Characterization of demethylating DNA glycosylase ROS1 from Nicotiana tabacum L.

One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of 5-methylcytosine (mC), which is further recognized by regulatory proteins. In mammals, methylation mainly occurs in CG dinucleotides, while in plants it targets CG, CHG, and CHH sequences (H is any base but G). Correct maintenance of the DNA methylation status is based on the balance of methylation, passive demethylation, and active demethylation. While in mammals active demethylation is based on targeted regulated damage to mC in DNA followed by the action of repair enzymes, demethylation in plants is performed by specialized DNA glycosylases that hydrolyze the N-glycosidic bond of mC nucleotides. The genome of the model plant Arabidopsis thaliana encodes four paralogous proteins, two of which, DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1), possess 5-methylcytosine-DNA glycosylase activity and are necessary for the regulation of development, response to infections and abiotic stress and silencing of transgenes and mobile elements. Homologues of DME and ROS1 are present in all plant groups; however, outside A. thaliana, they are poorly studied. Here we report the properties of a recombinant fragment of the ROS1 protein from Nicotiana tabacum (NtROS1), which contains all main structural domains required for catalytic activity. Using homologous modeling, we have constructed a structural model of NtROS1, which revealed folding characteristic of DNA glycosylases of the helix– hairpin–helix structural superfamily. The recombinant NtROS1 protein was able to remove mC bases from DNA, and the enzyme activity was barely affected by the methylation status of CG dinucleotides in the opposite strand. The enzyme removed 5-hydroxymethylcytosine (hmC) from DNA with a lower efficiency, showing minimal activity in the presence of mC in the opposite strand. Expression of the NtROS1 gene in cultured human cells resulted in a global decrease in the level of genomic DNA methylation. In general, it can be said that the NtROS1 protein and other homologues of DME and ROS1 represent a promising scaffold for engineering enzymes to analyze the status of epigenetic methylation and to control gene activity.

1. Agius F., Kapoor A., Zhu J.-K. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc. Natl. Acad. Sci. USA. 2006;103(31):11796-11801. DOI 10.1073/pnas.0603563103.

2. Ballestar E., Wolffe A.P. Methyl-CpG-binding proteins. Targeting specific gene repression. Eur. J. Biochem. 2001;268(1):1-6. DOI 10.1046/j.1432-1327.2001.01869.x.

3. Baubec T., Ivánek R., Lienert F., Schübeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell. 2013;153(2):480-492. DOI 10.1016/j.cell.2013.03.011.

4. Bochtler M., Kolano A., Xu G.-L. DNA demethylation pathways: additional players and regulators. Bioessays. 2017;39(1):1-13. DOI 10.1002/bies.201600178.

5. Branco M.R., Ficz G., Reik W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat. Rev. Genet. 2011;13(1):7-13. DOI 10.1038/nrg3080.

6. Choi C.-S., Sano H. Identification of tobacco genes encoding proteins possessing removal activity of 5-methylcytosines from intact tobacco DNA. Plant Biotechnol. 2007;24(3):339-344. DOI 10.5511/plantbiotechnology.24.339.

7. Choi W.L., Mok Y.G., Huh J.H. Application of 5-methylcytosine DNA glycosylase to the quantitative analysis of DNA methylation. Int. J. Mol. Sci. 2021;22(3):1072. DOI 10.3390/ijms22031072.

8. Choi Y., Gehring M., Johnson L., Hannon M., Harada J.J., Goldberg R.B., Jacobsen S.E., Fischer R.L. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell. 2002;110(1):33-42. DOI 10.1016/s0092-8674(02)00807-3.

9. Cléry A., Blatter M., Allain F.H.-T. RNA recognition motifs: boring? Not quite. Curr. Opin. Struct. Biol. 2008;18(3):290-298. DOI 10.1016/j.sbi.2008.04.002.

10. Devesa-Guerra I., Morales-Ruiz T., Pérez-Roldán J., Parrilla-Doblas J.T., Dorado-León M., García-Ortiz M.V., Ariza R.R., RoldánArjona T. DNA methylation editing by CRISPR-guided excision of 5-methylcytosine. J. Mol. Biol. 2020;432(7):2204-2216. DOI 10.1016/j.jmb.2020.02.007.

11. Fedorova O.S., Kuznetsov N.A., Koval V.V., Knorre D.G. Conformational dynamics and pre-steady-state kinetics of DNA glycosylases. Biochemistry (Moscow). 2010;75(10):1225-1239. DOI 10.1134/S0006297910100044.

12. Gong Z., Morales-Ruiz T., Ariza R.R., Roldán-Arjona T., David L., Zhu J.-K. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell. 2002;111(6): 803-814. DOI 10.1016/s0092-8674(02)01133-9.

