Reconfiguration of DNA methylation in aging

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MechanismsofAgeingandDevelopment151(2015)60–70

ContentslistsavailableatScienceDirect

Mechanisms of Ageing and Development

j o ur na l h o me p a g e:w w w . e l s e v i e r . c o m / l o c a te / m e c h a g e d e v

Reconﬁguration of DNA methylation in aging

Michele Zampieri

^a,b,1

, Fabio Ciccarone

^a,b,1

, Roberta Calabrese

^a,b

, Claudio Franceschi

^c

, Alexander Bürkle

^d

, Paola Caiafa

^a,b,∗

aDepartmentofCellularBiotechnologiesandHematology,“Sapienza”UniversityofRome,Rome00161,Italy

bPasteurInstitute-FondazioneCenciBolognetti,Rome00161,Italy

cDepartmentofExperimentalPathology,AlmaMaterStudiorum,UniversityofBologna,Bologna40126,Italy

dMolecularToxicologyGroup,DepartmentofBiology,UniversityofKonstanz,KonstanzD-78457,Germany

a r t i c l e i n f o

Articlehistory:

Availableonline20February2015

Keywords:

DNAmethylation Aging

Epigeneticreprogramming 5-Methylcytosine 5-Hydroxymethylcytosine

a b s t r a c t

Acomplexinterplaybetweenmultiplebiologicaleffectsshapestheagingprocess.Theadventofgenome- widequantitativeapproachesintheepigeneticﬁeldhashighlightedtheeffectiveimpactofepigenetic deregulation,particularlyofDNAmethylation,onaging.Age-associatedalterationsinDNAmethyla- tionarecommonlygroupedinthephenomenonknownas“epigeneticdrift”whichischaracterized bygradual extensive demethylation of genome and hypermethylation of a number of promoter- associatedCpGislands.Surprisingly,speciﬁcDNAregionsshowdirectionalepigeneticchangesinaged individualssuggestingtheimportanceoftheseeventsfortheagingprocess.However,theepigenetic informationobtaineduntilnowinagingneedsare-considerationduetotherecentdiscoveryof5- hydroxymethylcytosine,anewDNAepigeneticmarkpresentongenome.Arecapitulationofthefactors involvedintheregulationofDNAmethylationandthechangesoccurringinagingwillbedescribedin thisreviewalsoconsideringthedataavailableon5hmC.

Contents

1. Introduction... 61

2. GenomicDNAmethylationpatterns... 61

2.1. DNArepetitiveelements... 61

2.2. CpGislands... 61

3. IntroductionandremovalofDNAmethylationmark... 62

3.1. EstablishmentandmaintenanceofDNAmethylationpatterns... 62

3.2. DNAdemethylation... 62

4. EffectsofenvironmentalexposureandlifestyleonDNAmethylation... 62

4.1. SAM/SAHratio... 62

4.2. DNMTregulation... 63

5. ChangesofDNAmethylationpatternsinaging... 64

5.1. DNAhypomethylationevents... 64

5.2. DNAhypermethylationevents... 66

Abbreviations: DNMTs,DNAmethyltransferases;SAM,S-adenosyl-methionine;CGIs,CpGislands;5mC,5-methylcytosine;LTR,long-terminalrepeat;LINE,longinter- spersednuclearelement;SINE,shortinterspersednuclearelement;DMRs,differentiallymethylatedregions;5hmC,5-hydroxymethylcytosine;TET,ten-eleventranslocation;

5fC,5-formylcytosine;5caC,5-carboxylcytosine;BER,baseexcisionrepair;SAH,S-adenosylhomocysteine;ROS,reactiveoxygenspecies;WGBS,whole-genomebisulﬁte sequencing.

∗Correspondingauthorat:DepartmentofCellularBiotechnologiesandHematology,“Sapienza”UniversityofRome,Rome00161,Italy.Tel.:+39649976530;

fax:+39644231961.

E-mailaddress:caiafa@bce.uniroma1.it(P.Caiafa).

1 Theseauthorscontributedequallytothiswork.

http://dx.doi.org/10.1016/j.mad.2015.02.002

by-nc-nd/4.0/).

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-312460 Erschienen in: Mechanisms of Ageing and Development ; 151 (2015). - S. 60-70

https://dx.doi.org/10.1016/j.mad.2015.02.002

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6. 5hmCandaging ... 66

7. Conclusionsandperspectives... 67

Acknowledgements... 67

References... 67

1. Introduction

Longevityisnotuniformthroughouthumansanditislargely inﬂuencedbyenvironmentandlifestylechoices.Thereisanover- whelmingscientiﬁcconsensusthatdirectionalepigeneticchanges contributetoagingandalterthelifespan(Huidobroetal.,2013;

Issa,2014;VlamingandvanLeeuwen,2012).Infact,onlyabout 20–30% of humanlifespan seemsto be attributable togenetic effects,whichimpliesthatthemajorpartofthevariationisdue tonon-geneticfactors(Herskindetal.,1996;Mitchelletal.,2001;

Poulsenetal.,2007).Itisbecomingincreasinglyclearthatsome aging-associated diseases have an epigenetic origin (Huidobro etal.,2013;Issa,2014;VlamingandvanLeeuwen,2012).Undoubt- edly, consideringthatepigenetic regulationis indispensablefor many aspectsof genomefunctions,the alterationof epigenetic landscapescouldpotentiallyaccount forthecomplex natureof aging by explaining theperturbation of themultitude of gene families andcellularpathwaysthat areknowntocontributeto thisprocess(DeCarvalhoetal.,2010;Kirkwood,2005).Therefore, expectationsarerunninghighforinsightsintotheepigeneticbasis oftheagingprocess.

