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NATURE | ARTICLE

TumourhypoxiacausesDNAhypermethylationbyreducingTETactivity
BernardThienpont,JessicaSteinbacher,HuiZhao,FloraDAnna,AnnaKuchnio,AthanasiosPloumakis,BartGhesquire,Laurien
VanDyck,BramBoeckx,LucSchoonjans,ElsHermans,FredericAmant,VesselaN.Kristensen,KianPengKoh,Massimiliano
Mazzone,MathewL.Coleman,ThomasCarell,PeterCarmeliet&DietherLambrechts
Nature 537, 6368 (01September2016) doi:10.1038/nature19081
Received 19June2015 Accepted 05July2016 Publishedonline 17August2016

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Abstract
Hypermethylationofthepromotersoftumoursuppressorgenesrepressestranscriptionofthesegenes,conferringgrowthadvantagestocancer
cells.Howthesechangesariseispoorlyunderstood.Hereweshowthattheactivityofoxygendependentteneleventranslocation(TET)
enzymesisreducedbytumourhypoxiainhumanandmousecells.TETenzymescatalyseDNAdemethylationthrough5methylcytosine
oxidation.ThisreductioninactivityoccursindependentlyofhypoxiaassociatedalterationsinTETexpression,proliferation,metabolism,hypoxia
induciblefactoractivityorreactiveoxygenspecies,anddependsdirectlyonoxygenshortage.HypoxiainducedlossofTETactivityincreases
hypermethylationatgenepromotersinvitro.Inpatients,tumoursuppressorgenepromotersaremarkedlymoremethylatedinhypoxictumour
tissue,independentofproliferation,stromalcellinfiltrationandtumourcharacteristics.Ourdatasuggestthatuptohalfofhypermethylationevents
areduetohypoxia,withtheseeventsconferringaselectiveadvantage.Accordingly,increasedhypoxiainmousebreasttumoursincreases
hypermethylation,whilerestorationoftumouroxygenationabrogatesthiseffect.TumourhypoxiathereforeactsasanovelregulatorofDNA
methylation.
Subjectterms: Cancermicroenvironment

Tumourangiogenesis

Oncogenesis

DNAmethylation

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Authorinformation
Theseauthorscontributedequallytothiswork.
BernardThienpont,JessicaSteinbacher,HuiZhao&FloraDAnna
Affiliations
VesaliusResearchCenter,VIB,3000Leuven,Belgium.
BernardThienpont,HuiZhao,FloraDAnna,AnnaKuchnio,BartGhesquire,LaurienVanDyck,BramBoeckx,LucSchoonjans,Massimiliano
Mazzone,PeterCarmeliet&DietherLambrechts
LaboratoryofTranslationalGenetics,DepartmentofOncology,KULeuven,3000Leuven,Belgium
BernardThienpont,HuiZhao,FloraDAnna,LaurienVanDyck,BramBoeckx,VesselaN.Kristensen&DietherLambrechts
CenterforIntegrativeProteinScience,DepartmentfrChemieundPharmazie,LudwigMaximiliansUniversitt,81377Mnchen,
Germany
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TumourhypoxiacausesDNAhypermethylationbyreducingTETactivity:Nature:NatureResearch

JessicaSteinbacher&ThomasCarell
LaboratoryofAngiogenesisandVascularMetabolism,DepartmentofOncology,KULeuven,3000Leuven,Belgium
AnnaKuchnio,LucSchoonjans&PeterCarmeliet
InstituteofCancerandGenomicSciences,UniversityofBirmingham,BirminghamB152TT,UK
AthanasiosPloumakis&MathewL.Coleman
GynecologicOncology,UniversityHospitalsLeuven,DepartmentofOncology,KULeuven,3000Leuven,Belgium
ElsHermans&FredericAmant
DepartmentofGenetics,InstituteforCancerResearch,OsloUniversityHospitalRadiumhospitalet,N0310Oslo,Norway
VesselaN.Kristensen
DepartmentofClinicalMolecularBiology(EpiGen),AkershusUniversityHospitalandInstituteofClinicalMedicine,Facultyof
Medicine,UniversityofOslo,Postboks1171,Blindern0318Oslo,Norway
KianPengKoh
DepartmentofDevelopmentandRegeneration,andStemCellInstituteLeuven,KULeuven,3000Leuven,Belgium
MassimilianoMazzone
LaboratoryofMolecularOncologyandAngiogenesis,DepartmentofOncology,KULeuven,3000Leuven,Belgium
Contributions
B.T.andD.L.conceivedandsupervisedtheproject,designedexperimentsandwrotethemanuscript.B.T.andF.D.A.performedinvitro
experimentsandanalyseddata,helpedbyL.V.D.M.L.C.andA.P.analysedTetMichaelisMentenkineticsanimalmodelswereprovidedby
E.H.,F.A.(xenografts),M.M.(sFlk1),A.K.andP.C.(Phd2 +/)V.N.K.contributedideas,L.S.andK.P.K.providedreagentsJ.S.quantified
nucleotidesbyLCMS,supervisedbyT.C.B.G.quantifiedmetabolites.H.Z.analysedTCGAtumoursB.T.,H.Z.andB.B.performed
bioinformaticsandstatistics.
