Oxidative DNA demethylation mediated by Tet enzymes

REVIEW National Science Review2:318–328,2015doi:10.1093/nsr/nwv029Advance access publication8June2015 BIOLOGY&BIOCHEMISTRYOxidative DNA demethylation mediated by Tet enzymesGuo-Liang Xu1,∗and Jiemin Wong2,∗1Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences, Shanghai200031, China and2Institute of Biomedical Sciences and School of Life Sciences,East China Normal University, Shanghai200241, China∗Corresponding authors.E-mail:glxu@; jmweng@Received9March 2015;Revised20April2015;Accepted 20April2015ABSTRACTDNA modification,methylation of cytosine(5mC),and oxidation of5mC to5-hydroxymethylcytosine (5hmC),5-formylcytosine(5fC),and5-carboxylcytosine(5caC)can have profound effects on genome function in animals.These modifications are intricately involved in DNA methylation reprograming dynamics during mammalian development.Together,they contribute to cell lineage restriction and maintenance,while also undergoing dynamic changes during cellular transitions and induced reprograming. The last five years have seen an intense research focus on enzymatic DNA demethylation,triggered by the discovery of5hmC and Tet dioxygenases.In this review,we evaluate recent findings that have provided new insights into the mechanisms underlying DNA demethylation and its effect on developmental regulation. Keywords:DNA demethylation,5mC oxidation,Tet dioxygenase,TDG,epigenetic reprograming INTRODUCTIONAlthough a large number of modified nucleosides arepresent in RNA molecules,only5-methylcytosine(5mC;termed DNA methylation hereafter)hasbeen well characterized in mammalian DNA.DNA methylation occurs mainly in symmetric CGdinucleotides,of which∼70–80%is methylatedthroughout the mammalian genome.The remainingunmethylated CG dinucleotides are often foundin dense clusters termed CpG islands,which arefrequently located in or near gene promoters andcoding regions.In mammals,DNA methylationhas a critical role in the heritable silencing of manydifferent classes of repetitive DNA sequences.Theseinclude highly abundant retrotransposons and tan-dem repetitive sequences such as pericentromericminor and major satellite sequences,the methyla-tion of which helps to maintain genome stability bypreventing rearrangements.DNA methylation isalso required for regulation of developmental andtissue-specific gene expression,as well as for parent-of-origin genomic imprinting and X-chromosomeinactivation in females[1].DNA methylation ofpromoters results in transcriptional repressionthrough multiple mechanisms,including inhibitionof transcription factor binding and recruitment oftranscriptional repressor complexes[2].However,recent studies have provided evidence that DNAmethylation in gene bodies correlates positivelywith gene expression[3–7].Aberrant alterationsof genomic methylation patterns are implicated invarious human diseases,including cancers and im-printing syndromes such as Beckwith–Wiedemann,Prader–Willi,and Angelman syndromes[8,9].DNA methylation,one of the best-characterizedepigenetic modifications,is established and main-tained in mammals by three enzymatically activeDNA methyltransferases:DNMT1,DNMT3A,andDNMT3B.DNMT1knockout in mice results inembryonic lethality and a substantial reduction inglobal DNA methylation[10].DNMT1preferen-tially methylates hemimethylated DNA templates;it is the main enzyme responsible for maintainingDNA methylation patterns,following DNA replica-tion[11].Consistent with its maintenance activity,DNMT1localizes to DNA replication forks in theS phase of the cell cycle[12].Recent studies havedemonstrated that the correct targeting of DNMT1to replicating DNA requires an accessary protein,UHRF1,which binds to hemimethylated DNAthrough its unique SRA domain[13,14].The exis-tence of de novo DNA methyltransferase(DNMT)activity was discovered in an analysis of DNMT1-knockout mouse embryonic stem cells(ESCs)[15].Subsequent screening of expressed sequence tag li-braries for DNMT homologs led to the identifica-tion of DNMT3A and DNMT3B[16].Functionaland gene disruption studies have shown that bothC The Author(s)2015.Published by Oxford University Press on behalf of China Science Publishing&Media Ltd.All rights reserved.For Permissions,please email: journals.permissions@REVIEWXu and Wong319Figure 1.Dynamic changes in overall genomic methylation during mouse development.Note that the curves reflect the 5mC content of the whole genome.Individual genomic loci might follow a distinctive pattern of reprograming.enzymes are required for de novo methylation and mouse development [17].Although DNMT3A and DNMT3B are widely viewed as de novo DNA methy-lation enzymes,they also have a critical role in main-tenance methylation,as demonstrated by the fact that knockout of both DNMT3A and DNMT3B in mouse ESCs leads to a progressive reduction in global DNA methylation [18].Thus,in mammalian cells,DNMT1and DNMT3A/3B together are re-sponsible for establishing and maintaining global patterns of DNA methylation that can be faithfully transmitted through mitotic divisions.Given its potentially critical role in transcrip-tional regulation,epigenetic silencing,and other bi-ological processes,DNA methylation must be re-modeled during development.In support of the dy-namic nature of DNA methylation,the global lev-els of 5mC have been observed to undergo dras-tic changes in two developmental stages (Fig.1).In one instance,a global reduction in DNA methy-lation followed by remethylation occurs in primor-dial germ cells (PGCs)during male and female ga-metogenesis.In the other instance,a global reduc-tion in DNA methylation is observed in paternal and maternal DNA shortly after fertilization,long before the genome regains adult levels of methylation dur-ing gastrulation.In theory,DNA demethylation can occur through passive demethylation and/or active demethylation.The passive mode,in which methyla-tion of the newly synthesized DNA strand is blocked during DNA replication,is straightforward.Ge-netic and immunohistochemical evidence also sup-ports the existence of active demethylation.The bestevidence for active demethylation comes from the observation that the global reduction in DNA methylation in the paternal genome of one-cell stage mouse zygotes can occur in the absence of DNA replication [19,20].Although various proteins and molecular mechanisms have been described in an effort to understand the mechanism underlying active DNA demethylation [21],the recent find-ings that Tet family proteins can oxidize 5mC to three different oxidation products (5hmC,5fC,and 5caC)identify a pathway that could underpin ac-tive demethylation in mammals [22–24].In this review,we analyze recent findings that have pro-vided new insights into the mechanism underlying Tet-mediated DNA demethylation and its biological functions.DISTINCTIVE FUNCTIONAL CATEGORIES OF 5mC OXIDATION:METHYLATION ERASURE OR MAINTENANCE OF HYPOMETHYLATION5mC oxidation can decline through passive dilution.The maintenance DNA methyltransferase Dnmt1is unable to methylate an unmodified cytosine in the newly synthesized strand during DNA replication when the corresponding position on the template strand is an oxidized 5mC [25].Alternatively,oxi-dized 5mC bases can be actively removed by base excision repair (BER)or other unidentified path-ways to regenerate cytosine.We discuss these differ-ent mechanisms for the regeneration of unmodified320Natl Sci Rev,2015,Vol.2,No.3REVIEWFigure2.Two scenarios for5mC oxidation in active DNA demethylation.The first sce-nario(upper panel)conforms to the conventional notion that demethylation is a process of5mC erasure involving the conversion of5mCs(filled lollipops)into unmethylated cytosine residues(open lollipops)in a given genomic region.Without Tet-mediated 5mC oxidation(Tet KO),the hypermethylation state would be preserved.In the sec-ond scenario(lower panel),Tet enzymes tend to an unmethylated or hypomethylated region continuously to ensure the timely removal of5mC added by de novo methyl-transferases.In the absence of Tet,a normally unmethylated region becomes hyper-methylated through the activity of de novo methyltransferases(Dnmt).cytosine later in this review;however,it is im-portant to note here that5mC oxidation has twodistinguishable functions,depending on the regionalmethylation status of the particular genomic locusinvolved(Fig.2).The first function of5mC oxidation involveselimination of hypermethylation from a regulatoryregion commonly found in cells