Mapping the epigenetic modifications of DNA and RNA

Abstract

Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

Key words： DNA modification; DNA methylation; RNA modification; epitranscriptomics; epigenetics; long read sequencing

References

1 Adey A, Shendure J (2012) Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Res 22:1139–1143

2 Akichika S, Hirano S, Shichino Y, Suzuki T, Nishimasu H, Ishitani R, Sugita A, Hirose Y, Iwasaki S, Nureki O (2019) Cap-specific terminal N (6)-methylation of RNA by an RNA polymerase IIassociated methyltransferase. Science (New York, NY).https://doi.org/10.1126/science.aav0080

3 Alarcon CR, Goodarzi H, Lee H, Liu X, Tavazoie S,Tavazoie SF (2015) HNRNPA2B1 is a mediator of m(6)A-dependent nuclear RNA processing events. Cell 162:1299–1308

4 Arango D,Sturgill D, Alhusaini N, Dillman AA, Sweet TJ, Hanson G, Hosogane M, Sinclair WR, Nanan KK, Mandler MD (2018) Acetylation of cytidine in mRNA promotes translation efficiency. Cell 175:1872–1886.e1824

5 Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018) Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics. Nucleic Acids Res 46:2159–2168

6 Bartosovic M, Molares HC, Gregorova P, Hrossova D, Kudla G, Vanacova S (2017) N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3′-end processing. Nucleic Acids Res 45:11356–11370

7 Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, de Crecy-Lagard V,Ross R, Limbach PA, Kotter A (2018) MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res 46:D303–d307

8 Bokar JA, Rath-Shambaugh ME, Ludwiczak R, Narayan P, Rottman F (1994) Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex. J Biol Chem 269:17697–17704

9 Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W,Balasubramanian S (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science (New York, NY) 336:934–937

10 Booth MJ, Ost TW, Beraldi D, Bell NM, Branco MR, Reik W, Balasubramanian S (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8:1841–1851

11 Booth MJ, Marsico G, Bachman M, Beraldi D, Balasubramanian S (2014) Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem 6:435–440

12 Booth MJ, Raiber EA, Balasubramanian S (2015) Chemical methods for decoding cytosine modifications in DNA. Chem Rev 115:2240–2254

13 Boulias K, Toczydlowska-Socha D,Hawley BR, Liberman N, Takashima K, Zaccara S, Guez T, Vasseur JJ, Debart F, Aravind L (2019) Identification of the m(6)Am methyltransferase PCIF1 reveals the location and functions of m(6)Am in the transcriptome. Mol Cell 75(3):631.e8–643.e8

14 Cao G, Li HB (2016) Recent advances in dynamic m6A RNA modification. Open Biol 6:160003

15 Carlile TM, Rojas-Duran MF, Zinshteyn B, Shin H, Bartoli KM, Gilbert WV (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515:143–146

16 Chen K, Lu Z, Wang X, Fu Y, Luo GZ, Liu N, Han D, Dominissini D, Dai Q, Pan T(2015) High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinking-assisted m(6) A sequencing. Angew Chem Int Ed Engl 54:1587–1590

17 Chen X, Li A, Sun BF, Yang Y, Han YN, Yuan X, Chen RX, Wei WS, Liu Y, Gao CC(2019) 5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs. Nat Cell Biol 21(8):978–990

18 Chu JM, Ye TT, Ma CJ, Lan MD, Liu T, Yuan BF, Feng YQ (2018) Existence of Internal N7-methylguanosine modification in mRNA determined by differential enzyme treatment coupled with mass spectrometry analysis. ACS Chem Biol 13:3243–3250

19 Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G (2017) Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq). Nat Protoc 12:534–547

20 Clarke J, Wu HC, Jayasinghe L,Patel A, Reid S, Bayley H (2009) Continuous base identification for single-molecule nanopore DNA sequencing. Nat Nanotechnol 4:265–270

21 Courtney DG, Kennedy EM, Dumm RE, Bogerd HP, Tsai K, Heaton NS, Cullen BR (2017) Epitranscriptomic enhancement of influenza A virus gene expression and replication. Cell Host Microbe 22:377–386.e375