Competingfinancialinterests
Theauthorsdeclarenocompetingfinancialinterests.
Correspondingauthors
Correspondenceto:BernardThienpontorDietherLambrechts
MicroarrayandsequencingdataareavailableattheGeneExpressionOmnibusunderaccessionGSE71403.
ReviewerInformationNaturethanksR.S.Johnson,M.RehliandY.Xiongfortheircontributiontothepeerreviewofthiswork.

Extendeddatafiguresandtables
ExtendedDataFigures
1.ExtendedDataFigure1:Hypoxiainducedchangesin5hmC,5mCandTETexpression.(490KB)
Global5hmC/Cand5mC/CcontentofDNA,TET1,TET2andTET3mRNAexpressionandhypoxiamarkergeneexpressionin15cell
linesgrownfor24hundernormoxic(21%O 2,white)orhypoxic(0.5%O2,red)conditions.TETmRNAcopynumberisexpressed
relativetoB2Mforhumancelllines(HepG2,HT1080,MCF10A,H358,MCF7,Hep3B,A549,H1299,SKNBe2candSHSY5Y),and
toHprtformousecelllines(LLC,N2A,4T1,mESandTet1KOEScells).Shownarecelllinesderivedfromlivercancer(HepG2and
Hep3B),lungcancer(H358,A549,H1299andLLC),breastcancer(MCF7and4T1),fibrosarcoma(HT1080),neuroblastoma(SKN
Be2candSHSY5Y),normalbreastepithelium(MCF10A)andtheinnercellmassofblastocyststagemouseembryos(mESandTet1
KOEScells).ALDOAandBNIP3areexpectedtobeincreased,andHIF1Atobedecreaseduponhypoxia.Theglobal5fCcontentof
EScellsisdepicted,butwasundetectableincancercelllines.Barsrepresentthemeans.e.m.offivedifferentreplicatesamples.DNA
andRNAfromthesereplicateswereextractedfromcellsderivedfromthesamestockvialbutgrownondifferentdays.*P<0.05,**P<
0.01,***P<0.001bypairedttests.
2.ExtendedDataFigure2:ImpactofhypoxiaonTETexpression.(405KB)
a,ChangesinTet1,Tet2andTet3expressioninmousecelllines,attheproteinlevel(toprow,n=6)andthemRNAlevel(bottomrow,n
=5).Middlerow:representativeimmunoblotimagesofHif1a,Tet1,Tet2andTet3.Tubulinservesasloadingcontrol,andexpression
ofthecorrespondingcodinggene(Tuba1a)wasusedtonormalizemRNAexpression,enablingadirectcomparisonofrelativeprotein
andrelativemRNAexpressionchanges.Forthesamereason,mRNAexpressionwasdepictedrelativetocontrolconditions,incontrast
totheabsolutelevelsshowninExtendedDataFig.1.ChangesinTetmRNAandproteinexpressioncorrelatestrongly(PearsonsR=
0.855,P=410 4).Forexample,both4T1andN2AcellsdisplayedincreasedTet2expressionattheproteinandmRNAlevel.
