RNA Sequencing Reveals the Alteration of the Expression of Novel Genes in Ethanol-Treated ...

19 Pages·2016·2.85 MB·English

RESEARCHARTICLE
RNA Sequencing Reveals the Alteration of the
Expression of Novel Genes in Ethanol-Treated
Embryoid Bodies
ChanchalMandal1,SunHwaKim1,JinChoulChai1,SeonMiOh1,YoungSeekLee1,
KyoungHwaJung2*,YoungGyuChai1,3*
1 DepartmentofMolecularandLifeScience,HanyangUniversity,Ansan,RepublicofKorea,2 Instituteof
NaturalScienceandTechnology,HanyangUniversity,Ansan,RepublicofKorea,3 Departmentof
Bionanotechnology,HanyangUniversity,Seoul,RepublicofKorea
*[email protected](YGC);[email protected](KHJ)
Abstract
Fetalalcoholspectrumdisorderisacollectivetermrepresentingfetalabnormalitiesassoci-
atedwithmaternalalcoholconsumption.Prenatalalcoholexposureandrelatedanomalies
OPENACCESS
arewellcharacterized,butthemolecularmechanismbehindthisphenomenonisnotwell
Citation:MandalC,KimSH,ChaiJC,OhSM,Lee
characterized.Inthispresentstudy,ouraimistoprofileimportantgenesthatregulatecellu-
YS,JungKH,etal.(2016)RNASequencingReveals
theAlterationoftheExpressionofNovelGenesin lardevelopmentduringfetaldevelopment.Humanembryoniccarcinomacells(NCCIT)are
Ethanol-TreatedEmbryoidBodies.PLoSONE11(3): culturedtoformembryoidbodiesandthentreatedinthepresenceandabsenceofethanol
e0149976.doi:10.1371/journal.pone.0149976
(50mM).WeemployedRNAsequencingtoprofiledifferentiallyexpressedgenesintheeth-
Editor:ShihuiYang,NationalRenewableEnergy anol-treatedembryoidbodiesfromNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB
Lab,UNITEDSTATES
+EtOHdatasets.Atotalof632,205and517differentiallyexpressedgeneswereidentified
Received:July13,2015 fromNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB+EtOH,respectively.Functional
Accepted:February8,2016 annotationusingbioinformaticstoolsrevealsignificantenrichmentofdifferentialcellular
developmentanddevelopmentaldisorders.Furthermore,agroupof42,15and35tran-
Published:March1,2016
scriptionfactor-encodinggenesarescreenedfromallofthedifferentiallyexpressedgenes
Copyright:©2016Mandaletal.Thisisanopen
obtainedfromNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB+EtOH,respectively.We
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits validatedrelativegeneexpressionlevelsofseveraltranscriptionfactorsfromtheselistsby
unrestricteduse,distribution,andreproductioninany quantitativereal-timePCR.Wehopethatourstudysubstantiallycontributestotheunder-
medium,providedtheoriginalauthorandsourceare
standingofthemolecularmechanismunderlyingthepathologyofalcohol-mediatedanoma-
credited.
liesandeasefurtherresearch.
DataAvailabilityStatement:Geneexpressiondata
havebeensubmittedtotheNCBISequenceRead
Archive(SRA)repository(http://www.ncbi.nlm.nih.
gov/sra/)underaccessionnumbersSRX904625,
SRX904869andSRX1175001. Introduction
Funding:Thisworkwassupportedbypartofthe Prenatalexposuretoalcoholhasprofoundeffectsonmanyaspectsoffetaldevelopment.
