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.

  • Review Article
  • Published:

Means, mechanisms and consequences of adenine methylation in DNA

Nature Reviews Genetics volume 23, pages 411–428 (2022)Cite this article

Subjects

Abstract

N6-methyl-2′-deoxyadenosine (6mA or m6dA) has been reported in the DNA of prokaryotes and eukaryotes ranging from unicellular protozoa and algae to multicellular plants and mammals. It has been proposed to modulate DNA structure and transcription, transmit information across generations and have a role in disease, among other functions. However, its existence in more recently evolved eukaryotes remains a topic of debate. Recent technological advancements have facilitated the identification and quantification of 6mA even when the modification is exceptionally rare, but each approach has limitations. Critical assessment of existing data, rigorous design of future studies and further development of methods will be required to confirm the presence and biological functions of 6mA in multicellular eukaryotes.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Directed epigenetic DNA methylations.
Fig. 2: Potential modes of 6mA enrichment.
Fig. 3: Biological roles of DNA adenine methylation potentially conserved in unicellular and multicellular organisms.
Fig. 4: Emerging potential roles of 6mA in metazoa.

Similar content being viewed by others

References

  1. Janulaitis, A., Klimasauskas, S., Petrusyte, M. & Butkus, V. Cytosine modification in DNA by BcnI methylase yields N4-methylcytosine. FEBS Lett. 161, 131–134 (1983).

    Article  CAS  PubMed  Google Scholar 

  2. Ehrlich, M. et al. DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine. Nucleic Acids Res. 13, 1399–1412 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ehrlich, M., Wilson, G. G., Kuo, K. C. & Gehrke, C. W. N4-methylcytosine as a minor base in bacterial DNA. J. Bacteriol. 169, 939–943 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Grosjean, H. in DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution (ed Grosjean, H.) Ch. 1, 1–18 (CRC Press, 2009).

  5. Yu, M. et al. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing. Nucleic Acids Res. 43, e148 (2015).

    PubMed  PubMed Central  CAS  Google Scholar 

  6. Vanyushin, B. F., Tkacheva, S. G. & Belozersky, A. N. Rare bases in animal DNA. Nature 225, 948–949 (1970).

    Article  CAS  PubMed  Google Scholar 

  7. O’Brown, Z. K. & Greer, E. L. N6-methyladenine: a conserved and dynamic DNA mark. Adv. Exp. Med. Biol. 945, 213–246 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Iyer, L. M., Abhiman, S. & Aravind, L. Natural history of eukaryotic DNA methylation systems. Prog. Mol. Biol. Transl. Sci. 101, 25–104 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Bird, A. DNA methylation patterns and epigenetic memory. Genes. Dev. 16, 6–21 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Collins, M. & Myers, R. M. Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis. J. Mol. Biol. 198, 737–744 (1987).

    Article  CAS  PubMed  Google Scholar 

  13. Cheng, S. C., Herman, G. & Modrich, P. Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues. J. Biol. Chem. 260, 191–194 (1985).

    Article  CAS  PubMed  Google Scholar 

  14. Laddachote, S., Nagata, M. & Yoshida, W. Destabilisation of the c-kit1 G-quadruplex structure by N(6)-methyladenosine modification. Biochem. Biophys. Res. Commun. 524, 472–476 (2020).

    Article  CAS  PubMed  Google Scholar 

  15. Sternglanz, H. & Bugg, C. E. Conformation of N6-methyladenine, a base involved in DNA modification: restriction processes. Science 182, 833–834 (1973).

    Article  CAS  PubMed  Google Scholar 

  16. Peng, S., Padva, A. & LeBreton, P. R. Ultraviolet photoelectron studies of biological purines: the valence electronic structure of adenine. Proc. Natl Acad. Sci. USA 73, 2966–2968 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Engel, J. D. & von Hippel, P. H. Effects of methylation on the stability of nucleic acid conformations. Studies at the polymer level. J. Biol. Chem. 253, 927–934 (1978).

    Article  CAS  PubMed  Google Scholar 

  18. Gold, M., Hurwitz, J. & Anders, M. The enzymatic methylation of RNA and DNA. Biochem. Biophys. Res. Commun. 11, 107–114 (1963).

