真核生物基因组DNA甲基化和去甲基化分析
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摘要
DNA甲基化是真核生物的重要表观遗传修饰,如胞嘧啶C~5位甲基化5-甲基胞嘧啶(5mC)和腺嘌呤N~6位甲基化6-甲基腺嘌呤(6mA)。DNA 5mC可经Tet双加氧酶催化氧化形成5-羟甲基胞嘧啶(5hmC)、5-醛甲基胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)。这些氧化产物不仅是去甲基化过程的中间体,而且也可能存在各自特有的表观调控功能。其中,5hmC异常可能和癌症相关,有可能成为疾病诊断的生物标志物。发展可靠、高灵敏和抗干扰能力强的DNA甲基化和去甲基化检测技术和方法至关重要,有助于理解甲基化和去甲基化的分子机制以及提高肿瘤的诊断水平。现针对DNA甲基化和去甲基化检测技术进行简要介绍。
Eukaryotic DNA methylations, including methylation of C~5 position of cytosine 5-methylcytosine(5 m C) and N~6 position of adenine(6-methyladenine, 6 m A), are critical and important epigenetic markers. DNA 5 m C can be oxidized to form three intermediates, 5-hydroxymethylcytosine(5 hm C), 5-formylcytosine(5 f C), 5-carboxylcytosine(5 ca C), as catalized by Tet family dioxygenases. In addition to being as demethylation intermediates, 5 hm C content is negatively correlated with tumor occurrence, and thus is a potential biomarker indicating occurrence and development of tumors. To understand these events and underlying mechanisms, it is essential to develop reliable, sensitive and interference-free methods for detection of DNA methylation and demethylation. Here we briefly review and discuss analytical and sequencing technologies for detection of epigenetic modifications involved in DNA methylation and demethylation.
Eukaryotic DNA methylations, including methylation of C~5 position of cytosine 5-methylcytosine(5 m C) and N~6 position of adenine(6-methyladenine, 6 m A), are critical and important epigenetic markers. DNA 5 m C can be oxidized to form three intermediates, 5-hydroxymethylcytosine(5 hm C), 5-formylcytosine(5 f C), 5-carboxylcytosine(5 ca C), as catalized by Tet family dioxygenases. In addition to being as demethylation intermediates, 5 hm C content is negatively correlated with tumor occurrence, and thus is a potential biomarker indicating occurrence and development of tumors. To understand these events and underlying mechanisms, it is essential to develop reliable, sensitive and interference-free methods for detection of DNA methylation and demethylation. Here we briefly review and discuss analytical and sequencing technologies for detection of epigenetic modifications involved in DNA methylation and demethylation.
引文
[1]Cheng X.Structure and function of DNA methyltransferases.Annu Rev Biophys Biomol Struct,1995,24:293-318
[2]Wion D,Casadesus J.N6-methyl-adenine:an epigenetic signal for DNA-protein interactions.Nat Rev Microbiol,2006,4:183-92
[3]Goll MG,Bestor TH.Eukaryotic cytosine methyltransferases.Annu Rev Biochem,2005,74:481-514
[4]Cummings DJ,Tait A,Goddard JM.Methylated bases in DNA from Paramecium aurelia.Biochim Biophys Acta,1974,374:1-11
[5]Hattman S,Kenny C,Berger L,et al.Comparative study of DNA methylation in three unicellular eucaryotes.JBacteriol,1978,135:1156-7
[6]Pratt K,Hattman S.Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila.Mol Cell Biol,1981,1:600-8
[7]Gorovsky MA,Hattman S,Pleger GL.[6N]methyl adenine in the nuclear DNA of a eucaryote,Tetrahymena pyriformis.J Cell Biol,1973,56:697
[8]Zhang G,Huang H,Liu D,et al.N6-methyladenine DNAmodification in Drosophila.Cell,2015,161:893-906
[9]Fu Y,Luo GZ,Chen K,et al.N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.Cell,2015,161:879-92
[10]Greer EL,Blanco MA,Gu L,et al.DNA methylation on N6-Adenine in C.elegans.Cell,2015,161:868-78
