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.

A fungal dioxygenase CcTet serves as a eukaryotic 6mA demethylase on duplex DNA

Abstract

N6-methyladenosine (6mA) is a DNA modification that has recently been found to play regulatory roles during mammalian early embryo development and mitochondrial transcription. We found that a dioxygenase CcTet from the fungus Coprinopsis cinerea is also a dsDNA 6mA demethylase. It oxidizes 6mA to the intermediate N6-hydroxymethyladenosine (6hmA) with robust activity of 6mA-containing duplex DNA (dsDNA) as well as isolated genomics DNA. Structural characterization revealed that CcTet utilizes three flexible loop regions and two key residues—D337 and G331—in the active pocket to preferentially recognize substrates on dsDNA. A CcTet D337F mutant protein retained the catalytic activity on 6mA but lost activity on 5-methylcytosine. Our findings uncovered a 6mA demethylase that works on dsDNA, suggesting potential 6mA demethylation in fungi and elucidating 6mA recognition and the catalytic mechanism of CcTet. The CcTet D337F mutant protein also provides a chemical biology tool for future functional manipulation of DNA 6mA in vivo.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Enzymatic evaluation of CcTet 6mA demethylation.
Fig. 2: Overall structure of CcTet with bound NOG and Mn2+.
Fig. 3: Structure of CcTet bound to 6mA-containing dsDNA.
Fig. 4: Structural basis for substrate preference of CcTet in the active pocket.
Fig. 5: Substrate preference screen of CcTet mutants.

Data availability

The coordinates of the crystal structure have been deposited with the Protein Data Bank under the accession nos. 7VPN and 7W5P. All structures cited in this publication are available under accession nos. 4NM6, 5CG8, 5CG9, 5ZMD, 7CY8, 4JHT, 6IMC, 6KSF, 3BUC, 3BKZ, 2IUW, 4NRO, 4QKD, 3THT and 3LFM. Source data are provided with this paper.

References

  1. Luo, G.-Z., Blanco, M. A., Greer, E. L., He, C. & Shi, Y. DNA N6-methyladenine: a new epigenetic mark in eukaryotes? Nat. Rev. Mol. Cell Biol. 16, 705–710 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Fu, Y. et al. N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161, 879–892 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Beh, L. Y. et al. Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization. Cell 177, 1781–1796.e25 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Luo, G.Z. et al. N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA. Genome Biol. 19, 200 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Greer, E. L. et al. DNA methylation on N6-adenine in C. elegans. Cell 161, 868–878 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  8. Zhang, G. et al. N6-methyladenine DNA modification in Drosophila. Cell 161, 893–906 (2015).

    CAS  PubMed  Google Scholar 

  9. Zhou, C. et al. Identification and analysis of adenine N6-methylation sites in the rice genome. Nat. Plants 4, 554–563 (2018).

    CAS  PubMed  Google Scholar 

  10. Liang, Z. et al. DNA N6-adenine methylation in Arabidopsis thaliana. Dev. Cell 45, 406–416.e3 (2018).

    CAS  PubMed  Google Scholar 

  11. Luo, G.-Z. & He, C. DNA N6-methyladenine in metazoans: functional epigenetic mark or bystander? Nat. Struct. Mol. Biol. 24, 503–506 (2017).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Wu, T. P. et al. DNA methylation on N6-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  16. 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).

    PubMed  PubMed Central  Google Scholar 

  17. Douvlataniotis, K., Bensberg, M., Lentini, A., Gylemo, B. & Nestor, C. E. No evidence for DNA N6-methyladenine in mammals. Sci. Adv. 6, eaay3335 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  19. Hao, Z. et al. N6-deoxyadenosine methylation in mammalian mitochondrial DNA. Mol. Cell 78, 382–395.e8 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Li, Z. et al. N6-methyladenine in DNA antagonizes SATB1 in early development. Nature 583, 625–630 (2020).