2. GenomicDNAmethylationpatterns

DNAmethylationisthefirstepigeneticmodificationidentified onDNA.TheenzymesdeputedtotheDNAmethylationreaction belongtothefamilyoftheDNAmethyltransferases(DNMTs)which introduceontotheC5positionofcytosineresidueamethylgroup derivingfromS-adenosyl-methionine (SAM)(Fig.1A). In mam- mals,thethreeactiveDNMTenzymes,namelyDNMT1,DNMT3A andDNMT3B,preferentiallymodifycytosinefollowedbyagua- nineresidue,commonlyknownasCpGdinucleotide(Bird,2002;

Jurkowskaetal.,2011;MirandaandJones,2007;SuzukiandBird, 2008).However,inembryonicstemcellsmethylatedcytosinecan alsobepresentinnon-CpGpositions,mainlyinCpAcontext,thanks totheaction ofDNMT3A/3B enzymesinconcertwithDNMT3L (Arandetal.,2012;Zilleretal.,2011).

CpGdinucleotidedensityisgenerallylowinthebulkofgenome butlargelyincreasinginspeciﬁcregionsreferredtoasCpGislands

(CGIs). Notably, mammalian genome is dominated by methyl- atedDNAwhileCGIs,which accountforonly1–2%oftotal CpG dinucleotides,aregenerally depletedofDNAmethylation mark.

Methylatedcytosine,knownas5-methylcytosine(5mC),is con- sideredtheﬁfthbaseofDNAduetoitsnon-randomdistribution ingenomewhichintroducesanepigeneticcode(Bird,2002;Jones andTakai,2001;SuzukiandBird,2008;TakaiandJones,2002).

2.1. DNArepetitiveelements

CpGmethylationofthebulkofgenomeassuresgenomicsta- bility through the control of DNA repetitiveelements, suchas interspersedandtandemrepeats,whichcompriseatleasthalfof thehumanDNA(JonesandTakai,2001;Landeretal.,2001).The interspersedrepeats,includinglong-terminalrepeats(LTR),long interspersednuclearelements(LINE),shortinterspersednuclear elements(SINE),areretrotransposableelementsmaintainedinac- tive through DNA methylation to avoid the risk of insertional mutagenesis (Robertson and Wolffe, 2000; Yoder et al., 1997).

Tandem repeats are present in centromeric, pericentromeric, andsubtelomericregionsandarealsoconstitutivelymethylated (Jurkowskaetal.,2011;SuzukiandBird,2008)(Fig.1B).DNAmeth- ylationof centromericregions hasbeendemonstrated toavoid centromererecombinationandabnormalcentromerelength(Jaco etal.,2008).Inthesameway,DNAmethylationisanimportant repressorofDNArecombinationattelomeresandanegativereg- ulatoroftelomerelength(Blasco,2007;Gonzaloetal.,2006).In addition,CpGmethylationisfundamentalfortheinactivationof the X chromosome and the establishment and maintenance of mono-allelicexpressionofimprintedgenes(ChaligneandHeard, 2014;Changetal.,2006;MirandaandJones,2007;Weaverand Bartolomei,2014).

2.2. CpGislands

CGIsareDNAregionsofmorethan500basepairscharacter- ized byat least 50% of GC content.60% of CGIsare associated withannotatedgenepromotersand areunmethylatedallowing thetranscriptionofthecorrelatedgene(Jones,2012;Suzukiand

Fig.1.DNAmethylationreactionandpatterns.A.Conversionofcytosine(C)to5mCbyDNMTenzymeswhichcatalyzethetransferofamethylgroup(CH3)fromSAMto the5-carbonpositionofcytosine.Oxidationof5mCandformationof5hmCbytheFe(II)and␣-ketoglutarate(␣-KG)-dependentTETenzymes.B.Schematicrepresentation ofDNAmethylationpatternsthroughoutachromosome,showingrepetitiveelementsandanexampleofgenewithCGI-promoter.Arrowsindicatetranscriptionstartsites, withbluecolorindicatingactivetranscription,whileredcolorindicatesrepressedtranscription.Whitecircles,unmethylatedCpGs;blackcircles,methylatedCpGs.(For interpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

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Bird,2008;TakaiandJones,2002).Onthecontrary,themethyl- ationstateofCpGdinucleotideslocalizedingenebodydoesnot affectgenetranscriptionbut probablyimpedestheactivationof alternativepromoters(Maunakeaetal.,2010).Theremaining40%

ofCGIsis localizedinintergenicandintragenicregionsand are definedas “orphan CGIs” becausetheyare notassociated with annotatedpromotersalthoughpresentingpromoter-likefeatures (Illingworthetal.,2010).Upto2KbdistantfrompromoterCGIs, particularregionscharacterizedbyalowerdensityofCpGs(around 1/10ofCGI)anddefinedasCGIshoresarealsoinvolvedinreg- ulationofgeneexpression.CGIshoresoftenoverlapwithtissue specific-differentiallymethylatedregions(DMRs)innormaltissues (Doietal.,2009;Irizarryetal.,2009)(Fig.1B).

3. IntroductionandremovalofDNAmethylationmark

DNA methylation patterns – characterized by widespread genomicmethylation punctuatedwithunmethylatedregulative regions,suchasCGIs–needtobepreserved.Thelossofthebulkof genomemethylationand/ortheintroductionofanomalousmethyl groupsontogenerallyunmethylatedregionsoccurphysiologically inagingbutalsoinseveralpathologicalstates(Bacalinietal.,2014;

Calabreseetal.,2012;KulisandEsteller,2010;Luetal.,2013).