    Article  CAS  PubMed  Google Scholar 

  19. Chiang, P. K. et al. S-Adenosylmethionine and methylation. FASEB J. 10, 471–480 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Delk, A. S. & Rabinowitz, J. C. Biosynthesis of ribosylthymine in the transfer RNA of Streptococcus faecalis: a folate-dependent methylation not involving S-adenosylmethionine. Proc. Natl Acad. Sci. USA 72, 528–530 (1975).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Delk, A. S., Romeo, J. M., Nagle, D. P. Jr. & Rabinowitz, J. C. Biosynthesis of ribothymidine in the transfer RNA of Streptococcus faecalis and Bacillus subtilis. A methylation of RNA involving 5,10-methylenetetrahydrofolate. J. Biol. Chem. 251, 7649–7656 (1976).

    Article  CAS  PubMed  Google Scholar 

  22. Urbonavicius, J., Skouloubris, S., Myllykallio, H. & Grosjean, H. Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria — evolutionary implications. Nucleic Acids Res. 33, 3955–3964 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Kamat, S. S. et al. Enzymatic deamination of the epigenetic base N-6-methyladenine. J. Am. Chem. Soc. 133, 2080–2083 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Saparbaev, M. & Laval, J. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc. Natl Acad. Sci. USA 91, 5873–5877 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Greer, E. L. et al. DNA methylation on N(6)-adenine in C. elegans. Cell 161, 868–878 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang, G. et al. N(6)-methyladenine DNA modification in Drosophila. Cell 161, 893–906 (2015). This paper and that of Greer et al. (2015) are the first to demonstrate the presence of 6mA in metazoan genomes.

    Article  CAS  PubMed  Google Scholar 

  28. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu, J. et al. Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig. Nat. Commun. 7, 13052 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Liang, Z. et al. DNA N(6)-adenine methylation in Arabidopsis thaliana. Dev. Cell 45, 406–416.e403 (2018).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  33. Kigar, S. L. et al. N(6)-methyladenine is an epigenetic marker of mammalian early life stress. Sci. Rep. 7, 18078 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Mondo, S. J. et al. Widespread adenine N6-methylation of active genes in fungi. Nat. Genet. 49, 964–968 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Zhou, C., Liu, Y., Li, X., Zou, J. & Zou, S. DNA N(6)-methyladenine demethylase ALKBH1 enhances osteogenic differentiation of human MSCs. Bone Res. 4, 16033 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Liang, D. et al. The decreased N(6)-methyladenine DNA modification in cancer cells. Biochem. Biophys. Res. Commun. 480, 120–125 (2016).

    Article  CAS  PubMed  Google Scholar 

  38. Ma, C. et al. N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation. Nat. Cell Biol. 21, 319–327 (2019).

    Article  CAS  PubMed  Google Scholar 

  39. Pogolotti, A. L. Jr., Ono, A., Subramaniam, R. & Santi, D. V. On the mechanism of DNA-adenine methylase. J. Biol. Chem. 263, 7461–7464 (1988).

    Article  CAS  PubMed  Google Scholar 

  40. Macon, J. B. & Wolfenden, R. 1-Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry 7, 3453–3458 (1968).

    Article  CAS  PubMed  Google Scholar 

  41. Schiffers, S. et al. Quantitative LC-MS provides no evidence for m(6) dA or m(4) dC in the genome of mouse embryonic stem cells and tissues. Angew. Chem. Int. Ed. Engl. 56, 11268–11271 (2017). This paper provides strong evidence that questions the presence of 6mA in the mouse genome.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  43. 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  PubMed  PubMed Central  CAS  Google Scholar 

  44. Musheev, M. U., Baumgartner, A., Krebs, L. & Niehrs, C. The origin of genomic N(6)-methyl-deoxyadenosine in mammalian cells. Nat. Chem. Biol. 16, 630–634 (2020).

    Article  CAS  PubMed  Google Scholar 

  45. Liu, X. et al. N(6)-methyladenine is incorporated into mammalian genome by DNA polymerase. Cell Res. 31, 94–97 (2021).

    Article  CAS  PubMed  Google Scholar 

  46. Johnson, T. B. & Coghill, R. D. The discovery of 5-methyl-cytosine in tuberculinic acid, the nucleic acid of the tubercle bacillus. J. Am. Chem. Soc. 47, 2838–2844 (1925).