22 Cui Q, Shi H, Ye P,Li L, Qu Q, Sun G,Sun G, Lu Z, Huang Y, Yang CG (2017a) m(6)A RNA methylation regulates the selfrenewal and tumorigenesis of glioblastoma stem cells. Cell Rep 18:2622–2634

23 Cui X, Liang Z, Shen L, Zhang Q, Bao S,Geng Y,Zhang B, Leo V, Vardy LA, Lu T (2017b) 5-Methylcytosine RNA methylation in Arabidopsis thaliana. Molecular plant 10:1387–1399

24 Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S (2016) RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science (New York, NY) 351:282–285

25 Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206

26 Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530:441–446

27 Dong C, Niu L, Song W, Xiong X, Zhang X, Zhang Z, Yang Y, Yi F, Zhan J, Zhang H (2016) tRNA modification profiles of the fast-proliferating cancer cells. Biochem Biophys Res Commun 476:340–345

28 Douvlataniotis K, Bensberg M, Lentini A, Gylemo B, Nestor CE (2020) No evidence for DNA N (6)-methyladenine in mammals. Sci Adv 6:eaay3335

29 Dubin DT, Taylor RH (1975) The methylation state of poly A-containing messenger RNA from cultured hamster cells. Nucleic Acids Res 2:1653–1668

30 Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R (2013) Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet 9:e1003602

31 Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, Marques CJ, Andrews S, Reik W (2011) Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473:398–402

32 Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7:461–465

33 Frye M, Jaffrey SR, Pan T, Rechavi G,Suzuki T (2016) RNA modifications: what have we learned and where are we headed? Nat Rev Genet 17:365–372

34 Fu Y, Jia G, Pang X, Wang RN, Wang X, Li CJ, Smemo S, Dai Q, Bailey KA, Nobrega MA (2013) FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat Commun 4:1798

35 Fu L, Guerrero CR, Zhong N, Amato NJ, Liu Y, Liu S, Cai Q, Ji D, Jin SG, Niedernhofer LJ (2014) Tet-mediated formation of 5-hydroxymethylcytosine in RNA. J Am Chem Soc 136:11582–11585

36 Fu Y, Luo GZ, Chen K, Deng X, Yu M, Han D, Hao Z, Liu J, Lu X, Dore LC (2015) N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161:879–892

37 Garalde DR, Snell EA, Jachimowicz D, Sipos B, Lloyd JH, Bruce M, Pantic N, Admassu T, James P, Warland A (2018) Highly parallel direct RNA sequencing on an array of nanopores. Nat Methods 15:201–206

38 Garcia-Campos MA, Edelheit S, Toth U, Safra M, Shachar R, Viukov S, Winkler R, Nir R, Lasman L, Brandis A (2019) Deciphering the “m(6)A code” via antibody-independent quantitative profiling. Cell 178:731–747.e716

39 Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS (2015) Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science (New York, NY) 347:1002–1006

40 Gilbert WV, Bell TA, Schaening C (2016) Messenger RNA modifications: form, distribution, and function. Science (New York, NY) 352:1408–1412

41 Gokhale NS, McIntyre AB, McFadden MJ, Roder AE, Kennedy EM, Gandara JA, Hopcraft SE, Quicke KM, Vazquez C, Willer J (2016) N6-methyladenosine in flaviviridae viral RNA genomes regulates infection. Cell Host Microbe 20:654–665

42 Greenberg MVC, Bourc’his D (2019) The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 20:590–607

43 Greer EL, Blanco MA, Gu L, Sendinc E, Liu J, Aristizabal-Corrales D, Hsu CH, Aravind L, He C, Shi Y (2015) DNA methylation on N6-adenine in C. elegans. Cell 161:868–878

44 Guo F, Li X, Liang D, Li T, Zhu P, Guo H, Wu X, Wen L,Gu TP, Hu B (2014) Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote. Cell Stem Cell 15:447–459

45 Hao Z, Wu T, Cui X, Zhu P, Tan C, Dou X, Hsu KW, Lin YT, Peng PH, Zhang LS (2020) N(6)-deoxyadenosine methylation in mammalian mitochondrial DNA. Mol Cell 78(3):382–395.e8

46 Harper JE, Miceli SM, Roberts RJ, Manley JL (1990) Sequence specificity of the human mRNA N6-adenosine methylase in vitro. Nucleic Acids Res 18:5735–5741

