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