undergoing fatetransition.For instance,in early mouse embryostransitioning from gamete to soma,inactive de-velopmental genes silenced by DNA methylationare rapidly demethylated for reactivation.5mCs atpluripotency loci are oxidized by Tet3at as earlyas the pronuclear stage of one-cell embryos[26](Fig.1).In experimental reprograming of fibroblastsinto iPS cells,Tet-catalyzed5mC oxidation facili-tates reprograming,and is essential for demethyla-tion and reactivation of microRNA genes crucial formesenchymal to epithelial transition[27,28].Theseobservations highlight the role of5mC oxidationin removing repressive methylation and eliminatingepigenetic barriers established in an earlier processof lineage determination and commitment.The second function of5mC oxidation involvescounteracting ongoing de novo methylation.ESCs,adult stem cells,zygotes,and neurons express highlevels of de novo methyltransferases.To keep CpG is-lands,transcription start sites(TSS),and enhancersfree of5mC accumulation,constant oxidation byTet appears to be important.Deficiency in Tet leadsto the accretion of methylation and downregula-tion of the genes affected.In Tet1-depleted ESCs, a significant degree of methylation occurs at spe-cific loci such as Ecat1,Esrrb[29],Lefty1[30],and Nanog[31],which are unmethylated in wild-type cells.Conditional knockout of Tet1in the adult mouse brain resulted in hypermethylation and re-pression of neurogenesis-related genes[32].The methylation-antagonizing function is thus impor-tant for the maintenance of transcriptional activity to stabilize cellular identity.While the first function in principle requires a one-time reaction,persistent oxidation is necessary for the second function.Interestingly,genome-wide mapping of the genomic binding sites for Tet1and Tet2in mouse ESCs indicates that the enzymes are positioned in hypomethylated regions[33–35], consistent with their role in removing stochasti-cally added5mC to keep the binding targets free of methylation.Accordingly,5hmC is mostly asso-ciated with gene promoters(or TSSs)and CpG is-lands where5mC is underrepresented[29,33,36]. The antagonism of cytosine methylation provided by Tet-mediated oxidation preserves the genomic methylation landscape of ESCs.This model recon-ciles well with the counterintuitive associations of Tet proteins and5hmC with the5mC-depleted ge-nomic regions observed in ESCs.A sustained hy-pomethylated state maintains the expression of tran-scriptionally active genes as well as the repression of Polycomb target genes.Given that combined dele-tion of Tet1and Tet2had a subtle effect on embry-onic development in vivo[37]and that Tet triple knockout had a minor effect on ESC proliferation in vitro[27],it is unclear to what extent the mainte-nance of appropriate hypomethylation is linked with cell pluripotency or plasticity.5mC OXIDATION PROMOTES DEMETHYLATION THROUGHPASSIVE DILUTIONWhile Tet-mediated oxidation has been impli-cated in active demethylation,the inhibition of maintenance methylation by oxidized bases during DNA synthesis provides a mechanism for passive demethylation through dilution[38].The passive dilution mode is compatible with the chromosomal banding patterns of5hmC,5fC,and5caC,which are asymmetric in sister chromatids in pre-implantation mouse embryos[39,40].However,oxidation of5mC is,in principle,not essential for passive demethylation because5mC by itself can undergo progressive dilution when the newly synthesized strand in DNA replication is not methylated by a maintenance methyltransferase.In fact,passiveREVIEW Xu and Wong321dilution of5mC was suggested based on the ob-servation of asymmetric5mC banding patterns in sister chromatids during metaphase in early embryos[41].The locus-specific loss of5mC from one sister chromatid can occur when methylation by Dnmt1is actively blocked at specific loci during pronuclear DNA replication in one-cell embryos. However,prior oxidation of5mC can accelerate the loss of5mC at genomic regions that are accessible to maintenance methylation because Dnmt1cannot recognize oxidized5mC bases during DNA repli-cation.While5hmC-mediated passive dilution has been observed at the genomic level in both zygotes and PGCs[39,42],the genomic context that allows