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Likewise,EScellsshowednopronouncedchangesattheproteinormRNAlevel.TheoverallexpressionofTetenzymeswasnot
alteredinanyofthesecelllines.Forgelsourcedata,seeSupplementaryFig.1.b,Hif1ChIPseqatthepromotersofTET1,TET2
andTET3andathypoxiamarkersgenes(Bnip3andAldoa),withpeaksorpromoterregionshighlightedusingcolouredboxes.Green
andredboxescorrespondtooverexpressionandnooverexpression(specifiedinthefigurepanel)ofthecorrespondinggene,
respectively,asdeterminedusingTaqManinExtendedDataFig.1.Scale:readspermillionreadsandperbasepair.c,Left,TET2
expressioninMCF7cellstransfectedwithcontrol(white)orTET2targeting(purple)siRNAs.Right,corresponding5hmClevelsas
determinedusingLCMS.d,5hmClevelsasdeterminedbyLCMS,inwildtype(white)andTet1knockout(purple)EScellsgrown
under21%(left)and0.5%(right)O 2tensions.Barsincanddrepresentthemeans.e.m.offivereplicatesamplesfromcellsderived
fromthesamestockvialbutgrownondifferentdays.*P<0.05,**P<0.01,***P<0.001bypairedttests(a,c,d).
3.ExtendedDataFigure3:Effectssecondarytohypoxia.(547KB)
ae,ROSproductionandredoxstateofMCF7cellsculturedfor24hundercontrol(21%O2,white)orhypoxic(0.5%O2,red)
conditions.Shownarecapillarygaschromatographymassspectrometry(GCMS)quantificationsofchangesinthecellularenergy
stateasrepresentedbytheadenylateenergycharge(AEC)(calculatedas[ATP+0.5ADP]/[ATP+ADP+AMP])(a)thereducing
equivalentsofthecellasrepresentedbytherelativeNADHandNADPHlevels(calculatedasNADH/[NAD++NADH]and
NADPH/[NADP++NADPH])andthereductivecapacityofthecellasrepresentedbythelevelsofglutathione(calculatedasGSH/[GSH
+GSSG2]).b,c,Quantification(b)andrepresentativeFACSintensitytraces(c)oftotalROSlevelsinMCF7cellsexposedto
hypoxiaorH2O2,asassessedusing2`,7`dichlorodihydrofluoresceindiacetate(DCFDA).d,NuclearROSinMCF7cellsasassessed
usingthenuclearperoxyemerald1probe(NucPE1)39.MCF7cellswereexposedto21%(control)or0.5%(hypoxia)O2for24h,after
whichlivecellswereloadedwithNucPE1(5M)andHoechst33342(10gml1)inO2preequilibratedPBSfor15min.Afterwashing,
controlcellswereincubatedwithH2O2(0.5mMinPBS)asapositivecontrol,orwithwater(controlandhypoxiacells)inPBSfor20
min.Cellswerewashedagainandimmediatelyimagedbyconfocalmicroscopy.Representativeimagesareshown.Scalebar,50m.e,
ThenuclearNucPE1signal,averagedacross>100nucleiandexpressedrelativetocontrolconditions.f,LCMSquantificationof8
oxoGconcentrationsinDNAofcellslinesculturedfor24hundercontrol(21%O2,white)andhypoxic(0.5%O2,red)conditions.8
oxoGservesasamarkerofnuclearROS53.gi,GCMSquantificationofchangesintheindicatedmetabolitelevelsinmouseEScells
(g),MCF10Acells(h)andMCF7cells(i)grownfor24hundercontrol(21%O2,white),hypoxic(0.5%O2,red)orglutaminefree(21%
O2,black)conditions.j,Quantitiesofketoglutarateand2hydroxyglutarateinMCF7cells,expressedrelativetoketoglutaratelevels
inMCF7cellsgrownundercontrolconditions(21%O2).k,LCMSquantificationof5hmClevelsinresponsetohypoxia(0.5%O2)and
glutaminefreecultureconditions.l,Growthofcelllinesculturedfor24hundercontrol(21%O2,white)andhypoxic(0.5%O2,red)
conditions,asassessedusingasulforhodamineBcolorimetricassay.Changesincelldensityafter24haredepictedrelativetocontrol
conditions(21%O2).m,IOX2inducedchangesintheglobal5hmCcontentofDNA,inTETmRNAexpressionandinhypoxiamarker
geneexpressionoffivecelllinestreatedfor24hwithDMSO(carrier,white)orIOX2(50M,blue).n,5mChydroxylationactivityof
nuclearlysatesfromMCF7cellsgrownfor24hunder21%or0.5%O2(whiteorred).Barsrepresentthemeans.e.m.of5(b,k,m),
6(a,ce),16(gj)or24(l)samplespreparedondifferentdays.*P<0.05,**P<0.01,***P<0.001byttest(b,e,hm).