    Article  CAS  Google Scholar 

  47. Hotchkiss, R. D. The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography. J. Biol. Chem. 175, 315–332 (1948).

    Article  CAS  PubMed  Google Scholar 

  48. Wyatt, G. R. Occurrence of 5-methylcytosine in nucleic acids. Nature 166, 237–238 (1950).

    Article  CAS  PubMed  Google Scholar 

  49. Dunn, D. B. & Smith, J. D. Occurrence of a new base in the deoxyribonucleic acid of a strain of Bacterium coli. Nature 175, 336–337 (1955).

    Article  CAS  PubMed  Google Scholar 

  50. Mason, S. F. Purine Studies. Part II. The ultra-violet absorption spectra of some mono- and poly-substituted purines. J. Chem. Soc. 1954, 2071–2081 (1954).

    Article  Google Scholar 

  51. Achwal, C. W., Iyer, C. A. & Chandra, H. S. Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA. FEBS Lett. 158, 353–358 (1983).

    Article  CAS  PubMed  Google Scholar 

  52. Bird, A. P. & Southern, E. M. Use of restriction enzymes to study eukaryotic DNA methylation: I. The methylation pattern in ribosomal DNA from Xenopus laevis. J. Mol. Biol. 118, 27–47 (1978).

    Article  CAS  PubMed  Google Scholar 

  53. Geier, G. E. & Modrich, P. Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of DpnI endonuclease. J. Biol. Chem. 254, 1408–1413 (1979).

    Article  CAS  PubMed  Google Scholar 

  54. Krais, A. M., Cornelius, M. G. & Schmeiser, H. H. Genomic N(6)-methyladenine determination by MEKC with LIF. Electrophoresis 31, 3548–3551 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Yuki, H., Kawasaki, H., Imayuki, A. & Yajima, T. Determination of 6-methyladenine in DNA by high-performance liquid chromatography. J. Chromatogr. 168, 489–494 (1979).

    Article  CAS  PubMed  Google Scholar 

  56. Boulias, K. & Greer, E. L. Detection of DNA methylation in genomic DNA by UHPLC-MS/MS. Methods Mol. Biol. 2198, 79–90 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Douvlataniotis, K., Bensberg, M., Lentini, A., Gylemo, B. & Nestor, C. E. No evidence for DNA N (6)-methyladenine in mammals. Sci. Adv. 6, eaay3335 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Fu, Y. et al. N(6)-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161, 879–892 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Dohno, C., Shibata, T. & Nakatani, K. Discrimination of N6-methyl adenine in a specific DNA sequence. Chem. Commun. 46, 5530–5532 (2010).

    Article  CAS  Google Scholar 

  60. Posfai, G. & Szybalski, W. A simple method for locating methylated bases in DNA using class-IIS restriction enzymes. Gene 74, 179–181 (1988).

    Article  CAS  PubMed  Google Scholar 

  61. Weber, M. et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat. Genet. 37, 853–862 (2005).

    Article  CAS  PubMed  Google Scholar 

  62. Pomraning, K. R., Smith, K. M. & Freitag, M. Genome-wide high throughput analysis of DNA methylation in eukaryotes. Methods 47, 142–150 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Li, Z. et al. N(6)-methyladenine in DNA antagonizes SATB1 in early development. Nature 583, 625–630 (2020). This paper proposes that 6mA antagonizes SATB1 to enforce a boundary between heterochromatin and euchromatin.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Chen, K., Luo, G. Z. & He, C. High-resolution mapping of N(6)-methyladenosine in transcriptome and genome using a photo-crosslinking-assisted strategy. Methods Enzymol. 560, 161–185 (2015).

    Article  CAS  PubMed  Google Scholar 

  66. Koh, C. W. Q. et al. Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome. Nucleic Acids Res. 46, 11659–11670 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods 7, 461–465 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Ye, P. et al. MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing. Nucleic Acids Res. 45, D85–D89 (2017).

    Article  CAS  PubMed  Google Scholar 

  69. Zhu, S. et al. Mapping and characterizing N6-methyladenine in eukaryotic genomes using single molecule real-time sequencing. Genome Res. 28, 1067–1078 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Zhang, Q. et al. N(6)-methyladenine DNA methylation in japonica and indica rice genomes and its association with gene expression, plant development, and stress responses. Mol. Plant. 11, 1492–1508 (2018).