3.1. EstablishmentandmaintenanceofDNAmethylationpatterns

Inmammals,DNAmethylationpatternsareestablishedduring embryonicdevelopmentafterawaveofgenome-widedemethyla- tionoccurringinpreimplantationembryosoonafterfertilization aimedatrestoring totipotency (Feng et al.,2010; Santoset al., 2002). A second wave of DNA demethylation followed by re- methylationoccurs duringgermline developmentin primordial germcells forthegenerationoftotipotentgameteswithproper speciﬁcgenomicimprints(Ciccaroneetal.,2012;Fengetal.,2010;

Hajkova,2011).TheestablishmentofDNAmethylationseemsto bepreferentiallydue totheaction ofthedenovoDNMT3Aand DNMT3Benzymes,alsoincooperationwithDNMT3Lasobserved ingametogenesisforimprintedgenesandrepetitiveelements(Jia etal.,2007;Katoetal.,2007).Onceintroduced,DNAmethylation patternsmustbemaintainedduringcelldivisioninordertopre- serve cellidentity.DNMT1 isconsidered themaintenance DNA methyltransferasebeingrecruitedduringreplicationontheneo- synthetizedDNAﬁlamentbyPCNAandUHRF1/NP95tocopythe methylationpatterns(Bosticketal.,2007;Jurkowskaetal.,2011;

Schneideretal.,2013).However,recentevidencehasclariﬁedthat DNMT3AandDNMT3Barealsoinvolvedinmaintenanceofmeth- ylationpatternsacrossspeciﬁcDNAregionswhileDNMT1canalso functionasdenovomethyltransferase(Feltusetal.,2003;Jairetal., 2006;Liangetal.,2002;Sharmaetal.,2011).

TogetherwiththemaintenanceofDNAmethylationacrosscell division,thepreservationoftheunmethylatedstateofDNAregu- lativeregionsisalsonecessary.Inparticular,severalmechanisms have beenidentified for the control of unmethylatedCGIs and unmethylatedallelesof imprintedDMRs,suchasintrinsic DNA sequencesandstructures(Ginnoetal.,2012;Horietal.,2002),the presenceofepigeneticmarksassociatedwithactivechromatincon- formation(Jinetal.,2014;Ooietal.,2007;Williamsetal.,2011) andtheabilityofpost-translationalmodificationsandtranscrip- tionfactorstoavoidtheactionofDNMTenzymes(Brandeisetal., 1994;Ciccaroneetal.,2014;Fedoriwetal.,2004;Guastafierroetal., 2013;Zampierietal.,2012).

3.2. DNAdemethylation

FailureofDNAmethyltransferaseactionduringDNAreplication inducesa lossof 5mCmarkknownaspassiveDNA demethyla-

tionprocess(Fengetal.,2010;Jurkowskaetal.,2011).Multiple mechanismscanbeinvolvedinpassiveDNAdemethylationinclud- ingdown-regulationofDNMTenzymes(Odaetal.,2013;Reichard etal.,2007;Zampierietal.,2009),cytosoliclocalizationofDNMT (CardosoandLeonhardt,1999;Jurkowskaetal.,2011),impairment ofDNMTrecruitmentonDNA(Bosticketal.,2007;Odaetal.,2013), decreaseoftheDNMTsubstrateSAM(Ulreyetal.,2005),inhibition ofDNMTenzymaticactivity(Caiafaetal.,2009;Fangetal.,2007) (Fig.2A).

Onlyrecently,severalpieces ofevidence havesustainedthe existenceofmolecularmechanismsinvolvedintheactiveremoval of 5mC through the formation of 5-hydroxymethylcytosine (5hmC).5hmCis catalyzedby threeFe(II) and␣-ketoglutarate- dependentDNAhydroxylases,theten-eleventranslocation(TET) familyenzymes(Fig.1),anditisapivotalintermediateofactive DNA demethylation (Delatte et al., 2014; Pastor et al., 2013).

DNMT3AandDNMT3Barepossiblyabletoconvert5hmCdirectly intocytosine(Chenetal.,2012a),althoughmuchmoreevidence suggeststheneedoffurthermodiﬁcationsof5mCand5hmCin ordertotriggerDNAdamageresponseforthereintroductionof unmethylatedcytosynes(Pastoretal.,2013).Consistently,itera- tivemodiﬁcationsof5hmCbyTETproteinsinto5-formylcytosine (5fC)andthen5-carboxylcytosine(5caC)canbeactivelyremoved bythebaseexcisionrepair(BER)pathway(Hashimotoetal.,2012;

Itoetal.,2011).Anadditionalmechanism,althoughstillcontrover- sial,involvesthedeaminationof5mCand5hmCbythedeaminase enzymesAID/APOBEC(Hashimotoetal.,2012;Nabeletal.,2012;

Pastoretal.,2013)(Fig.2B).Notably,5hmCisnotrecognizedby DNMT1enzyme leadingalsoto apassive lossof 5mC.In addition, 5fC and 5caC can undergo replication-dependent dilution (Inoueetal.,2011;Pastoretal.,2013;ValinluckandSowers,2007) (Fig.2A).

4. EffectsofenvironmentalexposureandlifestyleonDNA methylation

DNA methylation studies carried out on monozygotic twins highlightedthatgenomeofyoungpairsisepigeneticallysimilar whilethepatternsofagedonesareclearlydissimilar(Fragaetal., 2005).Notably,aging-associatedDNAmethylationchangesarepar- ticularlyevidentinmonozygotictwinswhohadspentalongperiod oftheirlivesapart,suggestingwhytheirmedicalhistoriesaredif- ferent(FeilandFraga,2012;Poulsenetal.,2007).DNAmethylation changescanbemediatedbyseveralextrinsicfactorsderivingfrom lifestyle,dietandenvironmentalexposure.