4.ExtendedDataFigure4:Genomicprofilesof5mCand5hmC.(136KB)
ShownareresultsfromDIPseqofDNAfromMCF7cellsculturedfor24hunder21%or0.5%O2(controlandhypoxia),withexamples
of5hmCDIPseq(top)and5mCDIPseq(bottom)readdepths(FPM,fragmentsperbasepairpermillionfragments)atregions
surroundingthetranscriptionstartsiteofNSD1,FOXA1andCDKN2A.Theseshow5hmCloss(FDR<5%)anda5mCgainthatis
moresubtle,perhapsbecausetheresolutionof5mCDIPseqislimiting:regionsrichin5hmCtendtobepoorerin5mC 54,andthus
havelesssubstrateavailableforpulldown.5mCDIPseqmoreovercapturesallmethylatedsites,somostofthe5mCDIPseqsignal
doesnotderivefromsitesthatareactivelyturningover5hmC.
5.ExtendedDataFigure5:Effectofhypoxiaonhypermethylationfrequencyintumours.(948KB)
a,Immunofluorescenceanalysisofpatientderivedtumourxenografts,stainedforpimonidazole(white),5hmC(red),DNA(propidium
iodide,blue)andpancytokeratin(green).Shownarerepresentativeimagesofabreastandtwoendometrialtumourxenografts.The
insetontherightshowsboxplotsillustratingthesignalinnormoxicpimonidazolenegativenuclei(blue),andinhypoxicpimonidazole
positivenuclei(red).b,Hypoxiamarkergeneexpressionclusters,withthefirstthreeclustersusedtodefinenormoxic,intermediateand
hypoxictumours.c,Unsupervisedclusteringof1,000CpGsshowingthehighestaveragemethylationincreaseintumourversus
correspondingnormaltissues.Thefirstthreeclusterswereusedtodefinetumoursoflow,intermediateandhighhypermethylation.The
colourbarabovetheclustersannotateseachtumourasnormoxic,intermediateorhypoxic,asdeterminedinb.d,Boxplotsshowing
therelativeexpression(zscore)ofgenesintumoursinwhichtheyhaveeither0or1hypermethylationeventintheirpromoter,
stratifiedintonormoxic,intermediateandhypoxictumours(blue,greyandred,respectively).Diamondsindicatemean,boxplotwedges
indicate2thestandarderrorofthemedian.Geneswith1hypermethylationeventsintheirpromotershavealoweraverage
expressionlevel(P<0.01foreachtumourtype).e,Fractionofgeneshavingapromoterthatisrich,intermediateorpoorinCpGs,out
ofallgenepromotersthatareassessedonthe450karray,andoutofallgenepromotersthatarefrequentlyhypermethylatedinthe
indicatedtumourtypes.f,Fractionof1,742TETwildtypetumoursand39TETmutantthatarenormoxic,intermediateandhypoxic.P
>0.2forallcomparisons.g,Cellproliferationmarkergene46expressionclusters,withthefirsttwoclustersusedtodefinehigh
proliferativeandlowproliferativetumours.h,hypermethylationfrequenciesinlowandhighproliferativetumours,withasterisks
representingPvaluesfromlinearmodelscorrectingforvariablesspecifiedinSupplementaryTable8.i,Partialcorrelationcoefficient
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(partialR 2)estimatesoftherelativecontributionoftumourcharacteristics(annotatedinTCGA)tothevarianceinhypermethylation
observedinthesetumours.PartialR 2valueswereobtainedfromlinearmodelestimationusingordinaryleastsquares,andexpressed
asafractionofthetotalvariance(thatis,totalR 2)explainedbythemodelwhentakingintoaccountallindicatedvariables,asindicated
betweenbracketsundereachtumourtype.*P<0.05,**P<0.01,***P<0.001byttest(a)orbygeneralizedlinearmodel(h).
6.ExtendedDataFigure6:Functionalannotationofgenesmorefrequentlyhypermethylatedinhypoxictumours.(502KB)
a,Ontologytermsenrichmentanalysisofgenesthataremorefrequentlyhypermethylatedattheirgenepromotersinhypoxicthan
normoxictumours,foreighttumourtypescharacterizedintheTCGApancancereffort.Arepresentativesetoftermsisdisplayed,
selectedfromtermsenrichedinmosttumourtypes.Pvaluesasdefinedbythegreyscaleinsert.EnrichmentcalculatedusingtopGO.b,
SelectedexamplesofhypermethylationfrequenciesinthepromoterofkeyTSGs(PTEN,CDKN1C,ATM)morefrequently
hypermethylatedinnormoxicthanhypoxictumours.c,Hypermethylationfrequencyinthepromoterofselectedgenesinvolvedinthe
processesindicated.P<0.05forallgenes(asterisksarenotdisplayed).Barsinbandcrepresentthehypermethylationfrequency
s.e.m.PvaluesinabyFishersexacttest.