13. Gruber D.R., Toner J.J., Miears H.L., Shernyukov A.V., Kiryutin A.S., Lomzov A.A., Endutkin A.V., Grin I.R., Petrova D.V., Kupryushkin M.S., Yurkovskaya A.V., Johnson E., Okon M., Bagryanskaya E.G., Zharkov D.O., Smirnov S.L. Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics, and enzymatic demethylation. Nucleic Acids Res. 2018;46(20):10827- 10839. DOI 10.1093/nar/gky893.

14. Hong S., Hashimoto H., Kow Y.W., Zhang X., Cheng X. The carboxyterminal domain of ROS1 is essential for 5-methylcytosine DNA glycosylase activity. J. Mol. Biol. 2014;426(22):3703-3712. DOI 10.1016/j.jmb.2014.09.010.

15. Iyer L.M., Abhiman S., Aravind L. Natural history of eukaryotic DNA methylation systems. Prog. Mol. Biol. Transl. Sci. 2011;101:25-104. DOI 10.1016/B978-0-12-387685-0.00002-0.

16. Jang H., Shin H., Eichman B.F., Huh J.H. Excision of 5-hydroxymethylcytosine by DEMETER family DNA glycosylases. Biochem. Biophys. Res. Commun. 2014;446(4):1067-1072. DOI 10.1016/j.bbrc.2014.03.060.

17. Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., ŽídekA., PotapenkoA., BridglandA., Meyer C., Kohl S.A.A., Ballard A.J., Cowie A., Romera-Paredes B., Nikolov S., Jain R., Adler J., Back T., Petersen S., Reiman D., Clancy E., Zielinski M., Steinegger M., Pacholska M., Berghammer T., Bodenstein S., Silver D., Vinyals O., Senior A.W., Kavukcuoglu K., Kohli P., Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583-589. DOI 10.1038/s41586-021-03819-2.

18. Kapazoglou A., Drosou V., Argiriou A., Tsaftaris A.S. The study of a barley epigenetic regulator, HvDME, in seed development and under drought. BMC Plant Biol. 2013;13:172. DOI 10.1186/1471-2229-13-172.

19. Kapoor A., Agarwal M., Andreucci A., Zheng X., Gong Z., Hasegawa P.M., Bressan R.A., Zhu J.-K. Mutations in a conserved replication protein suppress transcriptional gene silencing in a DNAmethylation-independent manner in Arabidopsis. Curr. Biol. 2005; 15(21):1912-1918. DOI 10.1016/j.cub.2005.09.013.

20. Le T.-N., Schumann U., Smith N.A., Tiwari S., Au P.C.K., Zhu Q.-H., Taylor J.M., Kazan K., Llewellyn D.J., Zhang R., Dennis E.S., Wang M.-B. DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis. Genome Biol. 2014;15(9):458. DOI 10.1186/s13059-014-0458-3.

21. Lee T.-F., Zhai J., Meyers B.C. Conservation and divergence in eukaryotic DNA methylation. Proc. Natl. Acad. Sci. USA. 2010;107(20): 9027-9028. DOI 10.1073/pnas.1005440107.

22. Li X., Qian W., Zhao Y., Wang C., Shen J., Zhu J.-K., Gong Z. Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis. Proc. Natl. Acad. Sci. USA. 2012;109(28):11425-11430. DOI 10.1073/pnas.1208557109.

23. Li Y., Kumar S., Qian W. Active DNA demethylation: mechanism and role in plant development. Plant Cell Rep. 2018;37(1):77-85. DOI 10.1007/s00299-017-2215-z.

24. Liu R., How-Kit A., Stammitti L., Teyssier E., Rolin D., Mortain-Bertrand A., Halle S., Liu M., Kong J., Wu C., Degraeve-Guibault C., Chapman N.H., Maucourt M., Hodgman T.C., Tost J., Bouzayen M., Hong Y., Seymour G.B., Giovannoni J.J., Gallusci P. A DEMETERlike DNA demethylase governs tomato fruit ripening. Proc. Natl. Acad. Sci. USA. 2015;112(34):10804-10809. DOI 10.1073/pnas.1503362112.

25. Morales-Ruiz T., Ortega-Galisteo A.P., Ponferrada-Marín M.I., Martínez-Macías M.I., Ariza R.R., Roldán-Arjona T. DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. Proc. Natl. Acad. Sci. USA. 2006;103(18):6853-6858. DOI 10.1073/pnas.0601109103.