NationalResearchFoundationofKorea(NRF)grant Althoughalterationsinsomaticgrowthandspecificminormalformationsoffacialstructure
fundedbytheKoreaGovernment(MSIP)
aremostcharacteristic,theeffectsofalcoholonbraindevelopmentaremostsignificantinthat
(2013R1A1A3011026toK.H.J.,and2011-0030049to
theyleadtosubstantialproblemswithneurobehavioraldevelopment.Sincetheinitialrecogni-
Y.G.C.).Thefundprovidershadnoroleinstudy
tionofthefetalalcoholsyndrome(FAS),anumberofimportantobservationshavebeenmade
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. fromstudiesinvolvingbothhumansandanimals.Ofparticularimportance,anumberof
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ProfilingofEthanol-ResponsiveTranscriptionFactors
CompetingInterests:Theauthorshavedeclared maternalriskfactorshavebeenidentified,whichmaybeofrelevanceinthedevelopmentof
thatnocompetinginterestsexist. strategiesforthepreventionoftheFASandinterventionforthosewhohavebeenaffected.
Inrecentdecades,tremendousprogresshasbeenachievedintheresearcharearelatedto
alcoholictoxicityduringfetaldevelopment.Alcoholcancausedramaticandirreversibleeffects
onthefetus,suchasdevelopmentaldelay,headandfacialirregularities,seizures,hyperactivity,
attentiondeficits,cognitivedeficits,learningandmemoryimpairments,poorpsychosocial
functioning,facialirregularities,andmotorcoordinationdeficits[1].However,theexactdevel-
opmentalphasesinwhichalcoholhasthesespecificeffectsonthefetusarenotentirelyknown.
Severalfindingsrelatedtomolecularmechanismhavebeenpublishedrecently,includingstud-
iesimplicatingretinoicacidsignaling[2,3,4],glucocorticoidsignaling[4,5]stressresponse
genes[6,7],mitogen-activatedproteinkinase(MAPK)cascade[8],neurotransmitters[9,10],
phosphoinositide3-kinase[11],calciumsignaling[12],Wntsignaling[13,14],andtheNotch
andJAK/STATsignalingpathways[11,15].
Epigeneticmodifications,includingDNAmethylationinparticular,regulatekeydevelop-
mentalprocesses,includinggermcellimprintingandstemcellmaintenance/differentiation,
andplayacrucialroleintheearlyperiodsofembryogenesis[16,17,18].DNAmethylationis
alsoafundamentalaspectofprogrammedfetaldevelopment,determinationofcellfate,pattern
formation,terminaldifferentiationandmaintenanceofcellularmemoryrequiredfordevelop-
mentalstability[17,19].Moreover,aberrantepigeneticchangesinresponsetoenvironmental
stimulihavebeenshowntocontributetodevelopmentaldisorders[20].Recently,several
hypothesesinvolvingalcohol(ethanol)-inducedchangesingeneticandepigeneticregulation
ofcellsaspossiblemolecularmechanismsoffetalalcoholspectrumdisorders(FASDs)have
beenadvanced[21,22,23,24,25,26].However,theprecisemechanismsbywhichethanolalters
thetranscriptionallandscapearestilllargelyunknown.Inaddition,ethanolinfluencesthe
molecular,cellular,andphysiologicalregulationofadultstemcellsinadose-dependentman-
ner,whichlikelycontributestothedeleteriousconsequencesofexcessivealcoholconsumption
inadults[27,28,29,30].
Embryoniccarcinoma(EC)cellsexhibitpluripotentgeneexpressionprofilessimilarto
embryonicstemcellsandbothofthesecelltypesexhibitunlimitedself-renewalcapacityand
cangiverisetoderivativesofallthreeembryonicgermlayersasdemonstratedbyEBsincell
cultureandinthedevelopmentoftumorsafterinjectionintoadultmice[31,32].ECcellsare
derivedfrommalignantteratocarcinomaandcanproliferateindependentlyofgrowthfactors
andcytokines.Invitro,EBscandifferentiatespontaneouslyfromES,EGandECcellsasaggre-
gates.EBsarepartofawell-establishedmodeltoinvestigatecellulardifferentiationandgene
expressionpatternsduringES,ECandEGcelldifferentiationinvitro[33].Inthepresent
study,wehavesetupaninvitromodelforethanolexposureonNCCITcell-derivedembryoid
bodies(EBs),whichmimicearlyfetaldevelopment.Ourgoalwastoprofilenovelgenesthat
werealteredbyethanoltoaidfutureresearchregardingalcohol-relatedfetalabnormalities.We
comparethegeneexpressionprofilesinthepresenceandabsenceofethanolbyRNAsequenc-
inganalysis.Wealsocarriedoutextensivebioinformaticsanalysisonthegeneexpressiondata
andselectedagroupofgenesthatencodetranscriptionfactors(TFs)duringembryonic
development.