47 He C (2010) Grand challenge commentary: RNA epigenetics? Nat Chem Biol 6:863–865

48 He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q, Ding J, Jia Y, Chen Z, Li L (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333:1303–1307

49 Hussain S, Aleksic J, Blanco S, Dietmann S, Frye M (2013a) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol 14:215

50 Hussain S, Sajini AA, Blanco S, Dietmann S, Lombard P, Sugimoto Y, Paramor M, Gleeson JG, Odom DT, Ule J (2013b) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep 4:255–261

51 Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA, He C, Zhang Y (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science (New York, NY) 333:1300–1303

52 Jain M, Olsen HE, Paten B, Akeson M (2016) The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol 17:239

53 Jain M, Koren S, Miga KH, Quick J, Rand AC, Sasani TA, Tyson JR, Beggs AD, Dilthey AT, Fiddes IT (2018) Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol 36:338–345

54 Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG (2011) N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 7:885–887

55 Karijolich J, Yi C, Yu YT (2015) Transcriptome-wide dynamics of RNA pseudouridylation. Nat Rev Mol Cell Biol 16:581–585

56 Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, Haripal B, Zucker-Scharff I, Moore MJ, Park CY (2015) A majority of m6A residues are in the last exons, allowing the potential for 3’ UTR regulation. Genes Dev 29:2037–2053

57 Ke S, Pandya-Jones A, Saito Y, Fak JJ, Vagbo CB, Geula S, Hanna JH, Black DL, Darnell JE Jr, Darnell RB (2017) m(6)A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev 31:990–1006

58 Kennedy EM, Bogerd HP, Kornepati AV, Kang D, Ghoshal D, Marshall JB, Poling BC, Tsai K, Gokhale NS, Horner SM (2016) Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression. Cell Host Microbe 19:675–685

59 Khoddami V, Cairns BR (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat Biotechnol 31:458–464

60 Khoddami V, Yerra A, Mosbruger TL, Fleming AM, Burrows CJ, Cairns BR (2019) Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution. Proc Natl Acad Sci USA 116:6784–6789

61 Kobayashi H, Kono T (2012) DNA methylation analysis of germ cells by using bisulfite-based sequencing methods. Methods Mol Biol (Clifton, NJ) 825:223–235

62 Kobayashi H, Sakurai T, Miura F,Imai M, Mochiduki K, Yanagisawa E,Sakashita A, Wakai T, Suzuki Y, Ito T (2013) Highresolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome Res 23:616–627

63 Koch A, Joosten SC, Feng Z, de Ruijter TC, Draht MX, Melotte V, Smits KM, Veeck J, Herman JG, Van Neste L (2018) Author correction: analysis of DNA methylation in cancer: location revisited. Nat Rev Clin Oncol 15:467

64 Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930

65 Lei Z, Yi C (2017) A radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection. Angew Chem Int Ed Engl 56:14878–14882

66 Lentini A, Lagerwall C, Vikingsson S, Mjoseng HK, Douvlataniotis K, Vogt H, Green H, Meehan RR, Benson M, Nestor CE (2018) A reassessment of DNA-immunoprecipitation-based genomic profiling. Nat Methods 15:499–504

67 Li E, Zhang Y (2014) DNA methylation in mammals. Cold Spring Harb Perspect Biol 6:a019133

68 Li WW, Gong L, Bayley H (2013) Single-molecule detection of 5-hydroxymethylcytosine in DNA through chemical modification and nanopore analysis. Angew Chem Int Ed Engl 52:4350–4355

69 Li X, Zhu P, Ma S, Song J, Bai J, Sun F, Yi C (2015) Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat Chem Biol 11:592–597

70 Li X, Xiong X, Wang K, Wang L, Shu X, Ma S, Yi C (2016a) Transcriptome-wide mapping reveals reversible and dynamic N (1)-methyladenosine methylome. Nat Chem Biol 12:311–316

71 Li X, Xiong X, Yi C (2016b) Epitranscriptome sequencing technologies: decoding RNA modifications. Nat Methods 14:23–31

72 Li W, Zhang X, Lu X, You L, Song Y, Luo Z, Zhang J, Nie J, Zheng W, Xu D (2017a) 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res 27:1243–1257