    Article  CAS  PubMed  Google Scholar 

  71. Kong, Y. et al. Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution. Science 375, 515–522 (2022).

    Article  CAS  PubMed  Google Scholar 

  72. Xie, Q. et al. N(6)-methyladenine DNA modification in glioblastoma. Cell 175, 1228–1243.e1220 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Abakir, A. et al. N(6)-methyladenosine regulates the stability of RNA:DNA hybrids in human cells. Nat. Genet. 52, 48–55 (2020).

    Article  CAS  PubMed  Google Scholar 

  74. Pacini, C. E., Bradshaw, C. R., Garrett, N. J. & Koziol, M. J. Characteristics and homogeneity of N6-methylation in human genomes. Sci. Rep. 9, 5185 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. He, S. et al. 6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda. Nat. Commun. 10, 2219 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Li, X. et al. The DNA modification N6-methyl-2′-deoxyadenosine (m6dA) drives activity-induced gene expression and is required for fear extinction. Nat. Neurosci. 22, 534–544 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Xiao, C. L. et al. N(6)-methyladenine DNA modification in the human genome. Mol. Cell 71, 306–318 e307 (2018).

    Article  CAS  PubMed  Google Scholar 

  78. Liu, B., Liu, X., Lai, W. & Wang, H. Metabolically generated stable isotope-labeled deoxynucleoside code for tracing DNA N(6)-methyladenine in human cells. Anal. Chem. 89, 6202–6209 (2017).

    Article  CAS  PubMed  Google Scholar 

  79. & Kweon, S. M. et al. An Adversarial DNA N(6)-methyladenine-sensor network preserves polycomb silencing. Mol. Cell 74, 1138–1147 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Hao, Z. et al. N(6)-deoxyadenosine methylation in mammalian mitochondrial DNA. Mol. Cell 78, 382–395 e388 (2020). This paper suggests that 6mA occurs predominantly in mtDNA and demonstrates that METTL4 has activity both in vitro and ex vivo.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Goh, Y. T., Koh, C. W. Q., Sim, D. Y., Roca, X. & Goh, W. S. S. METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing. Nucleic Acids Res. 48, 9250–9261 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Chen, H. et al. METTL4 is an snRNA m(6)Am methyltransferase that regulates RNA splicing. Cell Res. 30, 544–547 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Gu, L. et al. CG14906 (mettl4) mediates m(6)A methylation of U2 snRNA in Drosophila. Cell Discov. 6, 44 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Liu, P. et al. Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality. Mol. Cell Biol. 30, 4245–4253 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Li, W., Shi, Y., Zhang, T., Ye, J. & Ding, J. Structural insight into human N6amt1-Trm112 complex functioning as a protein methyltransferase. Cell Discov. 5, 51 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  86. Woodcock, C. B., Yu, D., Zhang, X. & Cheng, X. Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112. Cell Discov. 5, 50 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  87. Woodcock, C. B. et al. Human MettL3-MettL14 complex is a sequence-specific DNA adenine methyltransferase active on single-strand and unpaired DNA in vitro. Cell Discov. 5, 63 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Fedeles, B. I., Singh, V., Delaney, J. C., Li, D. & Essigmann, J. M. The AlkB family of Fe(II)/alpha-ketoglutarate-dependent dioxygenases: repairing nucleic acid alkylation damage and beyond. J. Biol. Chem. 290, 20734–20742 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Wei, Y. F., Carter, K. C., Wang, R. P. & Shell, B. K. Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair. Nucleic Acids Res. 24, 931–937 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Wang, S. Y. et al. The demethylase NMAD-1 regulates DNA replication and repair in the Caenorhabditis elegans germline. PLoS Genet. 15, e1008252 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Zhang, M. et al. Mammalian ALKBH1 serves as an N(6)-mA demethylase of unpairing DNA. Cell Res. 30, 197–210 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Tian, L. F. et al. Structural basis of nucleic acid recognition and 6mA demethylation by human ALKBH1. Cell Res. 30, 272–275 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  93. Xiong, J. et al. N 6-Hydroxymethyladenine: a hydroxylation derivative of N6-methyladenine in genomic DNA of mammals. Nucleic Acids Res. 47, 1268–1277 (2019). This paper demonstrates the presence of 6hmA in mammalian DNA and demonstrates that ALKBH1 can demethylate 6mA both in vitro and ex vivo.