for a mixed mode of demethylation has yet to be determined.TDG-MEDIATED BER FOLLOWING5mC OXIDATIONRestoration of unmodified cytosine upon Tet-mediated oxidation of5mC cannot occur sponta-neously.This is in contrast to histone demethylation, in which single-step hydroxylation of the methyl group leads to its release as formaldehyde from the attached nitrogen atom of a lysine residue. The carboxyl group of5caC,the final oxidation product of5mC,is stably attached to a carbon atom of the pyrimidine ring,and a decarboxylase is needed to catalyze its release as CO2.An analo-gous biochemical reaction exists in the thymidine salvage pathway of some fungi,in which thymine is converted into uridine through oxidation of the methyl group.A carboxyl group is formed by thymine-7-hydroxylase;the final oxidation product isoorotate is then decarboxylated by a decarboxylase [43].The original methyl group of thymine is thus released as CO2.A mammalian5caC decarboxylase and the fungal isoorotate decarboxylase(IDCase) might use conserved biochemical mechanisms. To facilitate homology-based searches for a5caC decarboxylase in mammals,Xu et al.solved the crystal structures of two fungal IDCases[44]. IDCases possess decarboxylase activity against 5-carboxylcytosine,although their activity towards the cognate substrate isoorotate is much stronger. However,no enzyme capable of decarboxylating 5caC in DNA has been identified.While the existence of a5caC DNA decarboxy-lase remains uncertain,BER has been shown to par-ticipate in Tet-initiated demethylation.In plants, active DNA demethylation depends on BER[45]. While several plant glycosylases recognize and excise 5mC directly to initiate BER,no glycosylase enzyme that processes5mC has been identified in mammals. Instead,thymine DNA glycosylase(TDG)recog-nizes5fC and5caC,the higher oxidation products of5mC,to initiate BER[23,46](Fig.3).The5caC glycosylase activity of TDG was confirmed by the observation that Tdg knockout in iPS cells led to the accumulation of5caC but not of5hmC[23]. TDG was originally identified based on its ability to excise T from G/T mismatches,presumably gen-erated by spontaneous deamination of5mC[47]. However,TDG knockout in mouse did not produce a genome instability phenotype but surprisingly re-sulted in embryonic lethality that correlated with DNA hypermethylation and misregulation of devel-opmental genes[48,49].In the cocrystal structure of TDG and5caC-DNA,the two carboxyl oxygen atoms of5caC form distinctive hydrogen bonds with critical residues of the active site of TDG[50].These observations establish TDG as an enzyme with a prominent role in oxidative demethylation,in addi-tion to possible roles in mismatch repair.Several recent studies have confirmed TDG’s role in oxidative demethylation.Genome-wide map-ping of increased5fC and5caC in TDG-depleted mouse ESCs indicated that these two modified bases appeared as intermediates in genomic regions sub-jected to active demethylation and that these regions were different from those enriched in5hmC[51–53].5fC and5caC were also reported to accumulate in differentiating neural stem cells depleted of TDG [54].Furthermore,cell reprograming assays have in-dicated that TDG is directly involved in demethy-lation of microRNA genes crucial for the conver-sion of fibroblasts into iPS cells[27].By contrast, active demethylation in mouse zygotes is unaffected by TDG deletion[55](Fig.3).This surprising re-sult suggests the existence of an as-yet-unknown demethylation mechanism downstream of5mC ox-idation.Alternatively,a glycosylase other than TDG might process oxidized bases as a backup mecha-nism.Previous studies have detected signs of BER in male pronuclear DNA undergoing demethylation [56,57];however,it is unclear whether BER is a bystander occurrence of massive epigenetic repro-graming or a major step central to active demethy-lation.Given that BER threatens genomic integrity by generating strand breaks,especially when it is op-erating at numerous cytosine positions at once,a decarboxylase-based mechanism for resolving oxi-dation products during active DNA demethylation in zygotes seems more plausible. IMPORTANCE OF TET FUNCTIONIN MOUSE DEVELOPEMENTInitial attempts to gauge the functional