73 Li X, Xiong X, Zhang M, Wang K, Chen Y, Zhou J, Mao Y, Lv J, Yi D, Chen XW(2017b) Base-resolution mapping reveals distinct m(1)A methylome in nuclear- and mitochondrial-encoded transcripts. Mol Cell 68:993–1005.e1009

74 Li Z, Weng H, Su R, Weng X, Zuo Z,Li C, Huang H, Nachtergaele S, Dong L, Hu C (2017c) FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase. Cancer Cell 31:127–141

75 Li QY, Xie NB, Xiong J, Yuan BF, Feng YQ (2018) Single-nucleotide resolution analysis of 5-hydroxymethylcytosine in DNA by enzyme-mediated deamination in combination with sequencing. Anal Chem 90:14622–14628

76 Lichinchi G, Gao S, Saletore Y, Gonzalez GM (2016a) Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nat Microbiol 1:16011

77 Lichinchi G, Zhao BS, Wu Y, Lu Z, Qin Y, He C, Rana TM (2016b) Dynamics of human and viral RNA methylation during Zika virus infection. Cell Host Microbe 20:666–673

78 Lin S, Liu Q, Lelyveld VS, Choe J, Szostak JW, Gregory RI (2018) Mettl1/Wdr4-mediated m(7)G tRNA methylome is required for normal mRNA translation and embryonic stem cell self-renewal and differentiation. Mol Cell 71:244–255.e245

79 Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767–772

80 Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 10:93–95

81 Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T (2015) N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518:560–564

82 Liu F, Clark W, Luo G, Wang X, Fu Y,Wei J,Wang X, Hao Z, Dai Q, Zheng G (2016) ALKBH1-mediated tRNA demethylation regulates translation. Cell 167(3):816–828

83 Liu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T (2017) N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Res 45:6051–6063

84 Liu H, Begik O, Lucas MC, Ramirez JM, Mason CE, Wiener D, Schwartz S, Mattick JS, Smith MA, Novoa EM (2019a) Accurate detection of m6A RNA modifications in native RNA sequences. Nat Commun 10:4079

85 Liu J, Harada BT, He C (2019b) Regulation of gene expression by N (6)-methyladenosine in cancer. Trends Cell Biol 29(6):487–489

86 Liu J, Li K, Cai J, Zhang M, Zhang X, Xiong X, Meng H, Xu X, Huang Z, Peng J(2019c) Landscape and regulation of m(6)A and m (6)Am methylome across human and mouse tissues. Mol Cell 77 (2):426.e6–440.e6

87 Liu Y, You Y, Lu Z, Yang J, Li P,Liu L, Xu H, Niu Y, Cao X (2019d) N (6)-methyladenosine RNA modification-mediated cellular metabolism rewiring inhibits viral replication. Science (New York, NY) 365:1171–1176

88 Liu Y, Cheng J, Siejka-Zielinska P, Weldon C, Roberts H, Lopopolo M, Magri A, D’Arienzo V, Harris JM, McKeating JA (2020) Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS. Genome Biol 21:54

89 Louloupi A, Ntini E, Conrad T, Orom UAV (2018) Transient N-6-methyladenosine transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency. Cell Rep 23:3429–3437

90 Lovejoy AF, Riordan DP, Brown PO (2014) Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PLoS ONE 9:e110799

91 Lu X, Song CX, Szulwach K, Wang Z, Weidenbacher P, Jin P, He C (2013) Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc 135:9315–9317

92 Lu X, Han D, Zhao BS, Song CX, Zhang LS, Dore LC, He C(2015) Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics. Cell Res 25:386–389

93 Lu W, Tirumuru N, St Gelais C, Koneru PC, Liu C, Kvaratskhelia M, He C, Wu L (2018) N(6)-methyladenosine-binding proteins suppress HIV-1 infectivity and viral production. J Biol Chem 293:12992–13005

94 Malbec L, Zhang T, Chen YS, Zhang Y, Sun BF, Shi BY, Zhao YL, Yang Y,Yang YG (2019) Dynamic methylome of internal mRNA N (7)-methylguanosine and its regulatory role in translation. Cell Res.https://doi.org/10.1016/j.molp.2019.12.007

