The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells

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ContentslistsavailableatScienceDirect

Journal

of

Biotechnology

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j b i o t e c

The

DNA

methylation

landscape

of

Chinese

hamster

ovary

(CHO)

DP-12

cells

Anna

Wippermann

a,b

_,

_Oliver

_Rupp

c

_,

_Karina

_Brinkrolf

b

_,

_Raimund

_Hoffrogge

a,b

_,

Thomas

Noll

a,b,∗

a_Institute_of_Cell_Culture_Technology,_Bielefeld_University,_Bielefeld,_Germany b_Center_for_{Biotechnology,}_Bielefeld_University,_Bielefeld,_Germany

c_{Bioinformatics}_and_Systems_Biology,_{Justus-Liebig-University,}_Giessen,_Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18December2014

Receivedinrevisedform9February2015 Accepted11February2015

Availableonline19February2015 Keywords:

CHO Epigenetics DNAmethylation

Wholegenomebisulﬁtesequencing Geneexpression

a

b

s

t

r

a

c

t

Chinesehamsterovary(CHO)cellsrepresentthemostcommonlyusedproductioncelllinefortherapeutic

proteins.Byrecentgenomeandtranscriptomesequencingabasiswascreatedforfutureinvestigationsof

genotype–phenotyperelationshipsandforimprovementofCHOcellproductivityandproductquality.In

thiscontextinformationismissingaboutDNAcytosinemethylationasacrucialepigeneticmodiﬁcation

andanimportantelementinmammaliangenomeregulationanddevelopment.Here,wepresentthe

ﬁrstDNAmethylationmapofaCHOcelllineinsingle-baseresolutionthatwasgeneratedbywhole

genomebisulﬁtesequencingcombinedwithgeneexpressionanalysisbyCHOmicroarrays.WeshowCHO

DP-12cellstoexhibitglobalhypomethylationcomparedtoamajorityofmammalianmethylomesand

hypermethylationofCpG-denseregionsatgenepromoterscalledCpGislands.Wealsoobservedpartially

methylateddomainsthatcover62%oftheCHODP-12cellgenomeandcontainfunctionalclustersof

genes.Geneexpressionanalysisshowedtheseclusterstobeeitherhighlyorweaklyexpressedwith

regardtoCHO-speciﬁccharacteristicsandhenceprovesDNAmethylationinCHOcellstobeanimportant

linkbetweengenomicsandtranscriptomics.

license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Chinese hamster ovary (CHO) cells are the major

mam-malian production cell linefor therapeutic proteins. Therefore

improvementofCHO-basedbioprocessperformanceandproduct

characteristicsofbiopharmaceutics,e.g.bymolecularengineering

approaches,isofgreatinteresttoacademiaandindustry(Baikand

Lee,2014).AnessentialbasisforthispurposeistheCHOK1genomic

sequenceandtranscriptomedatathatbecamerecentlyavailable

andwillenableadeeperunderstandingofgenotype–phenotype

relationshipsintermsofproductqualityattributes(BaikandLee,

2014;Lewisetal.,2013;Ruppetal.,2014).Amissinglinkbetween

thesecellularlevelsinCHOcells,however,istheyetunknownCHO

cellepigenome.

The term epigenetics classically describes phenomena that

modify gene expression without altering the DNA sequence.

It comprises posttranslational histonemodiﬁcations, chromatin

∗ Correspondingauthorat:BielefeldUniversity,Universitätsstraße25,33615 Bielefeld,Germany.Tel.:+495211068700;fax:+495211066023.

E-mailaddress:thomas.noll@uni-bielefeld.de(T.Noll).

remodelingand DNA methylation (Ndlovuet al.,2011; Tessarz

andKouzarides,2014).Furthermore,miRNAswereshowntobe

involved in epigenetic regulation,e.g. bydouble-negative

feed-backloops(Osellaetal.,2014).DNAmethylationhasbeenknown

sincethe1970s(HollidayandPugh,1975;Riggs,1975)andwas

shown tocontribute toprocesses of irreversiblegene silencing

suchasXchromosomeinactivationorsilencingofretrotransposons

(Bestoretal.,2014;PanningandJaenisch,1996;Walshetal.,1998).

DNAmethylationtakesplaceatcytosinebasesinthecontextof

CpGdinucleotidesandeffectsthemajorityofvertebrategenomes,

whichforthemostpartcontainonlyfewregionsoflow

methyla-tion(Meissneretal.,2008).Theseregionsincludee.g.CpG-dense,

oftenunmethylatedareasatgene promoterscalledCpGislands

(CGIs)thathavebeenextensivelystudiedinlocus-speciﬁc

exper-iments(DeatonandBird,2011;IllingworthandBird,2009).The

advent of nextgeneration sequencing technologies enabled an

in-depthmapping of thedistributionof DNA methylation ona

genomescaleforseveralmammaliantissuesandcelltypes.This

led,for instance,tothediscoveryofglobaldifferences between

thegenomesofhumanembryonicstemcellsandfetalﬁbroblasts

regarding the composition and patterning of DNA methylation

(Listeretal.,2009).Fibroblastcellsturnedouttobecharacterized

http://dx.doi.org/10.1016/j.jbiotec.2015.02.014

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bywidespreadreducedmethylationassociatedwithlessactively

transcribedgenesandrepresentedtheﬁrstcelllineshownto

con-tainso-calledpartiallymethylateddomains(PMDs)asanexception

tothenotionofgenerallyhighlymethylatedmammaliangenomes

(Gaidatzisetal.,2014).

Severalreportshinttowardaninﬂuenceofepigeneticeffectson

productgeneexpressioninCHO-basedbioprocesses,e.g.regarding

intragenic CpG content of transgenes (Bauer et al., 2010) and

CMVpromotermethylation(Kimetal.,2011;Yangetal.,2010;

Osterlehner et al., 2011).To analyzeDNA methylation in CHO

cells,werecentlydevelopedaCpGislandmicroarrayandshowed

differentialDNAmethylation inresponse tobutyratetreatment

(Wippermann et al., 2013). However, a comprehensive

under-standingofepigeneticeffectsinbiopharmaceuticsproductionas

animportant linkbetweengenotype and phenotyperequires a

globalcharacterizationoftheyetunknownCHOcellepigenome.