signifi-cance of Tet-mediated5mC oxidation used mouse ESCs,in which Tet1and Tet2are highly expressed322Natl Sci Rev ,2015,Vol.2,No.3REVIEWOO O5mC5hmCROS1, DME, DML2 or DML3OOO NH 2N NO CH 35mCTetNH 2OCO ON NO In plantsIn mammalsAbasic siteBERReplicative dilution TDG-independant active removal?NH 2N NO CH 3OOO NH 2NN OCH 2OHOO ONH 2N NO CHO OOO NH 2N NO COOHOO OorTDGOOOOH BERNH 2NNOOOOC(In ESCs & neurons)(In zygotes)NH 2N NO OOO C Figure 3.Active demethylation in plants and mammals.Active demethylation in mammals relies on Tet-catalyzed 5mC ox-idation to trigger TDG-mediated BER in ESCs and neurons or an unidentified process in zygotes.In plants,5mC is directly recognized and processed by glycosylases to initiate BER.and 5hmC is readily detectable.Phenotypic alter-ations have been described in numerous studies.For example,knockdown of Tet1,but not of Tet2or Tet3,impaired ESC self-renewal and maintenance,partially because of increased promoter methyla-tion and downregulation of Nanog [31].However,knockout of one or even all three Tet genes in com-bination did not affect mouse ESCs self-renewal and maintenance in general [27,37,58].While double knockout of Tet1and Tet2in mouse is compatible with embryogenesis,triple knockout of Tet1/2/3in ESCs affects proper cell differentiation without abolishing the overall capacity to form three germ layers [27,59].The inconsistency among different studies suggests that due caution needs to be exer-cised when drawing conclusions about the in vivo relevance of observations made in ESCs.The vari-ous Tet-associated ESCs phenotypes might be in-fluenced by the culture conditions,passage number,and method of Tet depletion.For example,the 5mC and 5hmC contents in genomic DNA vary greatly when Erk1/2and Gsk3βinhibitors (2i)and vitamin C are included in the medium [60–62].ESCs are characterized by distinct functional states under dy-namic equilibrium and by the heterogeneous expres-sion of critical pluripotency factors and reversible epigenetic modifications.In vitro culture conditions might not maintain this equilibrium and balanced developmental plasticity.Nonetheless,ESC studies have been useful in elucidating the molecular pro-cess of 5mC oxidation and its role in gene regulation.Tet-knockout animal models have established the relationship between 5hmC and 5mC and the role of Tet proteins in their regulation.Mutant mice exhibit a general lack of overt developmental defects.Mice deficient in any individual Tet or Tet1/Tet2in combination develop to term [27,37,58].This is somewhat surprising because knockout of a single methyltransferase,in particular Dnmt1or Dnmt3b,causes embryonic lethality [63].The birth of live pups has nevertheless provided an opportunity to observe a wide spectrum of post-natal phenotypes,yielding insights into the physiological role of Tet proteins.Tet1regulates adult hippocampal neuro-genesis and cognition by controlling the prolifera-tion of neural stem cells [32].It is also implicated in the epigenetic regulation of germ cell develop-ment.Female mutant mice show impaired demethy-lation and activation of meiotic genes in PGCs and produce fewer oocytes [64].Similarly,male mutant mice display failed demethylation in a subset of im-printed genes in PGCs,potentially leading to de-velopmental abnormalities,including the growth re-tardation observed among some pups in the nextREVIEWXu and Wong 323generation [65].Tet2mutant mice are viable with normal fertility but tend to develop myeloid malig-nancies within 1year of age,similar to that observed in leukemia patients carrying TET2mutations [66].Similarly,somatic deletion of Tet3is compatible with embryonic development but leads to post-natal lethality.The mutant pups are born normal but al-most all die within the first day [26],partially owing to a suckling defect (unpublished).While Tet func-tions are clearly essential for mouse development,the factors that control phenotypic severity and in-dividual variation remain unidentified.DNA DEMETHYLATION IN ZYGOTESIn 2000,Mayer et al.reported a striking case of epi-genetic reprograming in mammalian development [20].In one-cell embryos (zygotes),at a few hours after fertilization,the 5mC signal level dropped sig-nificantly in the male pronucleus