    CAS  PubMed  PubMed Central  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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

    CAS  PubMed  Google Scholar 

  23. Yao, B. et al. Active N6-methyladenine demethylation by DMAD regulates gene expression by coordinating with polycomb protein in neurons. Mol. Cell 71, 848–857.e6 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang, M. et al. Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA. Cell Res. 30, 197–210 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Yi, C., Yang, C.-G. & He, C. A non-heme iron-mediated chemical demethylation in DNA and RNA. Acc. Chem. Res. 42, 519–529 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Fu, Y. et al. FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat. Commun. 4, 1798 (2013).

    PubMed  Google Scholar 

  29. Toh, J. D. W. et al. Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates. Proc. Natl Acad. Sci. USA 117, 25284–25292 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Qin, F. X., Jiang, C.-Y., Jiang, T. & Cheng, G. New targets for controlling Ebola virus disease. Natl Sci. Rev. 2, 266–267 (2015).

    PubMed  Google Scholar 

  31. 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 

  32. 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 

  33. 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 

  34. Hu, L. et al. Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation. Cell 155, 1545–1555 (2013).

    CAS  PubMed  Google Scholar 

  35. Hu, L. et al. Structural insight into substrate preference for TET-mediated oxidation. Nature 527, 118–122 (2015).

    CAS  PubMed  Google Scholar 

  36. Xue, J.-H. et al. A vitamin-C-derived DNA modification catalysed by an algal TET homologue. Nature 569, 581–585 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Li, W. et al. Molecular mechanism for vitamin C-derived C5-glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1. Nat. Commun. 12, 744 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhang, L. et al. A TET homologue protein from Coprinopsis cinerea (CcTet) that biochemically converts 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. J. Am. Chem. Soc. 136, 4801–4804 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang, L. et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat. Chem. Biol. 8, 328–330 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Zhu, C. & Yi, C. Switching demethylation activities between AlkB family RNA/DNA demethylases through exchange of active-site residues. Angew. Chem. Int. Ed. Engl. 53, 3659–3662 (2014).

    CAS  PubMed  Google Scholar 

  41. Zhang, X. et al. Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates. Proc. Natl Acad. Sci. USA 116, 2919–2924 (2019).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  43. Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution—from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    PubMed  Google Scholar 

  45. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    PubMed  Google Scholar 

  46. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

Download references

Acknowledgements

This project was supported by grants from the Major Research plan of the National Natural Science Foundation of China (no. 91853118 to Liang Zhang), NSFC (nos. 22107067 to Lin Zhang; 22077081 and 21722802 to Liang Zhang), Science and Technology Commission of Shanghai Municipality (nos. 20S11900300 and 22S11900600 to Liang Zhang), Shuguang Program supported by the Shanghai Education Development Foundation and Shanghai Municipal Education Commission (no. 20SG16 to Liang Zhang), innovative research team of high-level local universities in Shanghai (no. SSMU-ZLCX20180702 to Liang Zhang) and the Key Program of NSFC (no. 22137006 to H.-W.L.). We thank Professor Zhonghua Liu from Nanjing Normal University for generously providing the genomic DNA of C. cinerea. We thank the staff from the BL19U1 and BL18U beamlines of the NFPS in Shanghai at the SSRF for their assistance during data collection. We thank the staff from the Core Facility of Basic Medical Sciences at Shanghai Jiao Tong University School of Medicine for their assistance during the LC–MS/MS data collection. C.H. is a Howard Hughes Medical Institute Investigator.

Author information

Authors and Affiliations

Authors

Contributions

Liang Zhang and Lin Zhang designed the experiments. Y.M. and J.Z. performed the protein purification and crystallization. Lin Zhang and J.H. performed the LC–MS/MS-based activity assays and determined the kinetics. Lin Zhang performed the biophysical experiments. Liang Zhang, C.H., Lin Zhang, H.-W.L. and H.-Z.C. wrote the paper. All authors discussed and commented on the manuscript.

Corresponding authors

Correspondence to Lin Zhang, Hong-Zhuan Chen or Liang Zhang.

Ethics declarations

Competing interests

C.H. is a scientific founder and member of the scientific advisory board of Accent Therapeutics and Inferna Green. The other authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks the anonymous reviewers 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.

Extended data

Extended Data Fig. 1 In vitro enzymatic activity of human TET2 catalytic domain on 6 mA or 5mC containing 19 bp dsDNA.