MaterialsandMethods
CellCultureandEthanolTreatment
Weusedhumanembryoniccarcinoma(NCCIT),collectedfromtheAmericanTypeCulture
Collection(CRL-2073)cellsforourstudy.ThecellswereculturedinRPMI-1640mediasupple-
mentedwith10%fetalbovineserum(FBS),100IUml–1penicillinand10μgml–1
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ProfilingofEthanol-ResponsiveTranscriptionFactors
streptomycinat37°CinaCO incubator.ForEBformation,1×106NCCITcellswereplated
2
inthe90-mmbacterialculturedishes(non-adherentcultureconditions)inOpti-MEMgrowth
mediumfor24h.ThemorphologyofEBswasexaminedbyphasecontrastmicroscopeand
immunocytochemistryanalysis(S1Fig).After24h,theEBswereexposedtoethanolfor
another48h,whereascontrolcellsremaineduntreated.Wechangedculturemediaeveryday
toensureaconstantethanolconcentrationduringthecourseofthestudy.Topreventethanol
evaporationfromtheculturedishes,ethanol-treatedcellswereculturedinaseparateCO incu-
2
batorthatwassaturatedwith50mMethanol,aspreviouslydescribedbyourgroup[34].A
graphicalexperimentalschemeofourprotocolispresentedinFig1A.
IsolatingTotalRNAandcDNASynthesis
TotalRNAsampleswereextractedbyhomogenizationinRNAisoPlus(TakaraBIO,Shiga,
Japan)accordingtothemanufacturerinstructions.Briefly,aftermixingofchloroform(200
ml)itwasgentlyinvertedfor5minutesandthencentrifugedfor15minat14,000xgat4°C.
Theuppersolutionwascollectedand600μlofisopropanolwasaddedtoit.After1hincuba-
tionthelysismixturewascentrifugedat14,000xgfor15minat4°C,andtheisopropanolwas
decanted.Ice-cold70%ethanolwasaddedtotheRNApelletforgentlewashing.After
centrifugingfor10min,theethanolwasdiscarded.TheRNApelletsweredriedatroomtem-
peraturefor5minandthenadded20μlRNase-freewaterinit.Thequalityandquantityof
extractedtotalRNAwasmeasuredbyanAgilent2100Bioanalyzer(AgilentTechnologies,
Waldbronn,Germany)andaspectrophotometer(NanoDropTechnologies,Wilmington,DE,
USA),respectively.ReversetranscriptionoftheextractedRNAwasconductedaccordingto
Halderetal.,2014[2].Inbrief,thefirst-strandcDNAwassynthesizedwithSuperScriptII
ReverseTranscriptase(Invitrogen,Carlsbad,CA,USA).
RNASequencing(RNA-seq)
RibosomalRNAs(rRNAs)fromtotalRNA(5μg)wereremovedusingaRiboMinusTMTran-
scriptomeIsolationKit(Invitrogen).rRNA-depletedtotalRNAs(100ng)wereusedtocon-
structpaired-endtranscriptomelibrariesusingtheNEBNext1UltraTMDirectionalRNA
LibraryPrepKitforIllumina1(NewEnglandBiolabs,Ipswich,MA,USA).Briefly,first-
strandcDNAsweresynthesizedfromrRNA-depletedRNAsamples,followedbysecond-
strandsynthesiswithDNApolymeraseIandRNaseH.Thedouble-strandedcDNAswerethen
end-repairedandligatedtoadaptors.Ligatedlibrarieswerethenseparatedona2%agarosegel
(Duchefa,Haarlem,TheNetherlands),andfragmentswithsizesbetween300–400bpwere
purifiedusingtheMinEluteGelExtractionKit(Qiagen,Hilden,Germany).Fragmentswere
amplifiedforfurtherenrichmentandpurifiedwithethanolprecipitation.ThecDNAfragments
(101bp)weresequencedusingtheIlluminaHiSeq2500(101cyclesPElane)(NationalInstru-
mentationCenterforEnvironmentalManagementinSeoulNationalUniversity).Twobiologi-
calreplicateswerepreparedfromeachcondition.Atabulardescriptionofreadcountis
presentedintheS1Table.GeneexpressiondatahavebeensubmittedtotheNCBISequence
ReadArchive(SRA)repository(http://www.ncbi.nlm.nih.gov/sra/)underaccessionnumbers
SRX904625,SRX904869andSRX1175001.