formed from the sperm but not in the female pronucleus formed from the oocyte.Bisulfite sequencing analysis by Oswald et al.confirmed the partial erasure of 5mC at three representative genomic loci in the paternal genome [19].These observations of 5mC loss specifically in paternal DNA have since been accepted as evidence for the existence of active demethylation because it occurs before the first replication of the paternal genome.806040200806040200R e l a t i v e 5m C l e v e lR e l a t i v e 5h m C l e v e lG1G2SFigure 4.Overall changes in 5mC and 5hmC levels in the pronuclear stage of mouse zygotes.The discovery of Tet1and 5hmC in the ge-nomic DNA of mouse ESCs and neuronal cells [22,67]prompted the hypothesis that conversion of 5mC into 5hmC by Tet might be involved in zygotic ing immunofluorescence staining with 5hmC-specific antibodies,several labs have observed 5hmC formation in the male pronu-cleus,most noticeably from the PN3stage onwards [26,68,69](Fig.4).The gain of 5hmC correlates well with the loss of 5mC.Mouse genetic studies have established that Tet3,the only Tet enzyme inher-ited from oocytes,is responsible for 5mC loss and 5hmC gain in the paternal genome [26,69].In ac-cordance with its responsibility in global demethyla-tion,Tet3is required for the demethylation of pater-nally methylated genomic loci,including some key pluripotency genes such as Oct4and Nanog .Are unmodified cytosines restored in the DNA strand inherited from the gametes via an active demethylation mechanism?The detection of 5hmC,as well as the higher oxidation products 5fC and 5caC,in the male pronucleus supports the active ox-idative demethylation of 5mC.However,it remains unclear whether a complete conversion from 5mC to unmodified cytosine residues takes place in the pronuclear DNA of the one-cell ing an M.SssI-assisted bisulfite sequencing method that in-corporates in vitro methylation of the DNA sam-ple into the conventional bisulfite sequencing anal-ysis [27],Guo et al.demonstrated the generation of unmodified cytosine residues in the examined re-gions of selected genomic loci in late-stage zygotes [55].Thus,in zygotes,DNA regions that were orig-inally hypermethylated in sperm or oocytes can be restored to an unmodified state via a demethylation process that is fully independent of DNA replica-tion.However,it remains to be determined whether demethylation is dependent on the BER pathway.Strong evidence also supports the passive demethylation of zygotic DNA.The evidence includes two early observations:cytoplasmic seg-regation of the maintenance methyltransferase Dnmt1in the zygote [70]and an asymmetric 5mC banding pattern in sister chromatids in the two-cell embryo,indicative of hemimethylation in the replicated zygotic DNA [41].In addition,a recent study has directly demonstrated hemimethylation in early embryos and PGCs by using genome-wide hairpin bisulfite analysis [71].What then are the relative contributions of active and passive demethylation to zygotic demethylation?The time window of oxidative demethylation overlaps with the first round of DNA replication in the embryo (Fig.4).Newly incorporated cytosine residues during pronuclear DNA replication can be left unmethylated if maintenance methylation is324Natl Sci Rev,2015,Vol.2,No.3REVIEWsuppressed,reducing methylation to half the level in gametes.Consistent with the involvement of passive demethylation,Dnmt1exhibits cytoplasmic localization in ing genome-scale single nucleotide-resolution bisulfite sequencing analysis, three labs have compared the contributions made by active removal of5mC and replicative passive dilution in mouse one-cell embryos[55,72,73]. Demethylated CpG sites spread throughout the en-tire genome.While numerous sites depend on Tet3, most depend only on replication for demethylation. Many CpGs undergo both Tet3-mediated active and replication-dependent passive demethylation concurrently,thereby presenting a scenario of Tet3-assisted passive demethylation.It is noteworthy that actively demethylated loci have also been identified in maternal DNA,albeit in smaller numbers than in paternal DNA[55,72,74].Tet3-mediated oxidative demethylation not only erases pre-existing methy-lation but also counteracts de novo