[11]Liu J,Zhu Y,Luo GZ,et al.Abundant DNA 6mAmethylation during early embryogenesis of zebrafish and pig.Nat Commun,2016,7:13052
[12]Wu TP,Wang T,Seetin MG,et al.DNA methylation on N6-adenine in mammalian embryonic stem cells.Nature,2016,532:329-33
[13]Rougier N,Bourc’his D,Gomes DM,et al.Chromosome methylation patterns during mammalian preimplantation development.Gene Dev,1998,12:2108-13
[14]Cliffe LJ,Kieft R,Southern T,et al.JBP1 and JBP2 are two distinct thymidine hydroxylases involved in Jbiosynthesis in genomic DNA of African trypanosomes.Nucleic Acids Res,2009,37:1452-62
[15]Yu Z,Genest PA,Riet BT,et al.The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase.Nucleic Acids Res,2007,35:2107-15
[16]Tahiliani M,Koh KP,Shen Y,et al.Conversion of5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science,2009,324:930-5
[17]He YF,Li BZ,Li Z,et al.Tet-mediated formation of5-carboxylcytosine and its excision by TDG in mammalian DNA.Science,2011,333:1303-7
[18]Ito S,Shen L,Dai Q,et al.Tet proteins can convert5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science,2011,333:1300-3
[19]Yin R,Mao SQ,Zhao B,et al.Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.J Am Chem Soc,2013,135:10396-403
[20]Pfaffeneder T,Spada F,Wagner M,et al.Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.Nat Chem Biol,2014,10:574-81
[21]Nabel CS,Jia H,Ye Y,et al.AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation.Nat Chem Biol,2012,8:751-8
[22]Robertson AB,Dahl JA,Klungland A.DNA metabolism:bases of DNA repair and regulation.Nat Chem Biol,2014,10:487-8
[23]Sjolund AB,Senejani AG,Sweasy JB.MBD4 and TDG:multifaceted DNA glycosylases with ever expanding biological roles.Mutat Res,2013,743-744:12-25
[24]Bird AP.CpG-rich islands and the function of DNAmethylation.Nature,1986,321:209-13
[25]Wu H,Zhang Y.Reversing DNA methylation:mechanisms,genomics,and biological functions.Cell,2014,156:45-68
[26]Fuks F.DNA methylation and histone modifications:teaming up to silence genes.Curr Opin Genet Dev,2005,15:490-5
[27]Mohandas T,Sparkes R,Shapiro L.Reactivation of an inactive human X chromosome:evidence for X inactivation by DNA methylation.Science,1981,211:393-6
[28]Loenen WA,Dryden DT,Raleigh EA,et al.Highlights of the DNA cutters:a short history of the restriction enzymes.Nucleic Acids Res,2014,42:3-19
[29]Collier J,McAdams HH,Shapiro L.A DNA methylation ratchet governs progression through a bacterial cell cycle.Proc Natl Acad Sci USA,2007,104:17111-6
[30]Messer W,Noyer-Weidner M.Timing and targeting:the biological functions of Dam methylation in E.coli.Cell,1988,54:735-7
[31]Delhommeau F,Dupont S,Valle VD,et al.Mutation in TET2 in myeloid cancers.New Engl J Med,2009,360:2289-301
[32]Koh KP,Yabuuchi A,Rao S,et al.Tet1 and Tet2 regulate5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.Cell Stem Cell,2011,8:200-13
[33]Chen RZ,Pettersson U,Beard C,et al.DNA hypomethylation leads to elevated mutation rates.Nature,1998,395:89-93
[34]Ehrlich M.DNA methylation in cancer:too much,but also too little.Oncogene,2002,21:5400-13
[35]Kim YI,Giuliano A,Hatch KD,et al.Global DNAhypomethylation increases progressively in cervical dysplasia and carcinoma.Cancer,1994,74:893-9
[36]Ngo TT,Yoo J,Dai Q,et al.Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.Nat Commun,2016,7:10813
[37]Iurlaro M,Ficz G,Oxley D,et al.A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.Genome Biol,2013,14:R119