Factors that can positively or negatively affect lifespan are knowntoinﬂuenceDNAmethylationpatterns.Forexample,smok- ingattitudemayhavepro-agingeffectsinducingDNAmethylation changesofgenesinvolvedinage-associateddiseasessuchascar- diovascularpathologiesandcancer(BesingiandJohansson,2014;

Breitlingetal.,2011;LeeandPausova,2013;Noreenetal.,2014).

Onthecontrary,physicalactivity,antioxidantintakeandcaloric restrictionmayexertanti-agingactionalsobycounteractingdetri- mentalDNA methylationchanges(Fangetal., 2007;Ionsetal., 2013;LiandTollefsbol,2010;Miyamuraetal.,1993;Rönnetal., 2013).

Part of these factors inﬂuences DNA methylation patterns alteringtheavailabilityoftheDNMTcofactorSAMordirectlyinter- feringwiththeregulationofDNMTenzymes(Fangetal.,2007;Lee andPausova,2013;Martinez-ZamudioandHa,2011;Ulreyetal., 2005).

4.1. SAM/SAHratio

SAMis themethyldonorinthereactionof DNMTenzymes.

Astheﬁnal productofreactionistheS-adenosylhomocysteine

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Fig.2.DNAdemethylationprocesses.A.MechanismsaffectingDNMTactionduringDNAreplicationandthusinducingpassiveDNAdemethylation.B.Mechanismsinvolved intheactiveremovalof5mCviaenzymaticmodiﬁcationsandactivationofDNArepairpathways.

(SAH),theSAM/SAHratioisfundamentalforproperDNAmethyl- ationreactionsandisguaranteedbytheefﬁciencyofone-carbon metabolismcomprisingenzymesinvolvedinthesynthesisofSAM, e.g.,methioninesynthase(MTR),andcatabolismofSAH,e.g.,cys- tathioninesynthase(CS).Inappropriateintakeofdietarymolecules involvedinSAM synthesisorSAHdegradation,suchasmethio- nine,choline,betaine,folicacid,B6andB12vitamins,caninﬂuence DNAmethylationpatterns(Ulreyetal.,2005).Moreover,several environmentalandnutritionalcompoundsarealsoabletoaffect SAM/SAHratiobybecomingmethylatedthemselves(e.g.,arsenic) (ReichardandPuga,2010)orbyinhibitingone-carbonmetabolism enzymes(e.g.,theeffectofalcoholonMTRenzyme)(Varela-Rey etal.,2013).

Arsenic is a common toxic environmental contaminant of water, soiland food.Apartfromubiquitouspresence inearth’s crust,humanactivities,mainlymassiveindustrialization,havecon- tributed to increase arsenic contamination (Sharma and Sohn, 2009). After entering the cells, arsenic undergoes methylation reactionbythearsenitemethyltransferase(AS3MT)enzymepro- ducingseveralmethylatedarseniccompounds(Kojimaetal.,2009).

Arsenicmethylationwasinitiallybelieved tobeadetoxiﬁcation reaction(Gebel,2002),butmorerecentevidenceinvalidatedsuch anhypothesisshowinganincreasedtoxicityofspeciﬁcmethyl- ated intermediate metabolites (Kojima et al., 2009; Sun et al., 2014).ConsideringthatSAMisthemethyldonorcofactoralsofor arsenicmethylation,excessivearseniclevelsin acellcanlower theSAM/SAHratioinducingDNAhypomethylation(Reichardand Puga,2010).Besidesarsenic and othermetals suchas bismuth andselenium(Hirnerand Rettenmeier,2010), alsodietarycon- stituentscanundergoenzymaticmethylationinthecell.Thisis thecaseofcatecholstructure-containingcompoundssuchasthe mostabundantpolyphenolingreentea,the(−)-epigallocatechin- 3-gallate (EGCG), and the coffee polyphenols caffeic acid and

chlorogenicacid,which arereadilymethylatedbythecatechol- O-methyltransferase(COMT)(Fangetal.,2007;LeeandZhu,2006) (Fig.3).

CigarettesmokingisalsodeeplyinvolvedinalterationofDNA methylation patternsand it is an environmentalrisk factor for chronicdiseases(Leeand Pausova,2013;Zeilingeretal.,2013).

EventsofDNAhypomethylationcausedbycigarettesmokecanpar- tiallydependonarsenic,whichisoneofthecarcinogensderiving fromsmoking.Demethylationofspeciﬁcgeneshasbeensignif- icantlyassociatedwithcigarette smokingattitudesothatF2RL3 geneisconsideredapromisingbiomarkerofcurrentandlifetime smokingexposure(LeeandPausova,2013;Zhangetal.,2014).An associationbetweentobaccosmokingandanincreasedincidence ofaberrantpromotermethylationofthep16INK4AandMGMTgenes hasbeenalsodescribed(Liuetal.,2006).Itisinterestingthatthe samegeneshavebeenshowntoundergohypomethylationandre- expressionaftertreatmentwiththepolyphenolEGCG(Fangetal., 2003;LiandTollefsbol,2010)suggestingthatdifferentcompounds mayhavedifferentoutcomesonthemethylationstateofaspe- ciﬁcDNAlocus.Theseeventsarerelevantinthecaseofp16INK4A andMGMTconsideringthattheyarefrequentlyhypermethylated incancerbutalsoinaging(Liuetal.,2006;Matsubayashietal., 2005;Soetal.,2006)

4.2. DNMTregulation

Besides an indirect regulation of DNA methylation patterns through modulationof SAM pools,severalcompoundscan also directlyinﬂuencetheexpressionoractivityofDNMTs.Evidence ofacompetitiveinhibitionofDNMTactivityaffectingtheentryof cytosineintoactivesitehasbeendemonstratedforEGCG,curcumin andthesoy-beanisoﬂavongenistein(Fangetal.,2005,2003;Lee etal.,2005;Liuetal.,2009;Xieetal.,2014).Nutritional(e.g.,cur-