95 Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, Linder B, Pickering BF, Vasseur JJ, Chen Q (2017) Reversible methylation of m(6)Am in the 5′ cap controls mRNA stability. Nature 541:371–375

96 Meyer KD (2019) DART-seq: an antibody-free method for global m (6)A detection. Nat Methods 16:1275–1280

97 Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell 149:1635–1646

98 Molinie B, Wang J, Lim KS, Hillebrand R, Lu ZX (2016) m(6)A-LAICseq reveals the census and complexity of the m(6)A epitranscriptome. Nat Methods 13:692–698

99 Musheev MU, Baumgärtner A, Krebs L, Niehrs C (2020) The origin of genomic N6-methyl-deoxyadenosine in mammalian cells. Nat Chem Biol.https://doi.org/10.1038/s41589-020-0504-2

100 Neri F,Incarnato D, Krepelova A, Rapelli S, Anselmi F,Parlato C, Medana C, Dal Bello F, Oliviero S (2015) Single-base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals promoter DNA methylation dynamics. Cell Rep 10:674–683

101 O’Brown ZK, Boulias K, Wang J, Wang SY, O’Brown NM, Hao Z, Shibuya H, Fady PE, Shi Y, He C (2019) Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA. BMC Genom 20:445

102 Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M, Jaffrey SR (2016) m(6)A RNA methylation promotes XISTmediated transcriptional repression. Nature 537:369–373

103 Pendleton KE, Chen B, Liu K, Hunter OV, Xie Y, Tu BP, Conrad NK (2017) The U6 snRNA m(6)A methyltransferase METTL16 regulates SAM synthetase intron retention. Cell 169:824–835. e814

104 Pfaffeneder T, Hackner B, Truss M, Munzel M, Muller M, Deiml CA, Hagemeier C, Carell T (2011) The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem 50:7008–7012

105 Ping XL, Sun BF, Wang L, Xiao W, Yang X,Wang WJ, Adhikari S, Shi Y, Lv Y, Chen YS (2014) Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res 24:177–189

106 Raiber E-A, Hardisty R, van Delft P,Balasubramanian S (2017) Mapping and elucidating the function of modified bases in DNA. Nat Rev Chem 1:0069

107 Rand AC, Jain M, Eizenga JM, Musselman-Brown A, Olsen HE, Akeson M, Paten B (2017) Mapping DNA methylation with highthroughput nanopore sequencing. Nat Methods 14:411–413

108 Roundtree IA, Evans ME, Pan T,He C (2017) Dynamic RNA modifications in gene expression regulation. Cell 169:1187–1200

109 Safra M, Sas-Chen A, Nir R, Winkler R, Nachshon A, Bar-Yaacov D, Erlacher M, Rossmanith W, Stern-Ginossar N, Schwartz S (2017) The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution. Nature 551:251–255

110 Schaefer M, Pollex T, Hanna K, Lyko F (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res 37:e12

111 Schutsky EK, DeNizio JE, Hu P, Liu MY, Nabel CS, Fabyanic EB, Hwang Y, Bushman FD, Wu H, Kohli RM (2018) Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nat Biotechnol.https://doi.org/10.1038/nbt.4204

112 Schwartz S, Agarwala SD, Mumbach MR, Jovanovic M, Mertins P, Shishkin A, Tabach Y,Mikkelsen TS, Satija R, Ruvkun G (2013) High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell 155:1409–1421

113 Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, Leon-Ricardo BX, Engreitz JM, Guttman M, Satija R, Lander ES (2014a) Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159:148–162

114 Schwartz S, Mumbach Maxwell R, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D (2014b) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep 8:284–296

115 Sendinc E, Valle-Garcia D, Dhall A, Chen H, Henriques T, Navarrete-Perea J, Sheng W, Gygi SP, Adelman K, Shi Y (2019) PCIF1 catalyzes m6Am mRNA methylation to regulate gene expression. Mol Cell 75(3):620.e9–630.e9

116 Shafik A, Schumann U, Evers M, Sibbritt T, Preiss T (2016) The emerging epitranscriptomics of long noncoding RNAs. Biochim Biophys Acta 1859:59–70

117 Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y (2013) Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153:692–706