AnalysisoftheSequencingData
RawsequencefilesunderwentaqualitycontrolanalysisusingFastQC[35].Toavoidlow-qual-
itydata,weclippedtheadaptersandtrimmedthereadsusingFASTX-Toolkit[36].Paired-end
readswerealignmentstotheHomosapiencegenome(HomosapiensUCSChg19)usingTop
hat[37]usingdefaultparameter.Fortheanalysisofdifferentialexpressedanalysiswas
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ProfilingofEthanol-ResponsiveTranscriptionFactors
Fig1.Functionalannotationofdifferentialgeneexpression.A)Graphicalexperimentalschemefordifferentiation/treatmentprotocol.NCCITcellswere
stabilizedandsubculturedtoformEBs.Afterstabilization,theEBsweretreatedwithorwithoutEtOHfor48h.Thesampleswerethencollectedforfurther
analyses.B)Venndiagramrepresentingtheoveralldistributionofalldifferentiallyexpressedgenes.Atotalof632,205and517differentiallyexpressed
geneswereidentifiedfromNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB+EtOH,respectively.C)HeatmaprepresentingallDEGsthatfoundcommon
betweenNCCITvs.EBandEBvs.EB+EtOHdatasets.Geneswithspecificexpressionpatternswereclusteredinto4groups–cluster1,2,3and4represent
up-up,up-down,down-downanddown-up,respectively.Cluster2and4werelistedhere.D)Linegraphrepresentingrelativeexpressionpatternofthose
geneclustersdefinedfromthetranscriptomicprofiling.Geneswithspecificexpressionpatternswereclusteredaccordingtotheirrelativeexpressionvalues.
doi:10.1371/journal.pone.0149976.g001
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ProfilingofEthanol-ResponsiveTranscriptionFactors
performedwithCufflinks[38].Defaultsettingswereusedinaforementionedmethods.Differ-
entiallyexpressedgenesshowingmorethan1.0-foldchangeintheirlog fold-change(P-
2
value<0.05)wereselectedforfunctionalannotation.
FunctionalAnnotation,HeatMapConstructionandEnrichmentAnalysis
FunctionalannotationofsignificantgenesidentifiedbytheRNA-seqanalysiswassearched
usingthewebaccessibleprogramDatabaseforAnnotation,VisualizationandIntegratedDis-
covery(DAVID)(http://david.abcc.ncifcrf.gov).DAVIDcalculatesamodifiedFisher'sExact
P-valuetodemonstrategeneontology(GO),whereP-valueslessthan0.05areconsideredtobe
stronglyenrichedintheannotationcategory.Weconstructedheatmapstoviewtherelative
expressionpatternsofourarraydatausingTIGRMultiexperimentViewer(MeV).MeVisa
Java-basedmicroarraydataanalysistool(desktopapplication)thatallowsadvancedanalysisof
geneexpressiondatathroughanintuitivegraphicaluserinterface.Weuploadedourarraydata
intextfileformatandchosetwocolorarraystocreateheatmaps.Integrateddiseaseenrich-
mentanalysiswasperformedusingingenuitypathwayanalysis(IPA).TheDEGsweremapped
intheanalysistoolandobservedforsignificantdiseasepathwayenrichment.Thisanalysis
helpedusunderstandcausalconnectionsbetweendiseasesandgenes.