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Fig.3. Environment,dietandlifestyleinﬂuenceDNAmethylation.NutritionalandenvironmentalcompoundsareabletoaffectDNAmethylationreactiondiminishing SAM/SAHratioorregulatingexpression,activity,recruitmentofDNMTenzymes.

cumin,alchol)andenvironmentalfactors(e.g.,arsenic,cadmium) havealsobeendemonstratedtoderegulatetheexpressionofDNMT geneswithunknownmechanismformostofthem.Accordingto timeanddoseofexposuretospeciﬁccompounds,theeffectson DNAmethylationpatternscanvaryfromextensiveDNAhypometh- ylationtolocalizedhypermethylation(Martinez-ZamudioandHa, 2011;Reichardetal.,2007;Varela-Reyetal.,2013;Yuetal.,2013).

AmongthemechanismsabletoinduceDNAhypermethylation,the effectofDNAdamagemediatedbygenotoxicagentsandreactive oxygenspecies(ROS)hastobeconsidered.Accordingly,itiswell acceptedthatDNMTsarerecruitedtoDNArepairsitesinducing methylationofCpGsneighboringDNAbreaks(Cuozzoetal.,2007;

Moranoetal.,2014;Mortusewiczetal.,2005).Inthiscontext,sev- eralDNAdamagingcarcinogensalsopresentincigarettesmoking mayinduceaberrantDNAhypermethylation(Huangetal.,2013;

LeeandPausova,2013).Anindirectcontributiontothismecha- nismcanbemediatedbycadmium,aweakgenotoxiccarcinogen highlyaccumulatedintobaccoleaves(SatarugandMoore,2004).

Cadmiumexposurehasbeenassociatedwithhypermethylation- dependentsilencingofDNA repair enzymes(Zhou etal., 2012) andcadmiumcanalsocompetewithzincbindingofDNArepair enzymesdelayinginthiswaytheirturnover/activity(Faturetal., 2003;Lutzenetal.,2004)andpossiblyfavoringDNMTactionat DNAbreaks(Fig.3).

5. ChangesofDNAmethylationpatternsinaging

Severalstudiesfromthelastthreedecadeshaveclearlydemon- stratedthatchangesinDNAmethylationpatternsassociatewith chronologicalagingacrossnearlytheentirehumanlifespan(Alisch etal.,2012;Belletal.,2012;Bocklandtetal.,2011;Boksetal.,2009;

Bollatietal.,2009;Christensenetal.,2009;Florathetal.,2013;

Garagnanietal.,2012;Gentilinietal.,2013;Hannumetal.,2013;

Heynetal.,2012;McClayetal.,2014;Rakyanetal.,2010).This phenomenon,commonly deﬁned as“epigenetic drift”,accounts fortheevidencethatepigeneticsimilaritiesbetweenyoungindi-

viduals are lost over time leading to divergent methylomes in elderlypopulation(Boksetal.,2009;Fragaetal.,2005;Heynetal., 2012).DNAmethylationdriftisanon-directionalchangeofmethy- lomeasitinvolvesbothhypermethylationandhypomethylation eventsandithasbeenassociatedwiththeprogressiveaccumu- lationof epigenetic damagedue toenvironmentalfactorsorto spontaneousstochastic errorsin theprocess of transmissionof theepigenetic information.Thisphenomenon leadstobasically unpredictabledifferences in the methylome among aging indi- viduals.Theseobservationsseemtodenythepossibilitythatthe driftmayreﬂectaprogrammedchangeofthemethylationcode.

However,partofmethylationchangesthatareobservedwithage involvespeciﬁcregionsofthegenomeandaredirectional.Infact, severalstudiesindicatetheexistenceof aging-associateddiffer- entiallymethylatedregions(a-DMRs),clustersofconsecutiveCpG siteswhichexhibitchangeovertime inthesamedirection.The existenceofa-DMRsindicatesthatpartofmethylationchangesis notstochastic,butinsteadtheycouldbeassociatedwithbiological mechanismscloselyinvolvedintheagingprocessorlongevity.

Most of the initial analyses in aging have been focused on candidateloci withpotentialrelevanceforage-related diseases.

Collectively,thesestudiesshowedthataging, similartocancer, associateswithgradualbutprofoundchangesinDNAmethylation where epigenomeis markedby genome-widehypomethylation togetherwithsite-speciﬁchypermethylationpreferentiallyoccur- ringatCGIpromoters.Notably,althoughthefunctionalimportance of both events in aging progression and outcome remains to beascertained,theseobservationssupportthenotionthatthese changesmayconcomitantlycontributetodevelopmentofaging- associateddiseases.

5.1. DNAhypomethylationevents

GlobaldeclineofgenomicCpGmethylationisthepredominant eventin aging.Thischangeis widespreadasit typicallyoccurs atrepetitivesequencesdispersedthroughoutthegenomesuchas

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Fig.4.Age-associatedchangesofDNAmethylome.SchematicrepresentationofDNAmethylationpatternsinyoung(A)vsolder(B)people.ThetoplinerepresentsDNA containingthreegenesandthreeclassesofinterspersedrepeats.Arrowsindicatetranscriptionstartsites,withbluecolorindicatingactivetranscription,whileredcolor indicatesrepressedtranscription.Exonsareshownindarkblue.EachbottomlinerepresentsthemethylationstateofDNAasdetectedforasingleindividual.White circles,unmethylatedCpGs;blackcircles,methylatedCpGs.Hypomethylatedandhypermethylatedaging-associatedDMRsarehighlightedinblueandred,respectively.(For interpretationofthereferencestocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

SINEs(i.e.,Aluelements)andLTR(i.e.,HERV-K)(Bollatietal.,2009;

Christensenetal.,2009;JintaridthandMutirangura,2010).How- ever,age-relatedlossofmethylationdoesnotaffectrepetitiveDNA equally.HypomethylationofAluandHERV-Ksequencesoccursat differentageswhileitdoesnotaffectLINE-1repeatssigniﬁcantly (Fig.4)(Bollatietal.,2009;JintaridthandMutirangura,2010).It isreasonabletoassumethatthiseventaccountsfortheincreased genomeinstabilityobservedintheelderly(VijgandDolle,2007).