118 Shen Q, Zhang Q, Shi Y,Shi Q, Jiang Y, Gu Y, Li Z, Li X, Zhao K, Wang C (2018) Tet2 promotes pathogen infection-induced myelopoiesis through mRNA oxidation. Nature 554:123–127

119 Shirane K, Toh H, Kobayashi H, Miura F, Chiba H, Ito T, Kono T, Sasaki H (2013) Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet 9:e1003439

120 Shu X, Cao J, Cheng M, Xiang S, Gao M, Li T, Ying X, Wang F, Yue Y, Lu Z (2020) A metabolic labeling method detects m6A transcriptome-wide at single base resolution. Nat Chem Biol. https://doi.org/10.1038/s41589-020-0526-9

121 Simpson JT, Workman RE, Zuzarte PC, David M, Dursi LJ, Timp W (2017) Detecting DNA cytosine methylation using nanopore sequencing. Nat Methods 14:407–410

122 Smallwood SA, Lee HJ, Angermueller C,Krueger F, Saadeh H, Peat J, Andrews SR, Stegle O, Reik W, Kelsey G (2014) Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 11:817–820

123 Smith AM, Jain M, Mulroney L, Garalde DR, Akeson M (2019) Reading canonical and modified nucleobases in 16S ribosomal RNA using nanopore native RNA sequencing. PLoS ONE 14: e0216709

124 Song J, Yi C (2019) Reading chemical modifications in the transcriptome. J Mol Biol.https://doi.org/10.1016/j.jmb.2019.10.006

125 Song CX, Clark TA, Lu XY, Kislyuk A, Dai Q, Turner SW, He C, Korlach J (2011a) Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat Methods 9:75–77

126 Song CX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, Li Y, Chen CH, Zhang W, Jian X (2011b) Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat Biotechnol 29:68–72

127 Song CX, Szulwach KE, Dai Q, Fu Y, Mao SQ, Lin L, Street C, Li Y, Poidevin M, Wu H (2013) Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153:678–691

128 Song CX, Yin S, Ma L, Wheeler A, Chen Y, Zhang Y, Liu B, Xiong J, Zhang W, Hu J (2017) 5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res 27:1231–1242

129 Song J, Zhuang Y, Zhu C,Meng H, Lu B, Xie B, Peng J, Li M, Yi C (2019) Differential roles of human PUS10 in miRNA processing and tRNA pseudouridylation. Nat Chem Biol 16(2):160–169

130 Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res 40:5023–5033

131 Stroud H, Feng S, Morey Kinney S, Pradhan S, Jacobsen SE (2011) 5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells. Genome Biol 12:R54

132 Su R, Dong L, Li C, Nachtergaele S,Wunderlich M, Qing Y, Deng X, Wang Y, Weng X, Hu C (2018) R-2HG exhibits anti-tumor activity by targeting FTO/m(6)A/MYC/CEBPA signaling. Cell 172:90–105.e123

133 Sun H, Zhang M, Li K, Bai D, Yi C (2019) Cap-specific, terminal N(6)-methylation by a mammalian m(6)Am methyltransferase. Cell Res 29:80–82

134 Tahiliani M, Koh KP, Shen Y,Pastor WA, Bandukwala H, Brudno Y, Agarwal S, Iyer LM, Liu DR, Aravind L (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935

135 Tanaka K, Okamoto A (2007) Degradation of DNA by bisulfite treatment. Bioorg Med Chem Lett 17:1912–1915

136 Tirumuru N, Zhao BS, Lu W, Lu Z, He C, Wu L (2016) N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. Elife 5:e15528

137 Tsai K, Courtney DG, Cullen BR (2018) Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication. PLoS Pathog 14:e1006919

138 Ueda Y, Ooshio I, Fusamae Y, Kitae K, Kawaguchi M, Jingushi K, Hase H, Harada K, Hirata K, Tsujikawa K (2017) AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells. Sci Rep 7:42271

139 Unnikrishnan A, Freeman WM, Jackson J, Wren JD, Porter H, Richardson A (2019) The role of DNA methylation in epigenetics of aging. Pharmacol Therapeut 195:172–185