[38]Booth MJ,Marsico G,Bachman M,et al.Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.Nat Chem,2014,6:435-40
[39]Booth MJ,Branco MR,Ficz G,et al.Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.Science,2012,336:934-7
[40]Yu M,Hon GC,Szulwach KE,et al.Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.Cell,2012,149:1368-80
[41]Lu X,Han D,Zhao BS,et al.Base-resolution maps of5-formylcytosine and 5-carboxylcytosine reveal genomewide DNA demethylation dynamics.Cell Res,2015,25:386
[42]Kellinger MW,Song CX,Chong J,et al.5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription.Nat Struct Mol Biol,2012,19:831-3
[43]Williams K,Christensen J,Pedersen MT,et al.TET1 and hydroxymethylcytosine in transcription and DNAmethylation fidelity.Nature,2011,473:343-8
[44]Kawasaki F,Beraldi D,Hardisty RE,et al.Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.Genome Biol,2017,18:23
[45]Olinski R,Starczak M,Gackowski D.Enigmatic5-hydroxymethyluracil:Oxidatively modified base,epigenetic mark or both?Mutat Res Rev Mutat Res,2016,767:59-66
[46]Kriaucionis S,Heintz N.The nuclear DNA base5-hydroxymethylcytosine is present in Purkinje neurons and the brain.Science,2009,324:929-30
[47]Wang X,Song Y,Song M,et al.Fluorescence polarization combined capillary electrophoresis immunoassay for the sensitive detection of genomic DNA methylation.Anal Chem,2009,81:7885-91
[48]Tang Y,Gao XD,Wang Y,et al.Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry.Anal Chem,2012,84:7249-55
[49]Zhong S,Li Z,Jiang T,et al.Immunofluorescence imaging strategy for evaluation of the accessibility of DNA 5-hydroxymethylcytosine in chromatins.Anal Chem,2017:5702-6
[50]Zhang N,Liu B,Wang H.Technology and methodology of DNA adducts analysis.Sci Sin Chim,2017,47:1432-44
[51]Yin R,Mo J,Lu M,et al.Detection of human urinary5-hydroxymethylcytosine by stable isotope dilution HPLC-MS/MS analysis.Anal Chem,2015,87:1846-52
[52]Zhao C,Wang H,Zhao B,et al.Boronic acid-mediated polymerase chain reaction for gene-and fragment-specific detection of 5-hydroxymethylcytosine.Nucleic Acids Res,2014,42:e81
[53]Meissner A,Mikkelsen TS,Gu H,et al.Genome-scale DNA methylation maps of pluripotent and differentiated cells.Nature,2008,454:766-70
[54]Cokus SJ,Feng S,Zhang X,et al.Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNAmethylation patterning.Nature,2008,452:215-9
[55]Zhao C,Wang H.The progress on sequencing and detection of hydroxymethylated DNA.Acta Chim Sin,2013,71:26-35
[56]Song CX,Szulwach KE,Dai Q,et al.Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming.Cell,2013,153:678-91
[57]Shen L,Wu H,Diep D,et al.Genome-wide analysis reveals TET-and TDG-dependent 5-methylcytosine oxidation dynamics.Cell,2013,153:692-706
[58]Flusberg BA,Webster DR,Lee JH,et al.Direct detection of DNA methylation during single-molecule,real-time sequencing.Nat Methods,2010,7:461-5
[59]Wang X,Suo Y,Yin R,et al.Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms.J Chromatoger B,2011,879:1647-52
[60]Yin R,Liu S,Zhao C,et al.An ammonium bicarbonateenhanced stable isotope dilution UHPLC-MS/MS method for sensitive and accurate quantification of acrolein-DNAadducts in human leukocytes.Anal Chem,2013,85:3190-7
[61]Yin R,Mo J,Dai J,et al.Nickel(II)inhibits Tet-mediated5-methylcytosine oxidation by high affinity displacement of the cofactor iron(II).ACS Chem Biol,2017,12:1494-8
[62]Li C,Liu B,Zhong S,et al.MEK inhibitor PD0325901and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.Oncotarget,2016,7:39730-9