However,progressiveage-dependentlossofmethylationalsocon- cernsspeciﬁcgenepromoters,includingITGALandIL17RCwhose demethylation-dependenttranscriptionalactivationhasbeenpro- posedtotriggerautoimmuneresponses(Weietal.,2012;Zhang etal.,2002).

More recently, the application of novel next-generation sequencing technologies for genome-wide assessment of DNA methylation levels in aging research haspermitted the conﬁr- mationof theseearlier observations. Studiesinvestigating DNA methylationonthewholehavebeenparticularlyinstructivefor DNA hypomethylationevents(Heyn et al.,2012; McClayet al., 2014).Infact,bywhole-genomebisulﬁtesequencing(WGBS),Heyn etal.(2012)comparedtheDNAmethylationstateofmorethan90%

ofallCpGspresentinthegenomebetweennewbornandnonage- narian/centenariansamples.AsigniﬁcantlossofmethylatedCpGs wasfoundinthecentenarianvsnewbornDNAs.Thiswasobserved for allchromosomes and concernedall genomiccompartments suchaspromoters,exonic,intronicandintergenicregions.Most ofthesechangeswerefocalandtheagedgenomewasthusless homogeneouslymethylatedwithrespecttothenewbornaccording totheage-dependentepigeneticdrift.

However, a part of methylation changes involved several neighboringCpGs.Amorereﬁnedsearchofdomainscontaining consecutiveCpGunitsshowingunidirectionalmethylationchanges actuallyrevealedthatabout2.2%oftotalgenomicCGsiteswere locatedinregionsthatweredifferentiallymethylatedinthecente-

narianwithrespecttothenewborn.Inagreementwithprevious observations,most oftheseDMRswere locatedinintronic and intergenic regionsand commonly colocalizedwithinterspersed repetitiveDNAelements.Interestingly,DMRsoftencorresponded tolaminaassociateddomains(LADs),wherecancer-specificmeth- ylation changes had been observed (Berman et al., 2011).The functionalsignificanceofLADs’hypomethylationincancerandin agingremainstobedefined.However,afar-reachingimpactofthis eventontheepigeneticregulationofgenomecanbeenvisaged.

Infact,theLADsdeﬁnelargeheterochromatincompartmentsof thegenomewhichembedkeydevelopmentalandcell-typespe- ciﬁcgenesthataremaintainedinanepigeneticallyrepressedstate (Guelenetal.,2008;Harretal.,2015;Peric-Hupkesetal.,2010;

Reddyetal.,2008).Hence,aneventofhypomethylationofthese regionsmaycauseorreﬂectafault ofthemechanismsthatare centraltothedevelopmentandconservationofnormalstatesof differentiationandtissue-speciﬁcpatternsofgeneexpression.

Concerningregulatoryorcodingsequences,suchaspromoters and exons, onlyabout 20%of theDMRscolocalized withthese regions and more than 80% of them underwent hypomethyla- tionin theelderly.Thisevent,however, waslargelydependent on promoter CpG content being much more common in CpG- poor/tissue-speciﬁc gene promoters than in CGI/housekeeping genepromoters.Signiﬁcantly,someoftheseDMRscolocalizedwith promotersofgenesinvolvedintheagingprocesssuchasSirtuins andIGFsignalingpathwaycomponents(Heynetal.,2012).

Recently,this findinghasbroadlybeenvalidatedin acohort of morethan 700individualsaged 25–92years. In fact,alsoin thiscase,agingwaspredominantlyfoundassociatedwithmeth- ylationdeficit.About60%ofage-associatedDMRsactuallyshowed age-relatedhypomethylationandcolocalizedwithbindingsitesfor chromatinregulatoryproteins,suchasCTCFand Polycombpro- teins,orspecifichistonemodificationstypicallyassociated with active chromatin (McClay et al., 2014). These findings indicate

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thatage-relatedhypomethylationmayleadtoglobalchromatin changesofpotentialfunctionalrelevancefortranscriptionalregu- lation.

5.2. DNAhypermethylationevents

Besides extensive genome-wide hypomethylation, aging involvesaprogressivegainofDNAmethylationthatmarkstheloss ofexpressionofspeciﬁcgenes.Infact,themajorityofage-related hypermethylated sites corresponds to CGI-promoters, where methylation often correlates withsuppression of transcription.

Thisaspectis ofparticular interestasit conferstomethylation changes,which accompany aging,the featuresof an epigenetic reprogramming.

Drivenbytheaccidentalobservationthatcancer-relatedhyper- methylationoftheCGI-promoterofestrogenreceptor(ER)gene arisesasadirectfunctionofageinnormaltissues(Issaetal.,1994), themajorityofinitialinvestigationsintoCGImethylationinaging focusedonspeciﬁcgenesselectedonthebasisoftheirinvolve- mentinthepathogenesisofcancerorotherage-relateddiseases.