7.ExtendedDataFigure7:EffectofhypoxiaonTETactivityinhumantumours.(120KB)
a,ThetvalueofcorrelationbetweenhypermethylationandexpressionofTETorDNMTgenesacross3,141tumoursof8tumourtypes
(bladder,breast,colorectal,headandneck,kidney,lungadenocarcinoma,lungsquamous,anduterinecarcinoma)profiledinTCGAfor
geneexpressionandDNAmethylation,whilecorrectingfortumourtype,hypoxiaandproliferation.ThedottedlinerepresentsP<0.05,
negativetvaluesrepresentinversecorrelations.b,Hypoxiametagenesignatureappliedto63glioblastomamultiformetumoursfrom
TCGA.c,Boxplotsshowingtherelativeexpression(zscore)ofgenesintumoursinwhichtheyhaveeither0or1hypermethylation
eventintheirpromoter,stratifiedintowildtypeIHD1tumoursthatarenormoxic(n=19),intermediate(n=21)andhypoxic(n=17)
(blue,greyandred,respectively),andIDH1 R138mutatedtumours(n=4,yellow).Diamondsindicatemean,boxplotwedgesindicate
2thestandarderrorofthemedian.Geneswith1hypermethylationeventsintheirpromotershavealoweraverageexpressionlevel.
NohypermethylationeventsweredetectedinwildtypeIHD1normoxictumours.d,Hypoxiametagenesignatureappliedto12normoxic
and12hypoxicnonsmallcelllungtumours.*P<0.05,***P<0.001byttest(c).
8.ExtendedDataFigure8:5hmC,hypoxiaandTSGhypermethylationinmousebreasttumours.(610KB)
a,Frequencyofhypermethylationeventsinthepromotersofallgenes,alloncogenesandallTSGsasannotated28,in695human
breasttumoursavailablethroughTCGAandstratifiedintonormoxic,intermediateandhypoxicsubsets.b,c,DNAwasextractedfrom
53tumoursdevelopinginMMTVPyMTmiceoftheindicatedages(c)orweights(d)andsequencedtoadepthof~500.Plottedarez
scoresofhypermethylation(yaxis,exponential)for15TSGs,relativetothetumoursfrom11weekoldmice.Thedottedlinerepresents
thethresholdforaBonferroniadjustedP<0.05,andbolddarkerdotsareusedfortumoursdisplayingsignificantlyincreased
hypermethylationevents.d,DNAextractedfrom20normalmammaryglandsfrom14weekoldmice,PCRamplifiedfor15TSGsand
sequencedtoadepthof~500x.Plottedarezscoresofhypermethylationrelativeto11weekoldtumours.e,StainingofPyMTtumours
for5hmC(red),DNA(propidiumiodide,blue),pimonidazole(white)andPyMT(green),andfractionofPyMTpositivecellsinnormoxic
andhypoxicareas.Theareaoutlinedcorrespondstothehypoxic,pimonidazolepositivesection,arrowheadspointtoPyMTnegative
cells.Scalebar,25m.ThebarchartinsetillustratestherelativenumberofPyMTpositivecellsinnormoxicandhypoxicareas(grey
andred,respectivelyn=19).f,Ki67positivecellsinPyMTtumours:representativeimageofstainingforDNA(propidiumiodide,blue),
Ki67(red)andpimonidazole(green).Scalebar,50m.ThebarchartinsetillustratesthequantificationofKi67positivecellsinnormoxic
andhypoxicareas(greyandred,respectively)across6tumours,analysing3fieldsofviewwithover150cellsperfieldofview.g,
CD45positivecellsinPyMTtumours:representativeimageofstainingforDNA(propidiumiodide,blue),5hmC(red),pimonidazole
(green)andCD45(white).Scalebar,100m.ThebarchartinsetillustratesthequantificationofCD45positivecellsinnormoxicand
hypoxicareas(whiteandred,respectively)of11tumours,capturingonaverage~2,500nucleiperanalysis.***P<0.001in(a)by
Fishersexacttest,significancerelativetoallgenes.