In this study theDNA methylation landscapeof IgG-producing

CHODP-12cellswascharacterizedandputativeinﬂuencesofthe

methylation landscape on gene expression were analyzed. For

this purposewe appliedwhole genomebisulﬁtesequencing in

combinationwithgeneexpressionanalysesbycustom-designed

CHO-speciﬁcmicroarrays(Beckeretal.,2014).Weshowthatthe

globallyhypomethylatedCHODP-12cellgenomecontains

hyper-methylatedCGIsandregionsofpartialmethylationthatcontain

functionalgeneclustersexpressedinadifferentialfashion.

2. Materialsandmethods

2.1. Cellcultureandcultivationprocessanalysis

IgG-producingCHODP-12cells(clone#1934,ATCCCRL-12445)

werecultivatedinCHOMACSCDmedium (MiltenyiBiotec,

Ber-gischGladbach,Germany)supplementedwith6mMl-glutamine

(FagronGmbH&Co.KG,Barsbüttel,Germany),200nM

methotrex-ate (MTX; Wyeth, Madison, NJ) and 0.1mg×L−1 Insulin-like

growthfactor(LongR3IGF-I;Sigma–Aldrich,Munich,Germany).

Cellswerecultivatedin250mLshakerﬂasks(CorningInc.,Corning,

NY)withaseedingcelldensityof1×106_cells_×_mL−1_._Four

repli-catecultures werepreparedfromonepreculture.Cultureﬂasks

werekeptat37◦C,80%humidityand5%CO2onashakingplatform

at185rpm(50mmdiameterrotation).Twodaysafterinoculation

samplesof6.5×106_cells_were_taken_for_DNA_and_RNA_extractions.

Measurementsofcelldensitiesandviabilitieswereperformedon

aCedexAS20cellcounter(InnovatisAG,Bielefeld,Germany).IgG

concentrationsweremeasuredwithaHPLCmethodonproteinA

columnsaccordingtothemanufacturer’sprotocol(PorosA,Applied

Biosystems,Foster City,CA). Glucoseandlactate concentrations

weremeasuredonanYSI2700SelectAnalyzer(YellowSprings

InstrumentsInc.,YellowSprings,OH).

2.2. Libraryconstructionandsequencing

ReplicatesampleswerepooledandgenomicDNAwasextracted

from2.6×107_CHO_DP-12_cells_using_a_DNeasy_Blood_&_Tissue_Kit

(Qiagen, Venlo,The Netherlands).50ng of extracted DNAwere

spikedwith0.01%(w/w)ofunmethylatedcl857Sam7LambdaDNA

(Promega,Madison,WI)thatservedasanunmethylatedcontrol

andwassubjectedtobisulﬁtetreatment(ZymoResearch

Methyl-ationLightningKit;ZymoResearch,Irvine,CA).Bisulﬁteconverted

DNA was stored at −80◦_C _until _library _{construction.} _Library

generationfrombisulﬁtetreatedDNAand10ngofunconverted

genomic DNA was performed using the EpiGnome Methyl-Seq

Kit (Epicenter, Madison, WI) according to the manufacturer’s

instructions. To allow for multiplexed sequencing of libraries

frombisulﬁtetreatedand nativeDNA,individuallibrarieswere

barcoded using EpiGnome Index PCR Primers (Epicenter). The

quality of both libraries was checked on High Sensitivity DNA

ChipsontheBioanalyzer(AgilentTechnologies,SantaClara,CA).

Paired-endsequencingwasperformedonanIlluminaHiSeq1500

systemina2×100bprunwith1%PhiXControlv3(Illumina)in

eachlane.Oneentirelanewasusedasacontroltosequencethe

unconverted CHODP-12 genomic DNAlibrary and toallowfor

phasingandpre-phasing.Lanescontainingbisulﬁtelibrarieswere

additionallyloadedwith10%oftheunconvertedDNAlibrary.

2.3. Bioinformaticsanalysisofsequencingdata

Paired-endreadswerequalitycheckedusingFastQC(Andrews,

2010)andtrimmedusingTrimmomatic(Bolgeretal.,2014).The

referenceCHOK1genome(Xuetal.,2011)wascorrectedforsingle

nucleotidepolymorphisms(SNPs)withintheCHODP-12cellline.

FirstthereadsfromthesequencingofunconvertedCHODP-12DNA

weremappedtotheCHOK1genomewithBowtie2(Langmeadand

Salzberg,2012).SNPswerethencalledusingthesamtoolsmpileup

algorithm(Lietal.,2009)andﬁlteredusinginhouseperlscripts.

SNPswithaminimalcoverageof4readsandatleast25%ofthe

readscontainingtheSNPwereusedforcelllinecorrectionusing

thevcfutilsvcf2fqmethod.TheSmithlabMethylationData

Analy-sisPipeline(Songetal.,2013)wasusedformappingofreadsfrom

bisulﬁtetreatedlibrariestotheSNP-correctedreferencegenome,

removalofduplicatereads,calculationofsingle-sitemethylation

levels,estimationofbisulﬁteconversionratesandcalculationof

methylationlevelsinuntranslatedregions(UTRs),exons,introns

andCGIs(annotationofCGIsbasedonWippermannetal.,2013).