N = 3 biologically independent experiments. Data represent mean values ± s.d.

Source data

Extended Data Fig. 2 LC-MS/MS analysis of 6 mA demethylation catalyzed by overexpression of CcTet wild type and CcTet D337F in E. coli. in vivo.

The unpaired two-sided student’s t-test values were also labeled. P = 0.002 (CcTet), 0.000083 (CcTet D337F), **P < 0.01, ****P < 0.0001. N = 6 biologically independent experiments. Data represent mean values ± s.d.

Source data

Extended Data Fig. 3 Multiple sequence alignment of TETs and ALKBs family.

The structural comparison was generated in the DALI server (http://ekhidna2.biocenter.helsinki.fi/dali/), and the sequence alignment was generated through ESPript online server (https://espript.ibcp.fr/ESPript/ESPript/). The secondary structures of CcTet were shown and labeled. The two key loops of CcTet were shaded in cyan, the substrate selection region was shaded in orange, and the long-disordered loop in hTET2 was shaded in grey. The residues that involved in α-KG/Ion/dsDNA binding were labeled with spheres. The finger residue V232 was marked with a red star, and two key residues G331 and D337 involved in substrate selection were marked with blue stars.

Extended Data Fig. 4 Superposition of CcTet (green) and CcTet-6mA-dsDNA (cyan) complex.

The black arrows indicate the rotation direction of the residue sidechains.

Extended Data Fig. 5 The interactions between DNA bases and CcTet residues.

CcTet and DNA chains were colored in cyan and wheat/palegreen. The two water molecules were shown in spheres and labeled as W1 and W2. The yellow dashes indicate H-bonds between two atoms.

Extended Data Fig. 6 Superposition of CcTet-6mA-dsDNA (cyan/wheat) with FTO-6mA-ssDNA (green/purple, pdb code: 5ZMD) complexes.

(a) Superposition of 6 mA in CcTet active pocket and 6 mA (purple) in FTO (green) active pocket. The wheat region indicates the key FTO residue that was reported to play key roles in m6A catalysis. (b) LC-MS/MS analysis of 6 mA demethylation by CcTet mutations. N = 3 biologically independent experiments. Data represent mean values ± s.d.

Source data

Extended Data Fig. 7 Superposition of CcTet-dsDNA with NgTet1-dsDNA or CMD1-dsDNA complex.

The yellow dashes indicate the H-bonds, and the grey dashes indicate the distance between two atoms, and the distances were labeled. (a) Superposition of CcTet-6mA-dsDNA (cyan/wheat) with NgTet1-5mC-dsDNA (yellow/orange, pdb code: 5CG9) complex. (b) Superposition of CcTet-6mA-dsDNA (cyan/wheat) with CMD1-5mC-dsDNA (slate/red, pdb code: 7CY8) complex.

Extended Data Fig. 8

Dot Blot analysis of concentration-dependent 6 mA (a) and 5mC (b) demethylation on Coprinopsis cinerea genomic DNA (mycelium stage) catalyzed by CcTet or D337F mutant. Upper panels show the representative antibody (anti-5mC or 6 mA) dot blot for the purified Coprinopsis cinerea genomic DNA; lower panels show the methylene blue staining to validate the equal loading amount of DNA.

Source data

Extended Data Fig. 9

Dot Blot analysis of concentration-dependent 6 mA (a) and 5mC (b) demethylation on green alga (Chlamydomonas reinhardtii) genomic DNA catalyzed by CcTet or D337F mutant. Upper panels show the representative antibody (anti-5mC or 6 mA) dot blot for the purified Chlamydomonas reinhardtii genomic DNA; lower panels show the methylene blue staining to validate the equal loading amount of DNA.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Figs. 1–17.

Reporting Summary

Supplementary Data

Source data for the supplementary figures.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blots and gels.

Source Data Extended Data Fig. 9

Unprocessed western blots and gels.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mu, Y., Zhang, L., Hu, J. et al. A fungal dioxygenase CcTet serves as a eukaryotic 6mA demethylase on duplex DNA. Nat Chem Biol 18, 733–741 (2022). https://doi.org/10.1038/s41589-022-01041-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-022-01041-3

Further reading

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