[63]Li C,Wang F,Wang H.Tetrachloro-1,4-benzoquinone induces apoptosis of mouse embryonic stem cells.JEnviron Sci(China),2017,51:5-12
[64]Yin R,Zhang D,Song Y,et al.Potent DNA damage by polyhalogenated quinones and H2O2 via a metal-independent and Intercalation-enhanced oxidation mechanism.Sci Rep,2013,3:1269
[65]Liu S,Wang J,Su Y,et al.Quantitative assessment of Tetinduced oxidation products of 5-methylcytosine in cellular and tissue DNA.Nucleic Acids Res,2013,41:6421-9
[66]Huang Y,Pastor WA,Shen Y,et al.The behaviour of5-hydroxymethylcytosine in bisulfite sequencing.PLoSOne,2010,5:e8888
[67]Meissner A,Gnirke A,Bell GW,et al.Reduced representation bisulfite sequencing for comparative high-resolution DNAmethylation analysis.Nucleic Acids Res,2005,33:5868-77
[68]Lu X,Song CX,Szulwach K,et al.Chemical modification-assisted bisulfite sequencing(CAB-Seq)for5-carboxylcytosine detection in DNA.J Am Chem Soc,2013,135:9315-7
[69]Song CX,Clark TA,Lu XY,et al.Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine.Nat Methods,2011,9:75-7
[70]Chavez L,Huang Y,Luong K,et al.Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea.Proc Natl Acad Sci USA,2014,111:E5149-58
[71]Clark TA,Lu X,Luong K,et al.Enhanced 5-methylcytosine detection in single-molecule,real-time sequencing via Tet1 oxidation.BMC Biol,2013,11:4
[72]Fang G,Munera D,Friedman DI,et al.Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.Nat Biotechnol,2012,30:1232-9
[73]Zhu L,Zhong J,Jia X,et al.Precision methylome characterization of Mycobacterium tuberculosis complex(MTBC)using PacBio single-molecule real-time(SMRT)technology.Nucleic Acids Res,2016,44:730-43
[74]Shin SC,Ahn DH,Kim SJ,et al.Advantages of singlemolecule real-time sequencing in high-GC content genomes.PLoS One,2013,8:e68824
[75]Li W,Liu M.Distribution of 5-hydroxymethylcytosine in different human tissues.J Nucleic Acids,2011,3:870726
[76]Zarakowska E,Gackowski D,Foksinski M,et al.Are8-oxoguanine(8-oxoGua)and 5-hydroxymethyluracil(5-hmUra)oxidatively damaged DNA bases or transcription(epigenetic)marks?Mutat Res Genet Toxicol Environ Mutagen,2014,764-765:58-63
[77]Bullard W,Kieft R,Sabatini R.A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA.Biol Methods Protoc,2017,2:bpw006-bpw
[78]Clark TA,Spittle KE,Turner SW,et al.Direct detection and sequencing of damaged DNA bases.Genome Integr,2011,2:1-9
[79]Genest PA,Baugh L,Taipale A,et al.Defining the sequence requirements for the positioning of base in DNAusing SMRT sequencing.Nucleic Acids Res,2015,43:2102-15
[80]Harrison GS,Findly RC,Karrer K.Site-specific methylation of adenine in the nuclear genome of a eucaryote,Tetrahymena thermophila.Mol Cell,1986,6:2364-70
[81]Krais AM,Cornelius MG,Schmeiser HH.Genomic N6-methyladenine determination by MEKC with LIF.Electrophoresis,2010,31:3548-51
[82]Dohno C,Shibata T,Nakatani K.Discrimination of N6-methyl adenine in a specific DNA sequence.Chem Commun(Camb),2010,46:5530-2
[83]Hong T,Yuan Y,Wang T,et al.Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification.Chem Sci,2017,8:200-5
[84]Koziol MJ,Bradshaw CR,Allen GE,et al.Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.Nat Struct Mol Biol,2016,23:24-30
[85]Wang Y,Chen X,Sheng Y,et al.N6-adenine DNAmethylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol IItranscribed genes in Tetrahymena.Nucleic Acids Res,2017,45:11594-606