    Article  CAS  PubMed  Google Scholar 

  94. Liu, F. et al. ALKBH1-mediated tRNA demethylation regulates translation. Cell 167, 816–828.e816 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Haag, S. et al. NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation. EMBO J. 35, 2104–2119 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. 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  PubMed  PubMed Central  Google Scholar 

  97. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Yao, B. et al. Active N(6)-methyladenine demethylation by DMAD regulates gene expression by coordinating with polycomb protein in neurons. Mol. Cell 71, 848–857.e846 (2018). This paper examines possible mechanisms of direct communication between 6mA and other chromatin modifications.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Aravind, L., Zhang, D. & Iyer, L. M. in 2-Oxoglutarate-Dependent Oxygenases (eds Hausinger, R. & Schofield, C.) Ch. 11 (Royal Society of Chemistry, 2015).

  101. Welsh, K. M., Lu, A. L., Clark, S. & Modrich, P. Isolation and characterization of the Escherichia coli mutH gene product. J. Biol. Chem. 262, 15624–15629 (1987).

    Article  CAS  PubMed  Google Scholar 

  102. Su, S. S., Lahue, R. S., Au, K. G. & Modrich, P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J. Biol. Chem. 263, 6829–6835 (1988).

    Article  CAS  PubMed  Google Scholar 

  103. Lahue, R. S., Au, K. G. & Modrich, P. DNA mismatch correction in a defined system. Science 245, 160–164 (1989).

    Article  CAS  PubMed  Google Scholar 

  104. Iyer, R. R., Pluciennik, A., Burdett, V. & Modrich, P. L. DNA mismatch repair: functions and mechanisms. Chem. Rev. 106, 302–323 (2006).

    Article  CAS  PubMed  Google Scholar 

  105. Slater, S. et al. E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell 82, 927–936 (1995).

    Article  CAS  PubMed  Google Scholar 

  106. Brendler, T., Abeles, A. & Austin, S. A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene. EMBO J. 14, 4083–4089 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. von Freiesleben, U., Rasmussen, K. V. & Schaechter, M. SeqA limits DnaA activity in replication from oriC in Escherichia coli. Mol. Microbiol. 14, 763–772 (1994).

    Article  CAS  Google Scholar 

  108. Wold, S., Boye, E., Slater, S., Kleckner, N. & Skarstad, K. Effects of purified SeqA protein on oriC-dependent DNA replication in vitro. EMBO J. 17, 4158–4165 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Iyer, L. M., Zhang, D. & Aravind, L. Adenine methylation in eukaryotes: apprehending the complex evolutionary history and functional potential of an epigenetic modification. Bioessays 38, 27–40 (2016).

    Article  CAS  PubMed  Google Scholar 

  110. Iyer, L. M., Zhang, D., Burroughs, A. M. & Aravind, L. Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA. Nucleic Acids Res. 41, 7635–7655 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Iyer, L. M., Burroughs, A. M. & Aravind, L. The ASCH superfamily: novel domains with a fold related to the PUA domain and a potential role in RNA metabolism. Bioinformatics 22, 257–263 (2006).

    Article  CAS  PubMed  Google Scholar 

  112. Aravind, L. & Iyer, L. M. The HARE-HTH and associated domains: novel modules in the coordination of epigenetic DNA and protein modifications. Cell Cycle 11, 119–131 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Fazakerley, G. V., Gabarro-Arpa, J., Lebret, M., Guy, A. & Guschlbauer, W. The GTm6AC sequence is overwound and bent. Nucleic Acids Res. 17, 2541–2556 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Wang, B. et al. Structural insights into target DNA recognition by R2R3-MYB transcription factors. Nucleic Acids Res. 48, 460–471 (2020).