Methylationdatatrackswereintegratedintothein-houseGenDBE

genomebrowser(Ruppetal.,2014)toinspectselectedregionswith

knownmethylationpatternsforaccordanceofWGBSresults.For

this,branchedchainaminoacidaminotransferasecytosolic-likegene,

Bcat1(GeneID100763658),servedasamethylatedcontrol.The

beta-actingene,Actb(GeneID100689477),servedasan

unmethy-latedcontrol.Partiallymethylateddomains(PMDs)ofmorethan

10kband methylationlevelsbelow70%wereidentiﬁedusinga

slidingwindowapproachaccordingtoListeretal.(2009).Forthis,

SNP-correctedCHOK1scaffoldscontainingmorethan100CpGs

wereincludedintotheanalysis(5874 scaffoldscomprising96%

of the CHOgenome). For PMDs and hypermethylated domains

(HMDs)with>100CpGs,averagemethylationlevels,sizeand

cov-eragebycodingsequence(CDS)wereanalyzed.AssignmentofCHO

DP-12PMDstotherecentlysequencedChinesehamster(CH)

chro-mosomes(Brinkrolfetal.,2013)wasbasedonreadcoverages.

2.4. Geneexpressionproﬁlingbymicroarrays

Total RNA was extracted from 6.5×106 _cells _per _replicate

culture usingthe Direct-zolRNA MiniPrepKit (ZymoResearch).

Puriﬁed RNA samples were quantiﬁed on a NanoPhotometer

(IMPLEN, Munich, Germany). Integrity of RNA was checked on

denaturingagarosegelsstainedwithGelRed(BiotiumInc.,

Hay-ward,CA)and visualizedona FusionXL7(Vilber,Eberhardzell,

Germany). RNA sampleswere pooled and analyzed by4×44K

one-color microarrays (Agilent Technologies). Labeling and

hybridizationoffourreplicatecustomdesignedCHOmicroarrays

wasperformedasdescribedinBeckeretal.(2014).Arrayquality

was estimated based on the resulting QC reports and plots of

raw data. Data analysiswassubsequently performed using the

limmapackageinR(Smyth,2005)afterbackgroundcorrectionand

quantilenormalizationacrossthetechnicalreplicates.Forthe

eval-uationofourwholegenomebisulﬁtesequencingdataweaimed

tocollectthesubset ofgenes highlyexpressedin oursamples.

Allgeneswithexpressionvaluesabovethe75thpercentilewere

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Viab VCD qp Glc Lac Cultivation time [d] g L - 1 A 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 Viab le cell dens ity [10 6 cells mL -1] Cultivation time [d] Cell spec ific productiv ity [pg cell -1d -1] B 0 1 2 3 4 5 6 7 Glc Lac Sampling Sampling Viab ility [%]

Fig.1. Cellgrowth,IgGproduction,glucoseandlactateconcentrationsofCHODP-12 cellcultures.Errorbarsrepresentstandarddeviationsoffourreplicatecultures.The verticallinemarksthesamplingpointforwholegenomebisulﬁtesequencingand geneexpressionanalysis.(A)Meanviability(Viab),cellspeciﬁcproductivity(qp) andviablecelldensity(VCD).(B)Glucose(Glc)andlactate(Lac)concentrations.

Gene Ontology annotations were performed using the DAVID

software(Huang etal.,2009)withEASEscoresof<0.1andFDR

correctionofpvaluesaccordingtoBenjaminiandHochberg(1995).

3. Results

3.1. Generationofthebase-pair-resolutionCHODP-12DNA

methylationmap

Replicate(n=4)batchculturesweremonitoredregardingviable

celldensity,viability,cellspeciﬁcproductivity(Fig.1A),glucose

consumptionandlactateformation(Fig.1B)toaccountforoptimal

growthandproductionbehavior.Theculturesshowednormalcell

growthwithamaximumviablecelldensitysevendayspost

inoc-ulationandhighviabilitiesuntilglucosedepletion.Whenglucose

waslimitedondayeightthecells startedtometabolizelactate.

Samplesweretakeninthemiddleoftheexponentialgrowthphase

whenthecellsexhibitedaviabilityof99%andacellspeciﬁc

pro-ductivityof7.4pg/cell×d.

ReplicatesampleswerepooledpriortoextractionofDNAfor

analysisbywholegenomebisulﬁtesequencing.Isolatedgenomic

DNA was treated with bisulﬁte, which led to the conversion

ofunmethylated cytosines touracil bases, whereas methylated

cytosinesremainedintact.AstheCHODP-12genomesequenceis

notavailableyet,sequencingreadsfrombisulﬁtetreatedlibraries

weremappedtotheCHOK1genome.Forthis, asecondlibrary

wasgeneratedfromuntreatedgenomicCHODP-12DNAtoaccount

forSNPspresentbetweenthetwogenomes.Bothlibrarieswere

subjectedtonextgenerationIlluminasequencing.Aftertrimming

ofpaired-endsequencingreadsweobtained66.2Gb(402,802,819

reads)ofsequenceinformationforthebisulﬁtetreatedCHO

DP-12DNAand95.8Gb(575,986,927reads)fortheunconvertedCHO

DP-12genome.84%ofthelattercouldbemappedtotheCHOK1

referencegenomeand74%wereproperlypaired.SNPdetection

showed4,054,041SNPsintheCHODP-12genomecomparedto

theCHOK1cellline.

In the nextstep, reads frombisulﬁte treated libraries were

mappedtotheSNP-corrected CHOK1genome.Thisresultedin

anaveragesequencingdepthof 14-foldwith94% ofallknown

CpGsitescovered(15.3millionCpGs).Subsequently,methylation

levelsforeach CpGdinucleotidewithintheCHODP-12genome

weredeterminedbycalculatingtheratioofmappedthymines(as

uracil isampliﬁedas thymine)and cytosines atindividual CpG

positions.Toevaluatethequalityofthesequencingdataandthe

correctnessofitsprocessing,CHODP-12cellgenomicregionswith

knownmethylationlevelswerecomparedtopreviousexperiments

tocheckforconsistency(accordingtoWippermannetal.,2013).For

thispurposearegionlocatedwithinthepromoterCpGislandof

thebranchedchainaminoacidaminotransferasecytosolic-likegene

(Bcat1)servedasamethylatedcontrol(SupplementaryFig.S1A).