[86]Dabe EC,Sanford RS,Kohn AB,et al.DNA methylation in basal metazoans:insights from ctenophores.Integr Comp Biol,2015,55:1096-110
[87]Yao B,Cheng Y,Wang Z,et al.DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress.Nat Commun,2017,8:1122
[88]Wang Y,Wang Y,Sheng Y,et al.A comparative study of genome organization and epigenetic mechanisms in model ciliates,with an emphasis on Tetrahymena,Paramecium and Oxytricha.Eur J Protistol,2017,61:376-87
[89]Liang D,Wang H,Song W,et al.The decreased N6-methyladenine DNA modification in cancer cells.Biochem Biophys Res Commun,2016,480:120-5
[90]Luo GZ,Wang F,Weng X,et al.Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.Nat Commun,2016,7:11301
[91]Mondo SJ,Dannebaum RO,Kuo RC,et al.Widespread adenine N6-methylation of active genes in fungi.Nat Genet,2017,49:964-8
[92]Luo GZ,Blanco MA,Greer EL,et al.DNA N6-methyladenine:a new epigenetic mark in eukaryotes?Nat Rev Mol Cell Biol,2015,16:705-10
[93]Liu B,Liu X,Lai W,et al.Metabolically generated stable isotope-labeled deoxynucleoside code for tracing DNAN6-methyladenine in human cells.Anal Chem,2017,89:6202-9
[94]Fu Y,Jia G,Pang X,et al.FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.Nat Commun,2013,4:1798
[2]Wion D,Casadesus J.N6-methyl-adenine:an epigenetic signal for DNA-protein interactions.Nat Rev Microbiol,2006,4:183-92
[3]Goll MG,Bestor TH.Eukaryotic cytosine methyltransferases.Annu Rev Biochem,2005,74:481-514
[4]Cummings DJ,Tait A,Goddard JM.Methylated bases in DNA from Paramecium aurelia.Biochim Biophys Acta,1974,374:1-11
[5]Hattman S,Kenny C,Berger L,et al.Comparative study of DNA methylation in three unicellular eucaryotes.JBacteriol,1978,135:1156-7
[6]Pratt K,Hattman S.Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila.Mol Cell Biol,1981,1:600-8
[7]Gorovsky MA,Hattman S,Pleger GL.[6N]methyl adenine in the nuclear DNA of a eucaryote,Tetrahymena pyriformis.J Cell Biol,1973,56:697
[8]Zhang G,Huang H,Liu D,et al.N6-methyladenine DNAmodification in Drosophila.Cell,2015,161:893-906
[9]Fu Y,Luo GZ,Chen K,et al.N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.Cell,2015,161:879-92
[10]Greer EL,Blanco MA,Gu L,et al.DNA methylation on N6-Adenine in C.elegans.Cell,2015,161:868-78
[11]Liu J,Zhu Y,Luo GZ,et al.Abundant DNA 6mAmethylation during early embryogenesis of zebrafish and pig.Nat Commun,2016,7:13052
[12]Wu TP,Wang T,Seetin MG,et al.DNA methylation on N6-adenine in mammalian embryonic stem cells.Nature,2016,532:329-33
[13]Rougier N,Bourc’his D,Gomes DM,et al.Chromosome methylation patterns during mammalian preimplantation development.Gene Dev,1998,12:2108-13
[14]Cliffe LJ,Kieft R,Southern T,et al.JBP1 and JBP2 are two distinct thymidine hydroxylases involved in Jbiosynthesis in genomic DNA of African trypanosomes.Nucleic Acids Res,2009,37:1452-62
[15]Yu Z,Genest PA,Riet BT,et al.The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase.Nucleic Acids Res,2007,35:2107-15
[16]Tahiliani M,Koh KP,Shen Y,et al.Conversion of5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.Science,2009,324:930-5
[17]He YF,Li BZ,Li Z,et al.Tet-mediated formation of5-carboxylcytosine and its excision by TDG in mammalian DNA.Science,2011,333:1303-7
[18]Ito S,Shen L,Dai Q,et al.Tet proteins can convert5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.Science,2011,333:1300-3
[19]Yin R,Mao SQ,Zhao B,et al.Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.J Am Chem Soc,2013,135:10396-403
