-deoxyadenosine in mammalian cells

Abstract

The proposal that N6-methyl-deoxyadenosine (m6dA) acts as an epigenetic mark in mammals remains controversial. Using isotopic labeling coupled to ultrasensitive mass spectrometry, we confirm the presence of low-level m6dA in mammalian DNA. However, the bulk of genomic m6dA originates from ribo-N6-methyladenosine, which is processed via the nucleotide-salvage pathway and misincorporated by DNA polymerases. Our results argue against m6dA acting as a heritable, epigenetic DNA mark in mammalian cells.

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Fig. 1: Slow DNA methylation kinetics of m6dA compared to m5dC.
Fig. 2: m6A ribonucleotide is converted into m6dA and misincorporated into gDNA.
Fig. 3: Not a DNA methyltransferase but ribo-m6A misincorporation generates m6dA.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information file. Source Data for Figs. 1 and 2 are available online.

References

  1. Wion, D. & Casadesús, J. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat. Rev. Microbiol. 4, 183–192 (2006).

    Article  CAS  Google Scholar 

  2. Koziol, M. J. et al. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat. Struct. Mol. Biol. 23, 24–30 (2016).

    Article  CAS  Google Scholar 

  3. Wu, T. P. et al. DNA methylation on N 6-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

    Article  CAS  Google Scholar 

  4. Xie, Q. et al. N 6-methyladenine DNA modification in glioblastoma. Cell 175, 1228–1243.e20 (2018).

    Article  CAS  Google Scholar 

  5. Schiffers, S. et al. Quantitative LC–MS provides no evidence for m6dA or m4dC in the genome of mouse embryonic stem cells and tissues. Angew. Chem. Int. Ed. 6, 11268–11271 (2017).

    Article  Google Scholar 

  6. Ratel, D. et al. Undetectable levels of N6-methyl adenine in mouse DNA: cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase. FEBS Lett. 580, 3179–3184 (2006).

    Article  CAS  Google Scholar 

  7. Liu, B., Liu, X., Lai, W. & Wang, H. Metabolically generated stable isotope-labeled deoxynucleoside code for tracing DNA N6-methyladenine in human cells. Anal. Chem. 89, 6202–6209 (2017).

    Article  CAS  Google Scholar 

  8. O’Brown, Z. K. et al. Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genomics 20, 445 (2019).

    Article  Google Scholar 

  9. Huang, W. et al. Determination of DNA adenine methylation in genomes of mammals and plants by liquid chromatography/mass spectrometry. RSC Adv. 5, 64046–64054 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Patil, D. P., Pickering, B. F. & Jaffrey, S. R. Reading m6A in the transcriptome: m6A-binding proteins. Trends Cell Biol. 28, 113–127 (2018).

    Article  CAS  Google Scholar 

  12. Wang, L. Mitochondrial purine and pyrimidine metabolism and beyond. Nucl. Nucl. Nucl. 35, 578–594 (2016).

    Article  CAS  Google Scholar 

  13. Löffler, M., Fairbanks, L. D., Zameitat, E., Marinaki, A. M. & Simmonds, H. A. Pyrimidine pathways in health and disease. Trends Mol. Med. 11, 430–437 (2005).

    Article  Google Scholar 

  14. Nyhan, W. L. Nucleotide synthesis via salvage pathway. In Encyclopedia of Life Sciences (John Wiley & Sons, Ltd, 2001).

  15. Charles, M. P. et al. N 6-Methyldeoxyadenosine, a nucleoside commonly found in prokaryotes, induces C2C12 myogenic differentiation. Biochem. Bioph. Res. Co. 314, 476–482 (2004).

    Article  CAS  Google Scholar 

  16. Engel, J. D. & Hippel, P. Hvon D.(M6ATP) as a probe of the fidelity of base incorporation into polynucleotides by Escherichia coli DNA polymerase I. J. Biol. Chem. 253, 935–939 (1978).

    CAS  PubMed  Google Scholar 

  17. Chen, M. et al. m6A RNA degradation products are catabolized by an evolutionarily conserved N6-methyl-AMP deaminase in plant and mammalian cells. Plant Cell 30, 1511–1522 (2018).

    Article  Google Scholar 

  18. Xiao, C.-L. et al. N6-methyladenine DNA modification in the human genome. Mol. Cell. 71, 306–318.e7 (2018).

    Article  CAS  Google Scholar 

  19. Kweon, S.-M. et al. An adversarial DNA N6-methyladenine-sensor network preserves polycomb silencing. Mol. Cell. 74, 1138–1147.e6 (2019).

    Article  CAS  Google Scholar 

  20. Wu, C., Jin, X., Tsueng, G., Afrasiabi, C. & Su, A. I. BioGPS: building your own mash-up of gene annotations and expression profiles. Nucleic Acids Res. 44, D313–D316 (2016).

    Article  CAS  Google Scholar 

  21. Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    Article  CAS  Google Scholar 

  22. Yao, B. et al. DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress. Nat. Commun. 8, 1122 (2017).

    Article  Google Scholar 

  23. Thomson, J. P. et al. Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques. Nucleic Acids Res. 41, e206 (2013).

    Article  CAS  Google Scholar 

  24. Lentini, A. et al. A reassessment of DNA-immunoprecipitation-based genomic profiling. Nat. Methods 15, 499–504 (2018).

    Article  CAS  Google Scholar 

  25. Lane, A. N. & Fan, T. W.-M. Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res. 43, 2466–2485 (2015).

    Article  CAS  Google Scholar 

  26. Yang, N. C., Ho, W. M., Chen, Y. H. & Hu, M. L. A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP. Anal. Biochem. 306, 323–327 (2002).

    Article  CAS  Google Scholar 

  27. Knuckles, P. et al. RNA fate determination through cotranscriptional adenosine methylation and microprocessor binding. Nat. Struct. Mol. Biol. 24, 561–569 (2017).

    Article  CAS  Google Scholar 

  28. Ruvinsky, A. & Graves, J. A. M. Mammalian Genomics (CABI, 2005).

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Acknowledgements

We thank L. Schomacher for discussions, valuable suggestions, including experimental design and feedback during manuscript preparation. We thank M. Pradhan and G. Joshi for discussions, suggestions and critical reading of the manuscript. We are grateful for technical support by S. Ritz from the Institute of Molecular Biology Core Facility Microscopy.

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Authors

Contributions

M.U.M. and C.N. designed the experiments. M.U.M., A.B. and L.K. performed the experiments. C.N. and M.U.M. wrote the manuscript. All authors were involved in discussions, planning the experiments and editing the manuscript.

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Correspondence to Michael U. Musheev or Christof Niehrs.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Tables 1–7

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Source Data Fig. 1

Source Data Fig. 1c

Source Data Fig. 2

Source Data Fig. 2c–e

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Musheev, M.U., Baumgärtner, A., Krebs, L. et al. The origin of genomic N6-methyl-deoxyadenosine in mammalian cells. Nat Chem Biol 16, 630–634 (2020). https://doi.org/10.1038/s41589-020-0504-2

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