Partofthe5UTR-associatedCpGislandofthebeta-actingene(Actb)

servedasanunmethylatedcontrol(SupplementaryFig.S1B).Both

regionsexhibitedtheexpectedmethylationlevels.Also,thequality

ofbisulﬁteconversionwascontrolledbycalculatingtheconversion

rateofthespike-inLambdaDNA,whichwasabove99%.

3.2. GlobalassessmentoftheCHODP-12DNAmethylation

landscape

CHODP-12cellshadanaveragemethylationlevelof61%.As

globalmethylationlevelsof>70%werereportedforawidevariety

ofcells(e.g.82%methylationforHUES64cellsor78%methylation

forfetalbraincells)(Zilleretal.,2013),thisﬁndingsuggests

hypo-methylationforthesamplesanalyzed.AglobalanalysisofDNA

methylationonthelevelofsingleCpGdinucleotidesshoweda

gen-erallybimodaldistributionof methylationwith52% ofallCpGs

beinglargelymethylated(>70%ofreadsshowingmethylation)and

30%ofunmethylatedCpGs(≤10%ofreadsshowingmethylation)

(Fig.2A).TheremainderofCpGdinucleotidesshowedpartial

meth-ylation.

TogetaninitialoverviewoftheCHODP-12 cellDNA

meth-ylation landscape, we examined whether the observed overall

hypomethylationwasduetoauniformdistributionofCpGswith

lowandhighmethylationlevelsoriftherewerelocaldifferencesin

averagemethylationbetweengenomicregions.Therefore,the

dis-tributionofaveragemethylationlevelsfornon-overlapping20kb

windowstiledthroughthegenomewasanalyzed (accordingto

Schroederetal.,2011).Windowscontaininglessthan100CpGs

were excluded from the analysis. The purpose of this

calcula-tionwastodiminishtheeffectof putativelyunmethylatedCpG

islands.Thisanalysisshowedaverybroadmethylationspectrum

between20%and80%methylationwithapeakat60%,indicatinga

highdegreeofheterogeneityinlocalCHODP-12DNAmethylation

(Fig.2B).

TofurthercharacterizetheCHODP-12 cellmethylome with

regard tothe methylationof annotated genomic features, DNA

methylationlevelsforCGIs,untranslatedregions (UTRs),coding

sequences(CDSs)andintrons(CDSandintronsalsoreferredtoas

gene-bodies)weredetermined(Fig.2C).45%ofallCGIsintheCHO

DP-12cellgenomeprovedtobeunmethylatedandCGIsshoweda

meanmethylationlevelof45%.UTRsshowedameanmethylation

of56%,with26%ofthembeingunmethylated.CDSsandintronic

regionsexhibitedmeanmethylationlevelsof76%and68%,

respec-tively,alowpercentageofunmethylatedregions(9%and7%)and

ahighfrequencyofhighlymethylatedregions.

In summary, CHODP-12 cells showedhypomethylation and

evidence for local differences in averagemethylation levels. In

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0 5 10 15 20 25 30 35 0 100

C

Freq u ency of CpGs [%] 0 20 40 60 80 0 5 10 15

A

B

Freq u ency of 20 kb wind o ws [%] Freq u ency of regi o n [%]

Methylation level [%] Methylation level [%]

Methylation level [%] partial methylation hyper-methylation hypo-methylation 0 10 20 30 40 50 7 % 0 100 0 100 0 100 0-10 >10-20>20-30>30-40>40-50>50-60 >70-80>80-90_>90-1 00 >60-70 mean 45 % mean 56 % 45 % 26 % 9 % mean 76% mean 68 % 20 %

CGIs UTRs CDSs Introns

Fig.2.GlobaldistributionofDNAmethylationlevels.(A)PercentmethylationofCpGdinucleotides.(B)Percentmethylationof20kb,non-overlappingwindowstiledthrough theCHODP-12genome.(C)PercentmethylationofCpGislands(CGIs),untranslatedregions(UTRs),codingsequences(CDSs)andintrons.Lightgreybarsmarkthepercentage ofregionswithmethylationlevelsbelow10%.Alsoindicatedistheaveragemethylationlevelofeachgenomicfeature.

provedtohavethelowestmethylation levels,althougha larger

fractionofthemwasmethylatedase.g.incomparisontoCGIsin

humantissues(9%ofhypermethylatedCGIs)(Eckhardtetal.,2006).

Gene-bodiesinCHODP-12cellsexhibitedahigherlevelofDNA

methylationthanUTRs.

3.3. EvidenceforpartiallymethylateddomainsintheCHODP-12

genome

AswefoundindicationsofheterogeneousDNAmethylation

pat-ternsintheCHODP-12cellgenome,weplottedmethylationlevels

alongtheSNP-correctedCHOK1scaffoldstovisualizetheir

dis-tribution.Inthisanalysisweobservedheterogeneouspatternsof

DNAmethylationwithregionsshowingahighdegreeof

methyla-tionandneighboringregionswithpartialmethylation(exemplarily

shownin Fig.3 andSupplementaryFig.S2).Similarpatternsof

alternating methylation domains were e.g. found in colorectal

cancer,breastcancer,ﬁbroblastcultures,SH-SY5Yneuronalcells

andplacenta.Regionswithpartialmethylationinthesecellsand

tissues were termed partially methylated domains (PMDs)

(Schroederetal.,2011,2013;Gaidatzisetal.,2014;Listeretal., 2009;Honetal.,2012;Bermanetal.,2012).Accordingly,we

classi-ﬁedregionswithaminimumsizeof10kbandamethylationlevel

below70%asCHODP-12PMDs.CalculationofthePMD-coverageof

allSNP-correctedCHOK1scaffoldswithmorethan100CpGs(4874

scaffoldscomprising96%oftheCHOgenome)showedthat62%of

theanalyzedscaffoldswerepartiallymethylated.Accordingly,38%

oftheCHODP-12cellgenomewerecoveredbyhighlymethylated

domains(HMDs).