    CAS  PubMed  Google Scholar 

  115. Low, D. A., Weyand, N. J. & Mahan, M. J. Roles of DNA adenine methylation in regulating bacterial gene expression and virulence. Infect. Immun. 69, 7197–7204 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Wallecha, A., Munster, V., Correnti, J., Chan, T. & van der Woude, M. Dam- and OxyR-dependent phase variation of agn43: essential elements and evidence for a new role of DNA methylation. J. Bacteriol. 184, 3338–3347 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Zaleski, P., Wojciechowski, M. & Piekarowicz, A. The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection. Microbiology 151, 3361–3369 (2005).

    Article  CAS  PubMed  Google Scholar 

  118. Sarnacki, S. H. et al. Dam methylation participates in the regulation of PmrA/PmrB and RcsC/RcsD/RcsB two component regulatory systems in Salmonella enterica serovar Enteritidis. PLoS ONE 8, e56474 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Rogers, J. C. & Rogers, S. W. Comparison of the effects of N6-methyldeoxyadenosine and N5-methyldeoxycytosine on transcription from nuclear gene promoters in barley. Plant. J. 7, 221–233 (1995).

    Article  CAS  PubMed  Google Scholar 

  120. Graham, M. W. & Larkin, P. J. Adenine methylation at dam sites increases transient gene expression in plant cells. Transgenic Res. 4, 324–331 (1995).

    Article  CAS  PubMed  Google Scholar 

  121. Allamane, S. et al. Bacterial DNA methylation and gene transfer efficiency. Biochem. Biophys. Res. Commun. 276, 1261–1264 (2000).

    Article  CAS  PubMed  Google Scholar 

  122. Lizarraga, A. et al. Adenine DNA methylation, 3D genome organization, and gene expression in the parasite Trichomonas vaginalis. Proc. Natl Acad. Sci. USA 117, 13033–13043 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Tronche, F., Rollier, A., Bach, I., Weiss, M. C. & Yaniv, M. The rat albumin promoter: cooperation with upstream elements is required when binding of APF/HNF1 to the proximal element is partially impaired by mutation or bacterial methylation. Mol. Cell Biol. 9, 4759–4766 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Lichtsteiner, S. & Schibler, U. A glycosylated liver-specific transcription factor stimulates transcription of the albumin gene. Cell 57, 1179–1187 (1989).

    Article  CAS  PubMed  Google Scholar 

  125. Sugimoto, K., Takeda, S. & Hirochika, H. Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein AGP1 to the AG-motif (AGATCCAA) of the wound-inducible Myb gene NtMyb2. Plant. J. 36, 550–564 (2003).

    Article  CAS  PubMed  Google Scholar 

  126. Wang, W. et al. Epigenetic DNA modification N(6)-methyladenine causes site-specific RNA polymerase II transcriptional pausing. J. Am. Chem. Soc. 139, 14436–14442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Pratt, K. & Hattman, S. Nucleosome phasing in Tetrahymena macronuclei. J. Protozool. 30, 592–598 (1983).

    Article  CAS  PubMed  Google Scholar 

  128. Karrer, K. M. & VanNuland, T. A. Methylation of adenine in the nuclear DNA of Tetrahymena is internucleosomal and independent of histone H1. Nucleic Acids Res. 30, 1364–1370 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Beh, L. Y. et al. Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization. Cell 177, 1781–1796.e1725 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Jeddeloh, J. A., Stokes, T. L. & Richards, E. J. Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat. Genet. 22, 94–97 (1999).

    Article  CAS  PubMed  Google Scholar 

  131. Tan, F. et al. Analysis of chromatin regulators reveals specific features of rice DNA methylation pathways. Plant. Physiol. 171, 2041–2054 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Brzeski, J. & Jerzmanowski, A. Deficient in DNA methylation 1 (DDM1) defines a novel family of chromatin-remodeling factors. J. Biol. Chem. 278, 823–828 (2003).

    Article  CAS  PubMed  Google Scholar 

  133. Marinus, M. G. & Morris, N. R. Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12. J. Mol. Biol. 85, 309–322 (1974).

    Article  CAS  PubMed  Google Scholar 

  134. Rogers, S. D., Rogers, M. E., Saunders, G. & Holt, G. Isolation of mutants sensitive to 2-aminopurine and alkylating agents and evidence for the role of DNA methylation in Penicillium chrysogenum. Curr. Genet. 10, 557–560 (1986).