Thisstrategyhighlightedthatagingviolates,inatime-dependent manner,theunmethylatedstateofCGIsofgenesinvolvedintumor suppression(p16INK4A(Soetal.,2006),CHD1(Wakietal.,2003), RASSF1(Wakietal.,2003),LOX(Soetal.,2006),RUNX3(Soetal., 2006),N33(Ahujaetal.,1998)andTIG1(Soetal.,2006)),genome stabilityandrepair (hTERT(Silvaetal.,2008),MLH1(Nakagawa etal.,2001),MGMT(Matsubayashietal.,2005)andOGG(Madrigano etal., 2012)),metabolism (e.g., COX7A1 (Ronn etal., 2008) and CRAT(Madriganoetal.,2012)),differentiationandgrowth(MYOD1 (Ahuja et al.,1998), c-Fos (Choiet al., 1996), IGF-2 (Issa et al., 1996)), regulationofimmune response(INFG(Madrigano etal., 2012)), coagulation (F3 (Madrigano et al., 2012)) and connec- tivetissue homeostasis(CollagenI(Takatsu etal.,1999)).Taken together, these studies also suggested that physiological aging couldpredisposeonetoage-relatedpathologicalphenotypesvia amethylation-relatedgenesilencing.Itisinterestingtoobserve thathypermethylationinage-relateddiseasesaffectsessentially allCpGsites withina CGI, whereas themethylation pattern in physiologically-agednormalindividuals ispartialandheteroge- neous.Therefore,partialmethylationchangesaccumulatinginthe aginggenome maypredispose one tothedevelopmentof age- relateddisease,inwhichthemethylationchangesarethereafter aggravatedbyamethylationspreadingmechanism(Wongetal., 1999).Examplesofsuchaprecursor-productrelationshipofaging andage-relateddiseaseshavebeenproposedforprostate,bladder, coloncancersandAlzheimer’sdisease(Florletal.,2004;Neuhausen etal.,2006;Shenetal.,2005;Siegmundetal.,2007).

Later genome-wide investigations further conﬁrmed that age-associated hypermethylation happens preferentially at CGI promoters(Alischetal.,2012;Belletal.,2012;Bocklandtetal., 2011;Christensenetal.,2009;Florathetal.,2013;Garagnanietal., 2012;Gentilinietal.,2013;Hannumetal.,2013;Heynetal.,2012;

McClayetal.,2014;Rakyanetal.,2010;XuandTaylor,2014).Of note,thischangedoesnotequallyimpactCGIshoreregions,which insteadundergosimilarproportionsofhyper-andhypomethyla- tionevents(Florathetal.,2013;McClayetal.,2014).

Althoughtheuseofarray-basedtechniquesfocusingmainlyon genepromotersandCGIsindicatedhypermethylationasprevalent eventinaging(Alischetal.,2012;Belletal.,2012;Bocklandtetal., 2011;Boksetal.,2009;Christensenetal.,2009;Florathetal.,2013;

Hannumetal.,2013;Rakyanetal.,2010;XuandTaylor,2014),age- associatedhypermethylationseemsaphenomenon ofrelatively lowmagnitude.Fewerthana hundredCpGshaveactuallybeen observedtomethylatewithageoveraboutatotalamountof37,000 CGIsinthehaploidhumangenome,conﬁrmingthatCGIhyper- methylationinagingisrelativelyuncommon.Thisclearlyemerges

fromthestudiesthatinterrogatedthegenomeasawhole(Heyn etal.,2012;McClayetal.,2014).Forexample,astudybasedon WGBSshowedthatonlyabout13%ofageassociatedDMRswere hypermethylatedincentenarianscomparedtonewborns,thussug- gestingthathypomethylationispredominantinaging(Heynetal., 2012).

Despitethis, aging-associatedDMRsaremore likelytobein CGIsundergoingmethylationwithage(McClayetal.,2014).Consis- tently,hypermethylationfrequentlyoccursatgeneswithpotential relevance for age-related phenotypes/diseases including genes involvedindevelopment(protocadherins,homeoboxgenes)and signaling(MAPKpathways’members,ryanodinereceptors)asso- ciatedwithcancer,longevity,senescenceandneurodegeneration (Belletal.,2012;Hannumetal.,2013;McClayetal.,2014;Rakyan et al., 2010; Xu and Taylor, 2014). It is interesting toobserve that,althoughmostanalyseshavebeenperformedonbloodcells, hypermethylatedaging-relatedDMRsseemtobelargelyshared by multiple tissues (Horvath et al., 2012; Rakyan et al., 2010;

Teschendorffetal.,2010).ThissupportstheviewthatDNAmeth- ylationchangesdonotoccurrandomlyinthecontextofthehuman genomebutaredirectedtoregionssharingcommonfeatures.

Notably,age-associatedhypermethylatedDMRsin differenti- atedtissuesoftenoverlapwithpromotersofgenesthatinstemcells havebivalentchromatinmarks(H3K4me3andH3K27me3)andare targetofthepolycombrepressivecomplex2(PRC2)(Hannumetal., 2013;Heynetal.,2012;Rakyanetal.,2010;Teschendorffetal., 2010;XuandTaylor,2014).Manyofthesegenesencodetranscrip- tionfactorsnecessaryfordifferentiationandarealreadytargetof epigeneticderegulationinstemcells duringaging,likelyunder- lyingtheobserveddeclineofstemcellfunction(Beermanetal., 2013;Borketal.,2010;BrackandRando,2007).Thiswouldsuggest thattheage-associatedmethylationdefectsobservedindifferen- tiatedtissuesmayreflectchangesoccurringintheagedstemcell population.Moreover,thesamestemcell-likebivalentmarksand PRC2occupancyhavebeenrecognizedtopredisposetumorsup- pressorgenepromoterstoDNAhypermethylationincancer.Infact, thereissignificantcorrespondencebetweenthosegenesunder- goinghypermethylationinagingandgenesundergoingthesame eventincancer(Teschendorffetal.,2010)andincancer-associated conditionssuchasobesity,inflammationandsmokingaddiction (Issa,2011;Issaetal.,2001;Selamatetal.,2012;Suzukietal.,2009;

Xuetal.,2013).Theseobservationslinkstemcellagingtocancer riskandsuggestthatagingmayelicitanepigeneticswitchfromless stablehistone-basedgenerepressioninstemcellstopermanent DNAmethylation-basedgene repressionincancercells (Xuand Taylor,2014).Takingcancerasanexample,thisepigeneticmech- anismcouldbeamodelexplaininghowtheagingprocesscould predisposeonetoage-speciﬁcphenotypes/diseases.