AsitwasshownthatXchromosomeinactivationcomplicates

PMDanalyses(Schroederetal.,2011)andduetothefactthatCHO

cellsoriginatefromafemaledonor,wenextaskedwhetherCHO

DP-12PMDsshoweddifferencesintheirdistributiononthe

sin-glechromosomes.Wecheckedforthispossibilitybymappingthe

wholegenomebisulﬁtesequencingreadstotheChinesehamster

(CH)chromosomes sequencedandannotated byBrinkrolfetal.

(2013).Normalizedtochromosomesize,wedetectedonaverage

18.4PMDs/MbandaverysigniﬁcantdifferenceinPMDcountper

2500 3000 3500 4000 4500 5000 5500 6000 CGI Exon PMD Meth level >80% >60-80% 0-60%

Color coding of methylation level

[kb]

Partial sequence of scaffold JH000005

Fig.3. Exemplarydisplayof3750kbofSNP-correctedCHOK1scaffoldJH000005.CHODP-12DNAmethylationlevelsfor1kbwindowswereplottedalongthescaffoldand color-codedaccordingtothelegend.AlsoshownareexonsandCpGislands(CGI)inblack.RegionsidentiﬁedasCHODP-12PMDsaremarkedbybluebars.Completescaffolds JH000001toJH000009areshowninSupplementaryFigureS2.(Forinterpretationofthereferencestocolorinﬁgurelegend,thereaderisreferredtothewebversionofthe article.)

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Table1

Geneontology(GO)classiﬁcationoftotalCHODP-12PMD-genes(4265)withBenjaminipvalues<0.05.

GOterm(category‘biologicalprocess’) % Foldenrichment pValue Benjaminipvalue

Translationalelongation 1.7 3.07 5.59×10−29 _2.86_×₁₀−25

Translation 3.6 1.96 1.96×10−22 _5.01_×₁₀−19

Sensoryperceptionofsmell 4.3 1.80 5.95×10−21 _1.02_×₁₀−17

Sensoryperceptionofchemicalstimulus 4.6 1.71 3.16×10−19 _4.05_×₁₀−16

Neurologicalsystemprocess 9.1 1.35 1.31×10−13 _1.35_×₁₀−10

Cognition 7.2 1.41 2.90×10−13 2.48×10−10

Sensoryperception 6.5 1.43 6.29×10−13 4.60×10−10

G-proteincoupledreceptorproteinsignalingpathway 8.1 1.28 8.68×10−9 _5.56_×₁₀−6

Ribosomebiogenesis 1.3 1.88 9.95×10−8 _5.66_×₁₀−5

Ribonucleoproteincomplexbiogenesis 1.7 1.64 1.67×10−6 _8.54_×₁₀−4

ncRNAprocessing 1.7 1.60 4.47×10−6 _0.002

rRNAprocessing 1.0 1.87 5.89×10−6 0.003

ncRNAmetabolicprocess 2.0 1.52 8.94×10−6 0.004

rRNAmetabolicprocess 1.0 1.80 2.28×10−5 _0.008

RNAsplicing,viatransesteriﬁcationreactions 1.4 1.60 3.19×10−5 _0.011

RNAsplicing,viatransesteriﬁcationreactionswith

bulgedadenosineasnucleophile

1.4 1.60 3.19×10−5 _0.011

NuclearmRNAsplicing,viaspliceosome 1.4 1.60 3.19×10−5 _0.011

RNAprocessing 3.9 1.27 1.25×10−4 0.039

MbforchromosomeX,whichwasmuchhigherwith26.4PMDs/Mb

(SupplementaryFig.S3).AsimilarﬁndingwasreportedbyLister

etal.(2009)forIMR90PMDs.Therefore,PMDsontheX

chromo-somewereomittedfromfurtheranalysesofPMDandHMDsize,

methylationlevelandcodingsequence(CDS)coverage.

CHODP-12PMDsexhibited54%methylationonaverage,amean

sizeof64kbandamaximumsizeof2990kb(Fig.4AandB).In

con-trast,HMDsshowedameanmethylationof77%,ameansizeof

34kbandamaximumsizeof450kb.Next,wecalculatedthe

cov-erageofCHODP-12PMDsandHMDsbycodingsequence(Fig.4C).

CHODP-12 PMDs showeda mean CDScoverage of 1.8% and a

maximumcoverageof65%,whereasHMDsexhibitedameanCDS

coverageof3.4%andamaximumcoverageof90%.Inorderto

fur-thermoreobtainthenumberofCHODP-12PMD-localizedgenes,

wecategorizedallannotatedCHOgenesaccordingtotheirgenomic

positionwithinoroutsideofaCHODP-12PMD.Geneswith

par-tialPMDcoveragewereexcluded.4265uniquegenesco-localized

withaCHODP-12PMD,representingonly18%ofthetotalof24,383

genespredictedfortheCHOK1cellline(Xuetal.,2011).In

sum-mary,CHODP-12PMDsprovedtobelargerbutalsoshowedclear

evidenceforcontainingfewergenesthanCHODP-12HMDs.

3.4. FunctionalclassiﬁcationofgeneswithinCHODP-12partially

methylateddomains

Inanextstep,weexaminedPMD-geneexpressioninour

sam-plesusing a CHO-speciﬁcmicroarray (Beckeret al.,2014), as a

knowncharacteristicofPMDsistranscriptionalrepressionof

co-localizedgenes.Thismicroarray enabled theanalysisof 16,693

uniquegeneclustersandcovered85%oftheidentiﬁedCHO

DP-12PMD-genes.Theremaining(635)PMD-geneswerenotpresent

onthemicroarrayandwerethereforenotanalyzed(Fig.4D).Aswe

appliedaone-colorlabelingprotocol,anestimationofgene

expres-sionstrengthswaspossiblebythisexperimentandallowedforthe

identiﬁcationofthesubsetofgenesthatwerehighlyexpressed.