[20]Pfaffeneder T,Spada F,Wagner M,et al.Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.Nat Chem Biol,2014,10:574-81
[21]Nabel CS,Jia H,Ye Y,et al.AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation.Nat Chem Biol,2012,8:751-8
[22]Robertson AB,Dahl JA,Klungland A.DNA metabolism:bases of DNA repair and regulation.Nat Chem Biol,2014,10:487-8
[23]Sjolund AB,Senejani AG,Sweasy JB.MBD4 and TDG:multifaceted DNA glycosylases with ever expanding biological roles.Mutat Res,2013,743-744:12-25
[24]Bird AP.CpG-rich islands and the function of DNAmethylation.Nature,1986,321:209-13
[25]Wu H,Zhang Y.Reversing DNA methylation:mechanisms,genomics,and biological functions.Cell,2014,156:45-68
[26]Fuks F.DNA methylation and histone modifications:teaming up to silence genes.Curr Opin Genet Dev,2005,15:490-5
[27]Mohandas T,Sparkes R,Shapiro L.Reactivation of an inactive human X chromosome:evidence for X inactivation by DNA methylation.Science,1981,211:393-6
[28]Loenen WA,Dryden DT,Raleigh EA,et al.Highlights of the DNA cutters:a short history of the restriction enzymes.Nucleic Acids Res,2014,42:3-19
[29]Collier J,McAdams HH,Shapiro L.A DNA methylation ratchet governs progression through a bacterial cell cycle.Proc Natl Acad Sci USA,2007,104:17111-6
[30]Messer W,Noyer-Weidner M.Timing and targeting:the biological functions of Dam methylation in E.coli.Cell,1988,54:735-7
[31]Delhommeau F,Dupont S,Valle VD,et al.Mutation in TET2 in myeloid cancers.New Engl J Med,2009,360:2289-301
[32]Koh KP,Yabuuchi A,Rao S,et al.Tet1 and Tet2 regulate5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.Cell Stem Cell,2011,8:200-13
[33]Chen RZ,Pettersson U,Beard C,et al.DNA hypomethylation leads to elevated mutation rates.Nature,1998,395:89-93
[34]Ehrlich M.DNA methylation in cancer:too much,but also too little.Oncogene,2002,21:5400-13
[35]Kim YI,Giuliano A,Hatch KD,et al.Global DNAhypomethylation increases progressively in cervical dysplasia and carcinoma.Cancer,1994,74:893-9
[36]Ngo TT,Yoo J,Dai Q,et al.Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.Nat Commun,2016,7:10813
[37]Iurlaro M,Ficz G,Oxley D,et al.A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.Genome Biol,2013,14:R119
[38]Booth MJ,Marsico G,Bachman M,et al.Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution.Nat Chem,2014,6:435-40
[39]Booth MJ,Branco MR,Ficz G,et al.Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.Science,2012,336:934-7
[40]Yu M,Hon GC,Szulwach KE,et al.Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.Cell,2012,149:1368-80
[41]Lu X,Han D,Zhao BS,et al.Base-resolution maps of5-formylcytosine and 5-carboxylcytosine reveal genomewide DNA demethylation dynamics.Cell Res,2015,25:386
[42]Kellinger MW,Song CX,Chong J,et al.5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription.Nat Struct Mol Biol,2012,19:831-3
[43]Williams K,Christensen J,Pedersen MT,et al.TET1 and hydroxymethylcytosine in transcription and DNAmethylation fidelity.Nature,2011,473:343-8
[44]Kawasaki F,Beraldi D,Hardisty RE,et al.Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania.Genome Biol,2017,18:23
[45]Olinski R,Starczak M,Gackowski D.Enigmatic5-hydroxymethyluracil:Oxidatively modified base,epigenetic mark or both?Mutat Res Rev Mutat Res,2016,767:59-66
[46]Kriaucionis S,Heintz N.The nuclear DNA base5-hydroxymethylcytosine is present in Purkinje neurons and the brain.Science,2009,324:929-30