Allinall,itisevident thatsomespecificDNAregionsdirec- tionallyundergoDNAmethylationchangesacrossagedindividuals probablyas aconsequence ofsharedchromatinfeatures. How- ever,thesesite-specificeventsco-existwiththeepigeneticdrift accordingtowhichdeviationofinter-individualgenomicmethyl- ationpatternsoccursovertime(Fig.4).Onepossibleexplanation isthatagingmightprimarilyintroduceageneraldisorderinthe methylationpatternswhichcouldbethenfollowedbytheselection andclonalexpansionofthosecellsbearingmethylationdefectsin specificregionsofthegenomewherethesedefectswouldbetoler- atedandgivethecellsadvantagesofsurvivalorproliferation(Issa, 2014).

6. 5hmCandaging

TheidentiﬁcationofthenewDNAepigeneticmark5hmCopens newperspectivesforthestudyof epigeneticreprogrammingin

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aging.OurknowledgeofDNAmethylationpatternsinbothphys- iologicalandpathologicalconditionsindeedneedsarevaluation afterthediscoveryof5hmC.Thisisduetothefactthatconventional bisulfitesequencingmethod,whichhasbeengenerallyconsidered thegoldstandardmethodforDNAmethylationanalysis,isnotable todiscriminatebetween5mCand5hmC.Therefore,studiesbased onconventionalbisulfitemodificationactuallymaskthecontribu- tionof5hmC(Huangetal.,2010).

Information regarding 5hmC in aging process is currently limited.Onlyfewreportshaveaddressedthisissuefocusingatten- tion on 5hmC changes in aged mice. Mouse cerebellum and hippocampusshowanincreaseof5hmClevelswithagingwhich canbepreventedbycaloricrestriction,awell-knownphenomenon associated withlongevity(Chen etal., 2012b; Chouliarasetal., 2012; Szulwachet al.,2011).Anincrease in 5hmC signalswas observedingenesactivatedinoldmicewithrespecttoyoungones demonstratingthat5hmCisacquiredindevelopmentallyactivated genes(Szulwachetal.,2011).Age-dependentincreaseof5hmCin mousehippocampuswasalsoobservedonthe5-LOXgene,whose expressionisknowntoincreaseduringaging(Chenetal.,2012b).

Theinterestfor5hmCisnowgrowingenormouslyconsidering itsinvolvement not onlyin physiological states, suchasdevel- opmentand aging,butalsoinpathologicalconditionsincluding cancer,autoimmuneandneurodegenerativedisorders(Calabrese etal.,2014;Chenetal.,2012b;Chengetal.,2014;Pfeiferetal., 2014;Putirietal.,2014;Villar-Menendezetal.,2013).

7. Conclusionsandperspectives

Over the last years, a growing body of research has ledto the progressive and sharp description of the impact of aging onthemethylome,especially as a resultof therecent applica- tionof genome-wideanalyses.From thiswork,it emergesthat aging-relatedchangesinvolveverydifferentphenomenasuchas epigeneticdrifttogetherwithdirectionalmethylationchangesof specificgenomeregions,leadingtotheclearperceptionthatthe methylomeisadynamiclandscapethatreflectsavarietyofchrono- logicalcomplex changes.Relevantchallengefor futureresearch wouldbetodetermineifthesechangescanbemodeledtotrace underlyingmechanisms.In this context, it would beextremely importanttoshedlightontherelationshipsthatlinkage-associated methylationchanges withdeficitof themethylationmachinery (Casillasetal.,2003;Xiaoetal.,2008)andenvironmentalexpo- sure.Inthis researchframework, moreattentionshouldalsobe addressed tothe contributionof DNA hydroxymethylation and TETenzymes.Tounderstandthemechanisticbasisofage-related methylationchangesitwouldberelevantnotonlytoclarifythe molecularfeaturesofaging,butalsotosetthestageforthedevel- opmentofstrategiestocounteractitspathologicalphenotypes.

AnotherpromisingﬁeldofresearchseesDNAmethylationasa markerofagingtobeusedtopredictandmonitorage-associated physiologicaldeclineanddiseases.Consistently,pioneeringstud- iesrevealedthatmethylationchangesofcertaingenescanserve todetectdifferentratesofhumanaging(Bocklandtetal.,2011;

Garagnanietal.,2012;Hannumetal.,2013;Horvath,2013).From thispointofview,itwillbeinterestingtotestthesebiomarkersin relationtoclinicalandenvironmentalvariablesthatimpactaging rate.Thiswould allowfortheapplication ofDNA methylation- basedmarkerstoevaluatequalityoflifeintheagingpopulation.

Acknowledgements

Thisstudy wassupported bythe EuropeanUnion’s Seventh FrameworkProgramundergrantagreementN^◦HEALTH-F4-2008-

200880MARK-AGE and the Italian Ministry of University and Research(MIUR)(P.C.:FIRB-RBIN06E9Z8003).

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