    Article  CAS  PubMed  Google Scholar 

  135. Pukkila, P. J., Peterson, J., Herman, G., Modrich, P. & Meselson, M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics 104, 571–582 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Fukui, K. DNA mismatch repair in eukaryotes and bacteria. J. Nucleic Acids 2010, 260512 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  137. Bakker, A. & Smith, D. W. Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli. J. Bacteriol. 171, 5738–5742 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Boye, E. & Lobner-Olesen, A. The role of dam methyltransferase in the control of DNA replication in E. coli. Cell 62, 981–989 (1990).

    Article  CAS  PubMed  Google Scholar 

  139. Lu, M., Campbell, J. L., Boye, E. & Kleckner, N. SeqA: a negative modulator of replication initiation in E. coli. Cell 77, 413–426 (1994).

    Article  CAS  PubMed  Google Scholar 

  140. Yamaki, H., Ohtsubo, E., Nagai, K. & Maeda, Y. The oriC unwinding by dam methylation in Escherichia coli. Nucleic Acids Res. 16, 5067–5073 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Collier, J., McAdams, H. H. & Shapiro, L. A DNA methylation ratchet governs progression through a bacterial cell cycle. Proc. Natl Acad. Sci. USA 104, 17111–17116 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Berdis, A. J. et al. A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA. Proc. Natl Acad. Sci. USA 95, 2874–2879 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Kozdon, J. B. et al. Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proc. Natl Acad. Sci. USA 110, E4658–E4667 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Li, B. et al. Epigenetic DNA modification N(6)-methyladenine inhibits DNA replication by DNA polymerase of Pseudomonas aeruginosa phage PaP1. Chem. Res. Toxicol. 32, 840–849 (2019).

    Article  CAS  PubMed  Google Scholar 

  145. Du, K. et al. Epigenetically modified N(6)-methyladenine inhibits DNA replication by human DNA polymerase eta. DNA Repair. 78, 81–90 (2019).

    Article  CAS  PubMed  Google Scholar 

  146. Fuks, F., Hurd, P. J., Deplus, R. & Kouzarides, T. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res. 31, 2305–2312 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Johnson, L. M. et al. The SRA methyl-cytosine-binding domain links DNA and histone methylation. Curr. Biol. 17, 379–384 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Rajakumara, E. et al. PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression. Mol. Cell 43, 275–284 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Fuks, F. DNA methylation and histone modifications: teaming up to silence genes. Curr. Opin. Genet. Dev. 15, 490–495 (2005).

    Article  CAS  PubMed  Google Scholar 

  151. Rountree, M. R. & Selker, E. U. DNA methylation and the formation of heterochromatin in Neurospora crassa. Heredity 105, 38–44 (2010).

    Article  CAS  PubMed  Google Scholar 

  152. Sheng, X., Wang, J., Guo, Y., Zhang, J. & Luo, J. DNA N6-methyladenine (6mA) modification regulates drug resistance in triple negative breast cancer. Front. Oncol. 10, 616098 (2020).

    Article  PubMed  Google Scholar 

  153. Marinus, M. G. Adenine methylation of Okazaki fragments in Escherichia coli. J. Bacteriol. 128, 853–854 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Kamat, S. S. et al. Catalytic mechanism and three-dimensional structure of adenine deaminase. Biochemistry 50, 1917–1927 (2011).

    Article  CAS  PubMed  Google Scholar 

  156. Mahdavi-Amiri, Y., Chung Kim Chung, K. & Hili, R. Single-nucleotide resolution of N6-adenine methylation sites in DNA and RNA by nitrite sequencing. Chem. Sci. 12, 606–612 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  157. Shah, K., Cao, W. & Ellison, C. E. Adenine methylation in drosophila is associated with the tissue-specific expression of developmental and regulatory genes. G3 9, 1893–1900 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Fernandes, S. B. et al. N(6)-methyladenine in eukaryotic DNA: tissue distribution, early embryo development, and neuronal toxicity. Front. Genet. 12, 657171 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. Sun, Q. et al. N(6)-methyladenine functions as a potential epigenetic mark in eukaryotes. Bioessays 37, 1155–1162 (2015).