QuantitativeReverseTranscription-PolymeraseChainReaction
AllassayswererunonaABI7500Real-TimePCRSystem(AppliedBiosystems,Inc.,Foster
City,CA,USA)usingtheSYBRPremixExTaqTMII(Otsu-Shi,Shiga,Japan).Thereactionvol-
umewas20μLandthePCRconditionswereasfollows:30sec.at95°C,40cyclesof5sec.at
95°Cand34sec.at60°C,followedbyameltingcurveanalysisstep.Ifallampliconshoweda
singleTm,thePCRreactionswereconsideredspecific.Everysamplewasmeasuredintripli-
cate,andrelativequantificationwaseffectedbymeansofthecomparativeCT(ΔΔCT)method.
GAPDHwasusedasahousekeepinggenetonormalizetheexpressiondata.Theprimersused
forgenevalidationarelistedinS2Table.
TranscriptionFactorBindingMotifEnrichmentAnalysis
NCBIreferencesequencemRNAaccessionnumbersweresubjectedtotranscriptionfactor
bindingmotifanalysisusingtheweb-basedsoftwarePscan(http://159.149.109.9/pscan/)[39].
TheJASPAR[40]databaseoftranscriptionbindingfactorsequenceswasanalyzedusing
enrichedgroupsof−950basepair(bp)sequencesto+50bpofthe5’upstreampromoters.The
rangeof−950to+50wasselectedfromtherangeoptionsinPscantoobtainthebestcoverfor
a−1000to+50bprange.
StatisticalAnalysis
Inthisstudy,weranthreetechnicalreplicatestostudytherelativegeneexpressionsforcontrol
andtreatedsamples.ForqRT-PCRanalysis,resultsarepresentedasthemean±standarderror
ofthemean(SEM)(n=3).Forthestatisticalanalyses,Student’st-testwasperformedusingthe
MicrosoftOfficeExcel,2010programatthe0.05probabilitylevel.
Results
GeneExpressionProfileAnalysisofEthanolExposureDuringEarly
Development
WeperformedRNA-seqanalysisforNCCIT,EBandEBwithEtOHandcomparedeach
datasetwithanotherinthemannerofNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB
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ProfilingofEthanol-ResponsiveTranscriptionFactors
+EtOH.Afternormalizationofgeneexpressionprofilingdata,wefirstexaminedthenum-
berofallalteredgenes.Usingthethresholdofmorethan1.0-foldchangeintheirlog fold-
2
change(P-value<0.05),weidentified632,205and517differentiallyexpressedgenes
(DEGs)inNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB+EtOHdatasets,respectively.
NCCITvs.EB+EtOHandEBvs.EB+EtOHdatasetsrepresentDEGsthatwerealteredin
responsetoacutealcoholintoxication.IntheNCCITvs.EB+EtOHdataset122geneswere
up-regulatedand83weredown-regulatedwhereas,213and304geneswereup-anddown-
regulatedintheEBvs.EB+EtOHdataset,respectively.Atotalof75,219and51genesare
commonbetweenNCCITvs.EBandNCCITvs.EB+EtOH,NCCITvs.EBandEBvs.EB
+EtOH,andNCCITvs.EB+EtOHandEBvs.EB+EtOH,respectively.Thenumberof
alteredgenesshowedaclearviewofthedeteriorativeeffectofethanolduringearlydevelop-
ment.Toobserveaclearcomparisonbetweenthese3datasetswehavedrawnaVenndia-
gramwhereintersectionalcomparisonswererepresentedina2by2comparisonmanner
(Fig1B).WehaveconstructedaheatmapthatrepresentedallcommonDEGsbetween
NCCITvs.EBandEBvs.EB+EtOH.WehavedividedallDEGsinto4clusterswhereCluster
1,2,3and4representup-up,up-down,down-downanddown-uprelationshipsbetweenthe
compareddatasets,respectively.Unfortunately,wedidnotgetanygenesundercluster1
and3where124and82geneswerelistedundercluster2and4,respectively(Fig1C).We
alsodrewlinegraphtogeneralizetheexpressionpatternsoftheDEGsamongthesedatasets
(Fig1D).Thenumbersofalteredgenesrepresenttheeffectofalcoholintoxicationduring
earlyfetaldevelopmentwhichmayhaveaseriousconsequencesregardingproperembry-
onicdevelopment.