Bythismeans,3120highlyexpressedgeneswereidentiﬁedand

40%(1261)ofthemalsobelongedtotheCHODP-12PMD-localized

genes.

Interestingly,Gene Ontology (GO)analysisof thetotal 4265

genesassociatedwithaCHODP-12PMDshowedsigniﬁcant

enrich-mentof two different generalcategories. Thesegenes wereon

theonehandrelated togene expression(RNAmetabolism and

translation)and ontheotherhandrelated toneurological

pro-cesses(Table 1).Whenwe performedseparate GOannotations

for thehighly expressed PMD-genes(1261) and theweakly or

notexpressedPMD-genes(2360),respectively,wefoundthatthe

highlyexpressedPMD-genesmainlybelongedtoGOclasseswith

functionsrelatedtogeneexpression(Table2andSupplementary

TableS1).Additionally,genescontributingtoenergymetabolism

and cellcyclecontrolwereenriched. Withintheweakly ornot

expressedPMD-genesthosecontributingtoneurologicalprocesses

wereoverrepresented(Table3andSupplementaryTableS3).Also,

geneswithfunctionsincellularadhesionandcalciumion

homeo-stasisweresigniﬁcantlyenrichedinthisgroup.

4. Discussion

4.1. TheCHODP-12cellDNAmethylomeischaracterizedby

globalhypomethylationandhypermethylationofCpGislands

Thisstudy providestheﬁrst genome-wide, single-baseDNA

methylation map ofa CHO cellgenome in combination witha

functionalassessmentofitsinﬂuenceongeneexpression.To

exem-plarilyexamineDNAmethylationpatternsandgeneexpressionof

viable,antibody-producingCHOcells,weanalyzedsamplesoffour

replicateCHODP-12cellbatchculturesintheexponentialgrowth

phasebywholegenomebisulﬁtesequencing.Weshowthatthe

CHODP-12cellgenomeishypomethylated(61%CpGmethylation)

comparedtomostmammaliancelltypeslikeembryonicstem(ES)

cells,theirderivativesandprimarycellsthatexhibitglobal

methyl-ationlevelsof>70%(Zilleretal.,2013).Globalhypomethylationhas

beenreportedforcancerssuchascolontumorsandalsofor

long-termculturedcellslikeIMR90,foreskinﬁbroblastsandHepG2cells

andprovedtobeeitherrelatedtoauniformoralocally

concen-trateddistributionofhypomethylatedregions(Zilleretal.,2013;

Laurentetal.,2010;Bermanetal.,2012;SchroederandLaSalle, 2013).

Theanalysisofmethylationofgenomicfeaturesshowedthat

CGIsand UTRsin CHODP-12 cells are less frequently

methyl-atedthanso-called genebodies (codingsequencesandintrons)

that are predominantly methylated. Thisis in accordance with

previouslydescribedDNAmethylomesthatshowhigherlevelsof

DNAmethylationingene-bodiescomparedtoUTRs.Thiswase.g.

shown inbovine muscletissue (Huang et al.,2014).For

multi-plespeciesagenome-widenegativecorrelationbetweenpromoter

methylationandgene expressionwasdescribed,whereas

gene-body methylationhasbeenreportedtobepositivelycorrelated

(6)

Methylati o n level of do m ain [%] D M P HMD Size of do m ain [kb] 0 CDS cove rage of do m ain [%]

A

B

C

5 10 20 40 60 20 40 60 80 11,213 2,360 635 1,859 1,261 PMD-localized genes (4,265) Highly expressed genes (3,120) Genes analyzed by microarray (16,693)

D

100 80 15 D M P HMD 0 10 0 120 D M P HMD 20 0

Fig.4. CharacterizationofCHODP-12partiallymethylateddomains(PMDs)andhighlymethylateddomains(HMDs).(A–C)Meanvaluesofdataareindicatedbyblack circles.Outliersarenotshown.(A)Percentmethylation.(B)Sizeinkilobasepairs(kb).(C)Codingsequence(CDS)coverage.(D)4265genesco-localizedwithCHODP-12 PMDs.Microarrayanalysisenabledassessmentoftheexpressionlevelsof16,693genes.Ofthese,3120werehighlyexpressed.1261ofthehighlyexpressedgenesalso co-localizedwithCHODP-12PMDs.

Table2

Geneontology(GO)classiﬁcationofhighlyexpressedCHODP-12PMD-genes(1261)withBenjaminipvalues<0.05.Displayedarethetop20GOtermsaccordingtopvalues. AcompletelistcanbefoundinSupplementaryTableSI.