140 Vaisvila R, Ponnaluri VKC, Sun Z, Langhorst BW, Saleh L, Guan S, Dai N, Campbell MA, Sexton B, Marks K (2019) EM-seq: detection of DNA methylation at single base resolution from picograms of DNA. BioRxiv Dec 23:2019. https://doi.org/10.1101/2019.12.20.884692

141 Venkatesan BM, Bashir R (2011) Nanopore sensors for nucleic acid analysis. Nat Nanotechnol 6:615–624

142 Viehweger A, Krautwurst S, Lamkiewicz K, Madhugiri R, Ziebuhr J, Holzer M, Marz M (2019) Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis. Genome Res 29:1545–1554

143 Vilfan ID, Tsai YC, Clark TA, Wegener J, Dai Q, Yi C, Pan T, Turner SW, Korlach J (2013) Analysis of RNA base modification and structural rearrangement by single-molecule real-time detection of reverse transcription. J Nanobiotechnol 11:8

144 Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC (2014) N6- methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat Cell Biol 16:191–198

145 Wang L, Wen M, Cao X (2019) Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses. Science. https://doi.org/10.1126/science.aav0758

146 Wang Y, Xiao Y, Dong S,Yu Q,Jia G (2020) Antibody-free enzymeassisted chemical approach for detection of N6-methyladenosine. Nat Chem Biol.https://doi.org/10.1038/s41589-020-0525-x

147 Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37:853–862

148 Wei J, Liu F, Lu Z, Fei Q, Ai Y, He PC, Shi H,Cui X, Su R, Klungland A (2018) Differential m(6)A, m(6)Am, and m(1)A demethylation mediated by FTO in the cell nucleus and cytoplasm. Mol Cell 71:973–985.e975

149 Wen J, Lv R, Ma H, Shen H, He C, Wang J, Jiao F,Liu H, Yang P, Tan L (2018) Zc3h13 regulates nuclear RNA m(6)A methylation and mouse embryonic stem cell self-renewal. Mol Cell 69:1028–1038.e1026

150 Wenger AM, Peluso P, Rowell WJ, Chang PC, Hall RJ, Concepcion GT, Ebler J, Fungtammasan A, Kolesnikov A, Olson ND (2019) Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol 37:1155–1162

151 Wongsurawat T, Jenjaroenpun P, Wassenaar TM, Wadley TD, Wanchai V, Akel NS, Franco AT, Jennings ML, Ussery DW, Nookaew I (2018) Decoding the epitranscriptional landscape from native RNA sequences. bioRxiv. https://doi.org/10.1101/487819

152 Workman RE, Tang AD, Tang PS, Jain M, Tyson JR, Razaghi R, Zuzarte PC, Gilpatrick T, Payne A,Quick J (2019) Nanopore native RNA sequencing of a human poly(A) transcriptome. Nat Methods 16:1297–1305

153 Wu H, Wu X, Shen L, Zhang Y (2014) Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol 32:1231–1240

154 Wu H, Wu X, Zhang Y (2016) Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc 11:1081–1100

155 Xia B, Han D, Lu X, Sun Z, Zhou A, Yin Q, Zeng H, Liu M, Jiang X, Xie W (2015) Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods 12:1047–1050

156 Xiang Y, Laurent B, Hsu CH, Nachtergaele S, Lu Z, Sheng W, Xu C, Chen H, Ouyang J, Wang S (2017) RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response. Nature 543:573–576

157 Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, Sun HY, Li A, Ping XL, Lai WY (2016) Nuclear m(6)A reader YTHDC1 regulates mRNA splicing. Mol Cell 61:507–519

158 Xiao S, Cao S, Huang Q, Xia L, Deng M, Yang M, Jia G, Liu X, Shi J, Wang W (2019) The RNA N(6)-methyladenosine modification landscape of human fetal tissues. Nat Cell Biol 21:651–661

159 Xiong X, Li X, Wang K, Yi C (2018) Perspectives on topology of the human m(1)A methylome at single nucleotide resolution. RNA (New York, NY) 24:1437–1442

160 Xu L, Liu X, Sheng N, Oo KS, Liang J, Chionh YH, Xu J, Ye F, Gao YG, Dedon PC(2017) Three distinct 3-methylcytidine (m(3) C) methyltransferases modify tRNA and mRNA in mice and humans. J Biol Chem 292:14695–14703