    Article  CAS  PubMed  Google Scholar 

  160. Nikolskaya, I. I., Lopatina, N. G., Chaplygina, N. M. & Debov, S. S. The host specificity system in Escherichia coli SK. Mol. Cell Biochem. 13, 79–87 (1976).

    Article  CAS  PubMed  Google Scholar 

  161. Nikolskaya, I. I., Lopatina, N. G. & Debov, S. S. On heterogeneity of DNA methylases from Escherichia coli SK cells. Mol. Cell Biochem. 35, 3–10 (1981).

    Article  CAS  PubMed  Google Scholar 

  162. Reich, N. O. & Mashhoon, N. Kinetic mechanism of the EcoRI DNA methyltransferase. Biochemistry 30, 2933–2939 (1991).

    Article  CAS  PubMed  Google Scholar 

  163. Siksnys, V. et al. CAATTG-specific restriction-modification munI genes from Mycoplasma: sequence similarities between R.MunI and R.EcoRI. Gene 142, 1–8 (1994).

    Article  CAS  PubMed  Google Scholar 

  164. Duncan, T. et al. Reversal of DNA alkylation damage by two human dioxygenases. Proc. Natl Acad. Sci. USA 99, 16660–16665 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  165. Zhang, Z. et al. Regulation of adipocyte differentiation by METTL4, a 6mA methylase. Sci. Rep. 10, 8285 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  166. Butkus, V. et al. Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine. Nucleic Acids Res. 15, 8467–8478 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Fazakerley, G. V., Kraszewski, A., Teoule, R. & Guschlbauer, W. NMR and CD studies on an oligonucleotide containing N4-methylcytosine. Nucleic Acids Res. 15, 2191–2201 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Gartner, K. et al. Cytosine N4-methylation via M.Ssp6803II is involved in the regulation of transcription, fine- tuning of DNA replication and DNA repair in the cyanobacterium Synechocystis sp. PCC 6803. Front. Microbiol. 10, 1233 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The work from the Greer laboratory is supported by grants from the NIH (DP2AG055947 and R01AI151215). The authors thank C. He, E. Kool and N. Mosammaparast for helpful discussions. They apologize for the literature omitted owing to space limitations.

Author information

Authors and Affiliations

  1. Division of Newborn Medicine, Boston Children’s Hospital, Boston, MA, USA

    Konstantinos Boulias & Eric Lieberman Greer

  2. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Konstantinos Boulias & Eric Lieberman Greer

  3. Harvard Medical School Initiative for RNA Medicine, Boston, MA, USA

    Konstantinos Boulias & Eric Lieberman Greer

Authors
  1. Konstantinos Boulias
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eric Lieberman Greer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Both authors contributed to all aspects of the article.

Corresponding author

Correspondence to Eric Lieberman Greer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Reviews Genetics thanks G. Fang, M. Koziol and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Epigenetic

In the context of this article, this term refers to changes to the DNA that do not alter its nucleotide sequence.

Dimroth rearrangement

A passive process by which a methyl group is transferred from the N1 position to the N6 position of the same adenine base.

Nucleotide salvage pathway

The process by which intermediates of nucleotide degradation are recovered to be converted back into nucleotides, bypassing de novo synthesis.

Long-read sequencing

Third-generation sequencing approaches that can generate reads >10,000 bases in length, which improves mapping accuracy compared with short-read approaches; however, long-read approaches have higher error rates at individual bases than short-read methods.

RNA–DNA hybrids

Occur when nascent RNA transcripts hybridize with one strand of the DNA template creating a three-stranded structure called an R loop.

MT-A70 domain

This domain binds to S-adenosylmethionine and is present in a clade of RNA and DNA methyltransferases.

Hemimethylated DNA

Describes a DNA molecule in which only one of the two complementary DNA strands is methylated.

Triple negative breast cancer

Cancers that are negative for oestrogen receptors, progesterone receptors and excess HER2 protein and therefore do not respond to hormonal therapies or targeting of the HER2 receptor.

Epigenomic editing

Engineered changes to the epigenome that do not alter the DNA sequence that are accomplished using a modified Cas9 nuclease.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Boulias, K., Greer, E.L. Means, mechanisms and consequences of adenine methylation in DNA. Nat Rev Genet 23, 411–428 (2022). https://doi.org/10.1038/s41576-022-00456-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41576-022-00456-x

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