Translation 11.7 5.78 4.54×10−76 _1.41_×₁₀−72

Translationalelongation 6.1 9.92 7.25E×10−64 1.13×10−60

RNAprocessing 9.3 2.78 6.47×10−25 6.72×10−22

Ribosomebiogenesis 4.0 5.33 1.14×10−23 _8.88_×₁₀−21

Ribonucleoproteincomplexbiogenesis 4.8 4.34 6.73×10−23 _4.19_×₁₀−20

NuclearmRNAsplicing,viaspliceosome 4.0 4.25 9.30×10−19 _4.83_×₁₀−16

RNAsplicing,viatransesteriﬁcationreactions 4.0 4.25 9.30×10−19 _4.83_×₁₀−16

RNAsplicing,viatransesteriﬁcationreactionswith

bulgedadenosineasnucleophile

4.0 4.25 9.30×10−19 _4.83_×₁₀−16

rRNAprocessing 2.9 5.23 2.61×10−17 1.16×10−14

RNAsplicing 5.4 3.11 2.84×10−17 _1.11_×₁₀−14

rRNAmetabolicprocess 2.9 5.01 1.31×10−16 _3.84_×₁₀−14

mRNAprocessing 5.6 2.84 2.09×10−15 _6.57_×₁₀−13

Oxidativephosphorylation 2.9 4.78 2.32×10−15 _6.60_×₁₀−13

ncRNAmetabolicprocess 4.4 3.17 1.47×10−14 _3.83_×₁₀−12

Generationofprecursormetabolitesandenergy 5.2 2.74 7.95×10−14 1.91×10−11

mRNAmetabolicprocess 5.8 2.57 1.12×10−13 2.50×10−11

ncRNAprocessing 3.7 3.27 7.37×10−13 _1.53_×₁₀−10

Energycoupledprotontransport,down

electrochemicalgradient

1.4 5.85 1.61×10−9 _3.14_×₁₀−7

ATPsynthesiscoupledprotontransport 1.4 5.85 1.61×10−9 _3.14_×₁₀−7

Ribosomalsmallsubunitbiogenesis 0.8 11.83 4.30×10−9 _7.88_×₁₀−7

(7)

Table3

Geneontology(GO)classiﬁcationofweaklyornotexpressedCHODP-12PMD-genes(2360)withBenjaminipvalues<0.05.Displayedarethetop20GOtermsaccordingto

pvalues.AcompletelistcanbefoundinSupplementaryTableSII.

Celladhesion 6.3 1.63 6.01×10−10 _1.23_×₁₀−6

Synaptictransmission 3.1 1.85 2.02×10−7 _2.77_×₁₀−4

Transmissionofnerveimpulse 3.4 1.78 2.25×10−7 _2.31_×₁₀−4

Behavior 4.2 1.60 1.70×10−6 _0.001

Elevationofcytosoliccalciumionconcentration 1.4 2.37 2.23×10−6 0.002

Calciumionhomeostasis 2.1 2.00 2.45×10−6 0.001

Neurologicalsystemprocess 9.0 1.34 2.48×10−6 _0.001

Cellularcalciumionhomeostasis 2.0 2.01 2.61×10−6 _0.001

Cellulardi-,tri-valentinorganiccationhomeostasis 2.3 1.86 5.97×10−6 _0.002

Cellularmetalionhomeostasis 2.1 1.92 8.43×10−6 _0.003

Responsetowounding 4.5 1.52 8.63×10−6 0.003

Cytosoliccalciumionhomeostasis 1.4 2.21 1.18×10−5 0.004

Cellularchemicalhomeostasis 3.4 1.62 1.27×10−5 _0.004

Cellularionhomeostasis 3.4 1.62 1.28×10−5 _0.003

Metalionhomeostasis 2.1 1.87 1.38×10−5 _0.004

Di-,tri-valentinorganiccationhomeostasis 2.4 1.80 1.39×10−5 _0.003

Cell–cellsignaling 4.8 1.45 3.90×10−5 _0.009

Cellularcationhomeostasis 2.4 1.72 4.26×10−5 0.009

Regulationofsystemprocess 2.8 1.64 5.03×10−5 0.010

Positiveregulationofhydrolaseactivity 1.8 1.84 8.70×10−5 _0.017

Themeaningofevolutionaryconservedgene-bodymethylationis

notcompletelyunderstoodyet,butstudiesindicatethatintragenic

DNAmethylationmodulatesalternativeRNAsplicing(Maunakea

etal.,2013).CGIsprovedtohavethelowestoverallmethylation

levelsinCHODP-12cells,althoughalargerfractionofthemis

meth-ylatedcomparedtohumantissues,mouseembryomicstemcells

ordifferentiatedmousecells(Eckhardtetal.,2006;Meissneretal.,

2008;Stadleretal.,2011).Thisﬁndingisincontrasttotheconcept

ofCGIsbeingusuallycharacterizedbytheirlackofDNA

methyla-tion,butinaccordancewiththehypothesisthatagreatnumberof

CGIsbecomemethylatedinpermanentcelllines(Antequeraetal.,

1990).Cancercellsarealsoknowntoexhibitglobal

hypomethyla-tionthataccompaniesaberranthypermethylationofCGIs(Sproul

andMeehan,2013).Inmanyofsuchcaseshypermethylationof

pro-moterCGIscanberelatedtosilencingoftumorsuppressorgenes

regulatingcell growth,differentiation and apoptosis (Yang and

Zheng,2014;Liuetal.,2014).Huetal.(2015),e.g.identiﬁednovel

candidatetumorsuppressorgenesinnaturalkillercelllymphoma

(NKCL)bypromotermethylationanalysisandgeneexpression

pro-ﬁling,includingbcl-2-likeprotein11(BCL2L11).EctopicBCL2L11

expressionledtoincreasedapoptosisinnaturalkillercelllines.A

similarapproachcouldbeappliedtoﬁndkeyregulatorsofgrowth

andapoptosisinCHOcellsusefulforcelllineengineering.

4.2. TheCHODP-12cellDNAmethylomeshowsevidenceof

partiallymethylateddomains

Theglobal characterization of DNA methylation in theCHO

DP-12 cell genome indicated a heterogeneous arrangement of

hypomethylatedregionsinsteadofanevendistribution.Whenwe

plottedmethylationlevelsalongtheSNP-correctedCHOK1

scaf-folds,weobservedlargegenomicregionswithpartialmethylation.

Methylationlevelsof54%onaveragecouldbeassignedtothese

regionswhichclearlyseparatedthemfromtheneighboringregions

withameanmethylationof77%.WetermedthemCHODP-12PMDs

accordingtothePMDsfoundincolorectalcancer,breastcancer,

ﬁbroblastcultures,SH-SY5Yneuronalcellsandplacenta(Schroeder

etal.,2011,2013;Gaidatzisetal.,2014;SchroederandLaSalle, 2013;Listeretal.,2009;Honetal.,2012;Bermanetal.,2012).