[47]Wang X,Song Y,Song M,et al.Fluorescence polarization combined capillary electrophoresis immunoassay for the sensitive detection of genomic DNA methylation.Anal Chem,2009,81:7885-91
[48]Tang Y,Gao XD,Wang Y,et al.Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry.Anal Chem,2012,84:7249-55
[49]Zhong S,Li Z,Jiang T,et al.Immunofluorescence imaging strategy for evaluation of the accessibility of DNA 5-hydroxymethylcytosine in chromatins.Anal Chem,2017:5702-6
[50]Zhang N,Liu B,Wang H.Technology and methodology of DNA adducts analysis.Sci Sin Chim,2017,47:1432-44
[51]Yin R,Mo J,Lu M,et al.Detection of human urinary5-hydroxymethylcytosine by stable isotope dilution HPLC-MS/MS analysis.Anal Chem,2015,87:1846-52
[52]Zhao C,Wang H,Zhao B,et al.Boronic acid-mediated polymerase chain reaction for gene-and fragment-specific detection of 5-hydroxymethylcytosine.Nucleic Acids Res,2014,42:e81
[53]Meissner A,Mikkelsen TS,Gu H,et al.Genome-scale DNA methylation maps of pluripotent and differentiated cells.Nature,2008,454:766-70
[54]Cokus SJ,Feng S,Zhang X,et al.Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNAmethylation patterning.Nature,2008,452:215-9
[55]Zhao C,Wang H.The progress on sequencing and detection of hydroxymethylated DNA.Acta Chim Sin,2013,71:26-35
[56]Song CX,Szulwach KE,Dai Q,et al.Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming.Cell,2013,153:678-91
[57]Shen L,Wu H,Diep D,et al.Genome-wide analysis reveals TET-and TDG-dependent 5-methylcytosine oxidation dynamics.Cell,2013,153:692-706
[58]Flusberg BA,Webster DR,Lee JH,et al.Direct detection of DNA methylation during single-molecule,real-time sequencing.Nat Methods,2010,7:461-5
[59]Wang X,Suo Y,Yin R,et al.Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms.J Chromatoger B,2011,879:1647-52
[60]Yin R,Liu S,Zhao C,et al.An ammonium bicarbonateenhanced stable isotope dilution UHPLC-MS/MS method for sensitive and accurate quantification of acrolein-DNAadducts in human leukocytes.Anal Chem,2013,85:3190-7
[61]Yin R,Mo J,Dai J,et al.Nickel(II)inhibits Tet-mediated5-methylcytosine oxidation by high affinity displacement of the cofactor iron(II).ACS Chem Biol,2017,12:1494-8
[62]Li C,Liu B,Zhong S,et al.MEK inhibitor PD0325901and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.Oncotarget,2016,7:39730-9
[63]Li C,Wang F,Wang H.Tetrachloro-1,4-benzoquinone induces apoptosis of mouse embryonic stem cells.JEnviron Sci(China),2017,51:5-12
[64]Yin R,Zhang D,Song Y,et al.Potent DNA damage by polyhalogenated quinones and H2O2 via a metal-independent and Intercalation-enhanced oxidation mechanism.Sci Rep,2013,3:1269
[65]Liu S,Wang J,Su Y,et al.Quantitative assessment of Tetinduced oxidation products of 5-methylcytosine in cellular and tissue DNA.Nucleic Acids Res,2013,41:6421-9
[66]Huang Y,Pastor WA,Shen Y,et al.The behaviour of5-hydroxymethylcytosine in bisulfite sequencing.PLoSOne,2010,5:e8888
[67]Meissner A,Gnirke A,Bell GW,et al.Reduced representation bisulfite sequencing for comparative high-resolution DNAmethylation analysis.Nucleic Acids Res,2005,33:5868-77
[68]Lu X,Song CX,Szulwach K,et al.Chemical modification-assisted bisulfite sequencing(CAB-Seq)for5-carboxylcytosine detection in DNA.J Am Chem Soc,2013,135:9315-7
[69]Song CX,Clark TA,Lu XY,et al.Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine.Nat Methods,2011,9:75-7
[70]Chavez L,Huang Y,Luong K,et al.Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea.Proc Natl Acad Sci USA,2014,111:E5149-58