9.ExtendedDataFigure9:Manipulationoftumouroxygenationinmousebreasttumours,andeffectson5hmC,TSGhypermethylation
andconfounders.(564KB)
a,PlasmasFlk1concentrationsattheindicatedtimesafterhydrodynamicinjectionwithanempty(n=7)orsFlk1overexpression
plasmid(n=5)(greyandred,respectively).b,cQuantificationoftumourvesselnumber(b)andhypoxicareas(c)oftumoursfrom
transgenicMMTVPyMTmice,hydrodynamicallyinjectedwithanemptyorsFlk1overexpressionplasmid,withrepresentativeimagesof
bloodvesselsstainedforCD31(b)andhypoxicareasstainedforpimonidazoleadducts(c).Scalebar,100m.d,ChangesinRNA
expressionofhypoxiamarkergenesthatareknowntobedownregulated(Mrc1)orupregulated(Bnip3,Car9,Ddit4)inhypoxic
conditions.e,5hmClevels(yaxis)acrossmousechromosome18(xaxis)in400kbbins,withthelocationofRefSeqgenes(middle),
anddifferencesin5hmClevels(lower).5hmClevelsweredeterminedusingshallowTABseq,andchromosome18wasselected
becauseithaslargestretchesofgenedesertsthatillustratethe5hmCdepletionintheseareas(n=3).5hmClevelsdecreaseby12.4%
3.5aftersFlk1overexpression,althoughtechnicallimitationsofTABseq(incomplete5hmCprotectionorbisulfiteconversion)may
partiallyobscurethemagnitudeofeffects.f,Hypermethylationintumoursdevelopingin12weekoldmicereceivinghydrodynamic
injectionwithanempty(n=19)orsFlk1overexpressingplasmid(n=24)3weeksearlier.DNAwasbisulfiteconverted,PCRamplified
fortheindicatedoncogenes,andsequencedtoadepthof~500.Plottedarezscoresofhypermethylation(yaxis,exponential),relative
tothemorenormoxictumours(thatis,empty).Thedottedlinerepresentsthethresholdat5%FDR,andbolddarkerdotsthetumours
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TumourhypoxiacausesDNAhypermethylationbyreducingTETactivity:Nature:NatureResearch

displayingsignificantlyincreasedhypermethylationevents.gj,Relativeweightsoftumoursfromtg(MMTVPyMT)mice,
hydrodynamicallyinjectedwithanempty(grey,n=19)orsFlk1overexpressionplasmid(red,n=24)(g),andcorrespondingRNA
expressionofPtprc(thegeneencodingCD45,n=5)(h),ofTetenzymes(i,n=15foremptyplasmid,n=12forsFlk1overexpressing
plasmid)andofcellproliferationmarkers(j,n=5foreach).km,Asindfbutfor16weekoldtransgenicMMTVPyMTmiceofthe
indicatedgenotype.n=5(k),n=3forPhd2 +/+n=4forPhd2 +/(l)andn=9(m).n,Asind,butfor16weekoldTie2CreTg(MMTV
PyMT)miceoftheindicatedgenotypes(n=5).o,DNAwasextractedfrom17breasttumoursdevelopinginTie2
CrePhd2 fl/WT Tg(MMTVPyMT)mice(blue)and13breasttumoursdevelopinginTie2CrePhd2 WT/WT Tg(MMTVPyMT)mice(grey),
all16weeksold.DNAwasbisulfiteconverted,PCRamplifiedfortheindicatedTSGs(left)andoncogenes(middle)andsequencedtoa
depthof>500.Plottedarezscoresofhypermethylation(yaxis,exponential),relativetothemorenormoxic,Phd2 WT/fl,tumours.The
dottedlinerepresentsthethresholdforaBonferroniadjustedP<0.05,andbolddarkerdotsthetumoursdisplayingsignificantly
increasedhypermethylationevents.Right,5hmClevelss.e.m.acrossametageneintumoursof16weekoldmicewiththeindicated
genotype(n=3forPhd2 fl/fln=4forPhd2 WT/fl).pu,RelativeweightsoftumoursfromPhd2 +/tg(MMTVPyMT)miceand
Phd2 +/+Tg(MMTVPyMT)mice(n=10and9resp.)(pr)andfromTie2CrePhd2 fl/WT Tg(MMTVPyMT)andTie2
CrePhd WT/WT Tg(MMTVPyMT)mice(n=17and13,respectively)(su),andthecorrespondingRNAexpressionofcellproliferation
markers(n=5,p,s),ofTetenzymes(n=5,q,t)andofPtprc(n=5)(r,u).#P<0.10,*P<0.05,**P<0.01,***P<0.001byttest.

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