PMDscorrespondtogenomicdomainsofanaveragesizeof135kb

thatcoverapproximately40%of theabove-mentionedgenomes

andhavelowerlevelsofDNAmethylationthantheneighboring

genomic regions (Gaidatzis et al., 2014; Schroederand LaSalle,

2013).For CHODP-12PMDswe detectedameansizeof 64kb,

whichisprobablyduetothecurrentdraftstatusoftheCHOK1

genome.Weobserved acoverage ofallanalyzedSNP-corrected

CHOK1scaffoldsbyCHODP-12PMDsof62%.

AnothercharacteristicofPMDsistranscriptionalrepressionof

geneslocalizedwithintheseregionsthataregenerallygene-poor

(Schroederetal.,2013).Accordingly,only18%(4265)ofallgenes

werelocatedwithinCHODP-12PMDs.However,geneexpression

analysisshowedthat30%(1261)oftheCHODP-12PMD-geneswere

highlyexpressed,contradictingthenotionofgenerallyrepressed

expressionofPMD-localizedgenes.Inagreementwiththe

differ-encesinexpression,GOanalysisofallgeneslocatedwithinCHO

DP-12PMDsshowedasigniﬁcantenrichmentintwodifferent

func-tional groups that wererelated to gene expressionon theone

handandtoneurologicalprocessesontheotherhand.Whenwe

performedaseparateGOanalysisforthehighlyexpressed

PMD-genes,wefounda signiﬁcantoverrepresentationofthosegenes

with functions in gene expression and, additionally,in energy

metabolismand cellcycle,thus representingfunctionsvital for

CHOcell growth and productivity.Several reports have shown

strongexpression of genesrelated to transcription, translation,

chromatinmodiﬁcationandenergymetabolisminCHOcell

cul-tures,too(Carlageetal.,2009;Meleadyetal.,2011;Nissometal.,

2006).

WeaklyornotexpressedCHODP-12PMDgeneswere

signiﬁ-cantlyenrichedforthefunctionsrelatedtoneurologicalprocesses

suchassynaptictransmissionandalsotocelladhesionandcalcium

ionhomeostasis.ItisknownthatPMDsoftencontainrepressed

geneswithtissue-speciﬁcfunctionsunrelatedtothetissueoforigin

(SchroederandLaSalle,2013).Neurologicalprocessesclearlyare

unnecessaryfunctionsinCHOcells.Theseprocessesarestrongly

relatedtothecellularcalciumhomeostasis,as intracellular

cal-ciumplaysacriticalroleinneurondevelopment(GomezandZheng,

2006).Itisstrikingthatreducedcapacityofcellularadhesionis

rele-vantforadaptionofCHOcellstosuspensionculture,whichisaCHO

cellpropertynecessaryforscalabilityofindustrialprocesses.Itis

alsostronglyrelatedtotheconversionofbenigntumorsto

aggres-sivecancers(Tokudaetal.,2014).Interestingly,PMDsinplacenta

tissue,SH-SY5YandIMR90cellsaremostsigniﬁcantlyenriched

in genescontributing to neurological processes,too (Schroeder

etal.,2013).Thisﬁndingissupportedbythefactthatmethylation

patternsin PMDswereshowntobeconservedacrosscelltypes

(8)

5. Conclusions

Thisstudyprovidestheﬁrstglobalanalysisof theCHODNA

methylationlandscape.WeshowthattheCHODP-12cell

methy-lomeexhibitsgeneralpropertiesthataretypicalformammalian

cells, althoughit also turns outtobe heterogeneousand

char-acterized by global hypomethylation and hypermethylation of

CpG islands.It furthermore shows strong evidence of partially

methylateddomains.Similarepigenomicfeaturesareobservedfor

manycancertypesandalsoforplacenta(SchroederandLaSalle,

2013).In fact,placentaltrophoblastic andmalignantcells share

features such as highproliferation or their lack of cell-contact

inhibitionanditwasshownthatasubsetofmalignanttumors

acti-vateectopicexpressionofgermlineandplacentalgenes(Ferretti

etal., 2006;Rousseauxet al.,2013).CHOcellsare, similarlyto

cancerandplacentaltrophoblastcells,characterizedbytheir

pro-liferative properties and deregulatedapoptotic pathways. Here,

we additionally report that CHO DP-12 cells not only exhibit

epigenomicfeaturessimilartocancerandplacentacells,butthat

thereisevidenceofthesefeaturesbeinginvolvedinthe

repres-sionoftissue-speciﬁcgenes,asitwasshownforplacentaPMDs,

too.Interestingly,therepressiveeffectofCHODP-12PMDsdoes

notaffectgenesrelevanttoCHOcellproperties.Tofully

under-standthisrelationship,itstillneedstobeelucidatedhowPMDs

evolved,if theyare generated in a dynamic manner and what

theylooklikeonthecellularlevel.AcomparisonofdifferentCHO

celllines withrespecttothepresence andlocation ofPMDs is

also required. However, our ﬁndings show that DNA

methyla-tionmightsigniﬁcantlycontributetoCHOphenotypesandallow

for a deeperunderstandingofCHOcell properties.As a

conse-quence,futureexperimentstoidentifytargetsforrationaldesign

ofCHOcelllineswithimprovedproductioncharacteristicsshould

notonlyaddressgeneexpressionchanges,butalsothe

underly-ingepigeneticpatterns.ThepresenceofPMDsintheCHODP-12

cellgenomeforexampleunderlinesthenecessityofsite-speciﬁc

integration of recombinant genes in order toavoid potentially

unfavorable epigeneticenvironments leadingtolow expression

levels.

Acknowledgements

AWacknowledgesascholarshipfromtheCLIBGraduateCluster

IndustrialBiotechnology.WethankAndreasAlbersmeierandAnika

WinklerforexpertperformanceofIlluminasequencing.

AppendixA. Supplementarydata

Supplementary data associated with this article can be

found,intheonlineversion,athttp://dx.doi.org/10.1016/j.jbiotec.

2015.02.014.

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