methylation to maintain an unmethylated state[73].Thus,the establishment of a zygotic methylome involves both passive and active demethylation,likely in the face of de novo and maintenance methylation.Many factors likely regulate these processes to determine where and when demethylation occurs.One such factor,PGC7/Stella,binds to H3K9-dimethylated chromatin regions and protects them from Tet3-mediated5mC oxidation[75].Future studies will need to identify additional regulatory factors,in-cluding positive regulators similar to PRDM14that stimulate Tet1-and Tet2-mediated demethylation in na¨ıve pluripotent cells and developing PGCs [76],and address how they orchestrate methylation reprograming.A role for zygotic demethylation in establishing totipotency has long been suspected but has not been tested experimentally.Several promoters and enhancers of critical pluripotency genes are methy-lated in sperm but are unmethylated in oocytes and early embryos[77,78].Therefore,prompt demethy-lation is needed to reactivate paternal genes in early embryos.Gu et al.have demonstrated that genetic ablation of oocyte Tet3impairs reactivation of the paternal Oct4gene;over half of the embryos without maternal Tet3degenerate post-implantation[26]. Notably,oocyte Tet3is also required for demethy-lation and reactivation of Oct4in the donor DNA of embryos reconstructed by somatic nuclear trans-fer.Recently,the cullin-ring ffinger ligase-4(CRL4) ubiquitin ligase,which is essential for female fertil-ity,was found to promote Tet3activity[79].Ac-tive demethylation has been observed in human em-bryos as well[80,81].These observations reinforce the importance of zygotic demethylation in mam-malian development.ALTERNATIVE ACTIVE DNA DEMETHYLATION PATHWAYSTet triple knockout ESCs are devoid of5hmC in the genome,indicating an absence of other biochemical pathways for5mC hydroxylation[27,59,82].How-ever,these ESCs show only a mild increase in global 5mC levels when grown in a conventional medium. Such observations cannot be taken as evidence sup-porting the absence of other demethylation mecha-nisms unrelated to Tet.The Tet-TDG axis as a pathway for DNA demethylation is vigorously supported by biochem-ical and genetic analyses(Fig.3);however,sev-eral alternative demethylation routes might exist. Among the candidate proteins discussed elsewhere [83],DNMTs have been proposed to function as demethylases because they can activate the C5-position of the pyrimidine ring through nucleophilic addition of a conserved cysteine residue to the C6-position[84].The C5-activation mechanism involves the transfer of a methyl group from S-adenosylmethionine(SAM)to form5mC during the methylation reaction[85].While Liutkeviciute et al.have reported that bacterial and mammalian methyltransferases generate cytosine from5caC and 5hmC substrates[84,86],Shen and colleagues have observed that human DNMTs can transform5hmC and5mC into unmodified cytosine[87,88].In these studies,methyl removal or decarboxylation by DNMTs required the absence or‘muted presence’of the cofactor and the methyl group donor SAM. While observations of in vitro demethylation,de-hydroxymethylation,and decarboxylation by DN-MTs are interesting because they establish chemi-cal plausibility,evidence of their in vivo relevance is lacking.Given that depletion of SAM is unlikely in living cells,a crucial question that needs to be addressed is the mechanism underlying the poten-tial functional switch of a DNMT from methyla-tion to demethylation mode.In this context,it is worth noting that decarboxylation in vitro can also be achieved by adding high concentrations of mercap-toethanol and imidazole to imitate the active site of methyltransferases[89].Thus,the in vivo relevance of DNMT-based demethylation remains a crucial is-sue for future studies to address.Activation-induced cytidine deaminase(AID) has also been implicated in active demethylation. AID is more commonly known as a DNA-modifying enzyme required to initiate somatic hypermutation and class switch recombination in immunoglobu-lin genes in B cells.In vitro,the enzyme is able to deaminate5mC in DNA and thus convert5mC to thymine,albeit with a much lower efficiency com-pared to that of cytosine deamination[90].The。