161 Yamaguchi S, Hong K, Liu R, Shen L, Inoue A, Diep D, Zhang K, Zhang Y (2012) Tet1 controls meiosis by regulating meiotic gene expression. Nature 492:443–447

162 Yang X, Yang Y, Sun BF, Chen YS, Xu JW, Lai WY, Li A, Wang X, Bhattarai DP, Xiao W (2017) 5-methylcytosine promotes mRNA export- NSUN2 as the methyltransferase and ALYREF as an m(5)C reader. Cell Res 27:606–625

163 Yang Y, Wang L, Han X, Yang WL, Zhang M, Ma HL, Sun BF, Li A, Xia J, Chen J (2019) RNA 5-methylcytosine facilitates the maternal-to-zygotic transition by preventing maternal mRNA decay. Mol Cell 75:1188–1202.e1111

164 Yu M, Hon GC, Szulwach KE, Song CX, Jin P, Ren B, He C (2012a) Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat Protoc 7:2159–2170

165 Yu M, Hon GC, Szulwach KE, Song CX, Zhang L, Kim A, Li X, Dai Q, Shen Y, Park B(2012b) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149:1368–1380

166 Yu J, Chen M, Huang H, Zhu J, Song H, Zhu J, Park J, Ji SJ (2018) Dynamic m6A modification regulates local translation of mRNA in axons. Nucleic Acids Res 46:1412–1423

167 Yuan F, Bi Y, Siejka-Zielinska P, Zhou YL, Zhang XX, Song CX (2019) Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate. Chem Commun (Camb) 55:2328–2331

168 Yue Y, Liu J, Cui X, Cao J, Luo G, Zhang Z, Cheng T, Gao M, Shu X, Ma H (2018) VIRMA mediates preferential m(6)A mRNA methylation in 3’UTR and near stop codon and associates with alternative polyadenylation. Cell Discov 4:10

169 Zaringhalam M, Papavasiliou FN (2016) Pseudouridylation meets next-generation sequencing. Methods (San Diego, Calif) 107:63–72

170 Zeng H, He B, Xia B, Bai D, Lu X, Cai J, Chen L, Zhou A, Zhu C, Meng H (2018) Bisulfite-free, nanoscale analysis of 5-hydroxymethylcytosine at single base resolution. J Am Chem Soc 140:13190–13194

171 Zhang G, Huang H, Liu D, Cheng Y, Liu X, Zhang W, Yin R, Zhang D, Zhang P,Liu J(2015) N6-methyladenine DNA modification in Drosophila. Cell 161:893–906

172 Zhang LS, Liu C, Ma H, Dai Q, Sun HL, Luo G, Zhang Z, Zhang L, Hu L, Dong X (2019a) Transcriptome-wide mapping of internal N(7)-methylguanosine methylome in mammalian mRNA. Mol Cell 4(6):1304.e8–1316.e8

173 Zhang Z, Chen LQ, Zhao YL, Yang CG, Roundtree IA, Zhang Z, Ren J, Xie W, He C, Luo GZ (2019b) Single-base mapping of m(6)A by an antibody-independent method. Sci Adv 5:250

174 Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, Hao YJ, Ping XL, Chen YS, Wang WJ (2014) FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res 24:1403–1419

175 Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vagbo CB, Shi Y, Wang WL, Song SH (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 49:18–29

176 Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB (2015) Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526:591–594

177 Zhou J, Wan J, Shu XE, Mao Y, Liu XM, Yuan X, Zhang X, Hess ME, Bruning JC, Qian SB (2018) N(6)-methyladenosine guides mRNA alternative translation during integrated stress response. Mol Cell 69:636–647.e637

178 Zhou H, Rauch S, Dai Q, Cui X, Zhang Z, Nachtergaele S, Sepich C, He C, Dickinson BC (2019) Evolution of a reverse transcriptase to map N(1)-methyladenosine in human messenger RNA. Nat Methods 16:1281–1288

179 Zhu C, Gao Y, Guo H, Xia B, Song J, Wu X, Zeng H, Kee K, Tang F, Yi C (2017) Single-Cell 5-formylcytosine landscapes of mammalian early embryos and ESCs at single-base resolution. Cell Stem Cell 20:720–731.e725