PathwayAnalysisofDifferentiallyExpressedGenes
ToobtainaglobalviewofthebiologicalprocessesrepresentedintheseDEGs,wecarriedout
GOtermenrichmentanalysisusingafalsediscoveryrate(FDR)cutoffof0.05.Wehave
short-listedthetotalenrichedGOtermsandshowedintheS2Fig.Toexploretheethanol
responsivecategoriesweconsideredthecomparisonbetweenEBvs.EB+EtOHdataset.GO
termenrichmentanalysisrevealedpreferentialincreasesintheexpressionofgenesin
responsetoethanolexposureinvolvedindiversecellularactivitiesincluding“positiveregula-
tionofcellproliferation”,“carbohydratebiosyntheticprocess”,“embryonicorgandevelop-
ment”,“regulationofcelldevelopment”,“negativeregulationofcelldifferentiation”and
others(S2CFig).Inaddition,therewasadramaticdecreaseinthelevelsoftranscriptsinthe
GOcategoriesof“intracellularsignalingcascade”,“regulationofcellproliferation”,
“responsetoorganicsubstance”,“positiveregulationofmolecularfunction”,“negativeregu-
lationofsignaltransduction”,andothersbetweenthecontrolandethanol-treatedEBs(S2C
Fig).Wehavealsoprovidedtheenrichedtermsforanothertwosetsofcomparisongroupsin
S2AFigandS2BFig.
Ingenuitypathwayanalysis(IPA)revealed71differentenrichedcanonicalpathways(P-
value<0.05,atleast5DEGslisted)fromtheEBvs.EB+EtOHdataset.Importantpathways
areILKsignaling,axonalguidancesignaling,RhoAsignaling,mTORsignalingetc.Thetop
15pathwaysthataresignificantlyenrichedtothedifferentiallyenrichedgenesarelistedin
Table1.Wealsolistedenrichedcanonicalpathwaysforothertwodatasets.Inbrief,DEGs
weresignificantlyinvolvedinatotalof119and17pathwaysinNCCITvs.EBandNCCIT
vs.EB+EtOH,respectively.Top15enrichedpathwaysfromeachdatasetwerelistedinS3
TableandS4Table,respectively.Theenrichmentofdifferentpathwaysprovideaclearidea
thatethanolhastheabilitytoaltercellstateortomisleaddirectedpathsduringearly
development.
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ProfilingofEthanol-ResponsiveTranscriptionFactors
Table1. Top15enrichedcanonicalpathwaysofalldifferentiallyexpressedgenesintheEBvs.EB+EtOHdatasets.
IngenuityCanonicalPathways -log(p- Molecules
value)
Remodelingofepithelialadherens 4.04E00 TUBB3,ACTB,ACTN3,TUBB2A,TUBG1,TUBB4A,ACTG1,MAPRE3
junctions
Sertolicell-sertolicelljunction 3.37E00 TUBB3,CLDN19,ACTB,ACTN3,TUBB2A,TUBG1,TUBB4A,JUP,CLDN6,ELK1,ACTG1,OCLN
signaling
Germcell-sertolicelljunction 3.21E00 TUBB3,RHOG,CFL1,RHOB,ACTB,ACTN3,TUBB2A,TUBG1,TUBB4A,JUP,ACTG1
signaling
ILKsignaling 2.68E00 FN1,RHOG,CFL1,RHOB,LIMS1,ACTB,ACTN3,PPP2R5B,TMSB10,PPP1R14B,ACTG1
Glioblastomamultiformesignaling 2.4E00 FZD10,PLCB4,RHOG,RHOB,PDGFA,PLCG2,IGF1R,CDK6,FZD6
Epithelialadherensjunction 2.4E00 TUBB3,ACTB,ACTN3,TUBB2A,TUBG1,ACVR1,TUBB4A,JUP,ACTG1
signaling
Phagosomematuration 2.39E00 ATP6V0C,TUBB3,VPS28,TUBB2A,TUBG1,TUBB4A,DYNLRB1,PRDX2