[71]Clark TA,Lu X,Luong K,et al.Enhanced 5-methylcytosine detection in single-molecule,real-time sequencing via Tet1 oxidation.BMC Biol,2013,11:4
[72]Fang G,Munera D,Friedman DI,et al.Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.Nat Biotechnol,2012,30:1232-9
[73]Zhu L,Zhong J,Jia X,et al.Precision methylome characterization of Mycobacterium tuberculosis complex(MTBC)using PacBio single-molecule real-time(SMRT)technology.Nucleic Acids Res,2016,44:730-43
[74]Shin SC,Ahn DH,Kim SJ,et al.Advantages of singlemolecule real-time sequencing in high-GC content genomes.PLoS One,2013,8:e68824
[75]Li W,Liu M.Distribution of 5-hydroxymethylcytosine in different human tissues.J Nucleic Acids,2011,3:870726
[76]Zarakowska E,Gackowski D,Foksinski M,et al.Are8-oxoguanine(8-oxoGua)and 5-hydroxymethyluracil(5-hmUra)oxidatively damaged DNA bases or transcription(epigenetic)marks?Mutat Res Genet Toxicol Environ Mutagen,2014,764-765:58-63
[77]Bullard W,Kieft R,Sabatini R.A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA.Biol Methods Protoc,2017,2:bpw006-bpw
[78]Clark TA,Spittle KE,Turner SW,et al.Direct detection and sequencing of damaged DNA bases.Genome Integr,2011,2:1-9
[79]Genest PA,Baugh L,Taipale A,et al.Defining the sequence requirements for the positioning of base in DNAusing SMRT sequencing.Nucleic Acids Res,2015,43:2102-15
[80]Harrison GS,Findly RC,Karrer K.Site-specific methylation of adenine in the nuclear genome of a eucaryote,Tetrahymena thermophila.Mol Cell,1986,6:2364-70
[81]Krais AM,Cornelius MG,Schmeiser HH.Genomic N6-methyladenine determination by MEKC with LIF.Electrophoresis,2010,31:3548-51
[82]Dohno C,Shibata T,Nakatani K.Discrimination of N6-methyl adenine in a specific DNA sequence.Chem Commun(Camb),2010,46:5530-2
[83]Hong T,Yuan Y,Wang T,et al.Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification.Chem Sci,2017,8:200-5
[84]Koziol MJ,Bradshaw CR,Allen GE,et al.Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications.Nat Struct Mol Biol,2016,23:24-30
[85]Wang Y,Chen X,Sheng Y,et al.N6-adenine DNAmethylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol IItranscribed genes in Tetrahymena.Nucleic Acids Res,2017,45:11594-606
[86]Dabe EC,Sanford RS,Kohn AB,et al.DNA methylation in basal metazoans:insights from ctenophores.Integr Comp Biol,2015,55:1096-110
[87]Yao B,Cheng Y,Wang Z,et al.DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress.Nat Commun,2017,8:1122
[88]Wang Y,Wang Y,Sheng Y,et al.A comparative study of genome organization and epigenetic mechanisms in model ciliates,with an emphasis on Tetrahymena,Paramecium and Oxytricha.Eur J Protistol,2017,61:376-87
[89]Liang D,Wang H,Song W,et al.The decreased N6-methyladenine DNA modification in cancer cells.Biochem Biophys Res Commun,2016,480:120-5
[90]Luo GZ,Wang F,Weng X,et al.Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.Nat Commun,2016,7:11301
[91]Mondo SJ,Dannebaum RO,Kuo RC,et al.Widespread adenine N6-methylation of active genes in fungi.Nat Genet,2017,49:964-8
[92]Luo GZ,Blanco MA,Greer EL,et al.DNA N6-methyladenine:a new epigenetic mark in eukaryotes?Nat Rev Mol Cell Biol,2015,16:705-10
[93]Liu B,Liu X,Lai W,et al.Metabolically generated stable isotope-labeled deoxynucleoside code for tracing DNAN6-methyladenine in human cells.Anal Chem,2017,89:6202-9
[94]Fu Y,Jia G,Pang X,et al.FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.Nat Commun,2013,4:1798