26. Ono A., Yamaguchi K., Fukada-Tanaka S., Terada R., Mitsui T., Iida S. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny. Plant J. 2012;71(4):564-574. DOI 10.1111/j.1365-313X.2012.05009.x.

27. Ortega-Galisteo A.P., Morales-Ruiz T., Ariza R.R., Roldán-Arjona T. Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks. Plant Mol. Biol. 2008;67(6):671-681. DOI 10.1007/s11103-008-9346-0.

28. Parrilla-Doblas J.T., Roldán-Arjona T., Ariza R.R., Córdoba-Cañero D. Active DNA demethylation in plants. Int. J. Mol. Sci. 2019; 20(19):4683. DOI 10.3390/ijms20194683.

29. Pastor W.A., Aravind L., Rao A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nat. Rev. Mol. Cell Biol. 2013;14(6):341-356. DOI 10.1038/nrm3589.

30. Pastor W.A., Pape U.J., Huang Y., Henderson H.R., Lister R., Ko M., McLoughlin E.M., Brudno Y., Mahapatra S., Kapranov P., Tahiliani M., Daley G.Q., Liu X.S., Ecker J.R., Milos P.M., Agarwal S., Rao A. Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells. Nature. 2011;473(7347):394-397. DOI 10.1038/nature10102.

31. Penterman J., Uzawa R., Fischer R.L. Genetic interactions between DNA demethylation and methylation in Arabidopsis. Plant Physiol. 2007;145(4):1549-1557. DOI 10.1104/pp.107.107730.

32. Ponferrada-Marín M.I., Parrilla-Doblas J.T., Roldán-Arjona T., Ariza R.R. A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine. Nucleic Acids Res. 2011;39(4): 1473-1484. DOI 10.1093/nar/gkq982.

33. Ponferrada-Marín M.I., Roldán-Arjona T., Ariza R.R. ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion. Nucleic Acids Res. 2009;37(13):4264-4274. DOI 10.1093/nar/gkp390.

34. Porello S.L., Leyes A.E., David S.S. Single-turnover and pre-steadystate kinetics of the reaction of the adenine glycosylase MutY with mismatch-containing DNA substrates. Biochemistry. 1998;37(42): 14756-14764. DOI 10.1021/bi981594+.

35. Roldán-Arjona T., Ariza R.R., Córdoba-Cañero D. DNA base excision repair in plants: an unfolding story with familiar and novel characters. Front. Plant Sci. 2019;10:1055. DOI 10.3389/fpls.2019.01055.

36. Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., Heer F.T., de Beer T.A.P., Rempfer C., Bordoli L., Lepore R., Schwede T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296- W303. DOI 10.1093/nar/gky427.

37. Wen S., Wen N., Pang J., Langen G., Brew-Appiah R.A.T., Mejias J.H., Osorio C., Yang M., Gemini R., Moehs C.P., Zemetra R.S., Kogel K.-H., Liu B., Wang X., von Wettstein D., Rustgi S. Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health. Proc. Natl. Acad. Sci. USA. 2012;109(50):20543-20548. DOI 10.1073/pnas.1217927109.

38. Wu X., Zhang Y. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat. Rev. Genet. 2017;18(9):517-534. DOI 10.1038/nrg.2017.33.

39. Yamamuro C., Miki D., Zheng Z., Ma J., Wang J., Yang Z., Dong J., Zhu J.-K. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation. Nat. Commun. 2014;5:4062. DOI 10.1038/ncomms5062.

40. Yu M., Hon G.C., Szulwach K.E., Song C.-X., Zhang L., Kim A., Li X., Dai Q., Shen Y., Park B., Min J.-H., Jin P., Ren B., He C. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 2012;149(6):1368-1380. DOI 10.1016/j.cell.2012.04.027.

41. Zahid O.K., Zhao B.S., He C., Hall A.R. Quantifying mammalian genomic DNA hydroxymethylcytosine content using solid-state nanopores. Sci. Rep. 2016;6:29565. DOI 10.1038/srep29565. Zemach A., Zilberman D. Evolution of eukaryotic DNA methylation and the pursuit of safer sex. Curr. Biol. 2010;20(17):R780-R785. DOI 10.1016/j.cub.2010.07.007.

42. Zharkov D.O. Base excision DNA repair. Cell. Mol. Life Sci. 2008; 65(10):1544-1565. DOI 10.1007/s00018-008-7543-2.

43. Zhu J., Kapoor A., Sridhar V.V., Agius F., Zhu J.-K. The DNA glycosylase/lyase ROS1 functions in pruning DNA methylation patterns in Arabidopsis. Curr. Biol. 2007;17(1):54-59. DOI 10.1016/j.cub.2006.10.059.