Semaphorinsignalinginneurons 2.29E00 CRMP1,RHOG,CDK5,CFL1,RHOB
Gapjunctionsignaling 2.23E00 PLCB4,TUBB3,GUCY2C,PLCG2,ACTB,TUBB2A,TUBG1,TUBB4A,ACTG1
Axonalguidancesignaling 1.94E00 FZD10,TUBB3,PLXNC1,PFN1,CFL1,PDGFA,TUBG1,TUBB2A,PTCH1,PLCB4,SEMA6D,CDK5,
NGFR,PLCG2,FZD6,TUBB4A,GLI1
14-3-3-mediatedsignaling 1.91E00 PLCB4,TUBB3,PLCG2,TUBB2A,TUBG1,TUBB4A,ELK1
D-myo-inositol-5-phosphate 1.9E00 PLCB4,PPP1R1A,PLCG2,PPP2R5B,NUDT14,PPP1R14B,PXYLP1,DUSP14
metabolism
RhoAsignaling 1.82E00 NEDD4,PFN1,CFL1,ACTB,IGF1R,ARHGEF1,ACTG1
Actincytoskeletonsignaling 1.75E00 FN1,PFN1,CFL1,PDGFA,ACTB,ACTN3,ARHGEF1,TMSB10,ACTG1,FGF19
PI3KsignalinginBlymphocytes 1.72E00 PLCB4,ATF3,NFKBIA,ATF5,PLCG2,PIK3AP1,ELK1
doi:10.1371/journal.pone.0149976.t001
NeuronalDevelopmentisActivatedbyEthanolDuringEarly
Development
FromtheGOanalysis,wegainedanideaoffunctionalcategoriesoftheDEGs.Again,toobtain
moremolecularinformation,weexaminedourgenelistforcellularandmolecularfunctional
analysisusingIPA.Underthethresholdof<0.05,atotalof21categoriesinEBvs.EB+EtOH
datasetarelisted.Thesecondtopmostenrichedcategoryis“cellulardevelopment”inwhich
155genesarealteredbyethanolexposure(Fig2C).These155genesareinvolvedinthedevel-
opmentofneurons,proliferationofcarcinomacelllines,differentiationofcells,neuritogenesis,
developmentofcentralnervoussystemcellsandalsootherimportantdevelopmentalfunctions
(datanotshown).Thus,anyalterationofthesedevelopmentalprocesseswillraisemajordisor-
ders.WealsoanalyzedenrichedcategoriesforNCCITvs.EBandNCCITvs.EB+EtOH.In
brief,atotalof24and25categoriesarelisted,respectively.Thetop10functionsofeachcom-
parisongrouparepresentedinFig2Aand2B.
FromthelistshowninFig2Cwehavenoticedthatalargeamountofdifferentially
expressedgenes(37)wereenrichedto“developmentofneurons”.Thefindingswereinteresting
andwedrewanetworkfordevelopmentofneuronsandfoundthat11geneswereinvolvedto
activateneuronaldevelopment(markedasorangelinesinthenetwork,Fig3A).Itwasreported
thatknockingdownofratPcyt1bgenedecreasedsproutingandbranchingofneuritesinPC-12
cells[41].Additionally,inducedexpressionofmouseLrrn1,Ddah1andAtf3increasedsynap-
togenesis[42],increasedformationofneurite[43]andincreasedsproutingofaxons[44],
respectively.Zineetal.[45]havereportedthatmouseHes1isinvolvedindifferentiationmor-
phogenesisofneuronsanddecreasingofitsexpressionresultsincreasedhaircellsformation
[46].KnockingdownofmouseRhobgeneincreasedbranchingofdendritesandincreased
lengthofdendriticspinesinpyramidalcells[47].Additionally,knockingdownofratElk1
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ProfilingofEthanol-ResponsiveTranscriptionFactors
Fig2.Functionalenrichmentanalysisofdifferentiallyexpressedgenes.A).B)andC)representPiechartsofcellularandmolecularfunctionsofthe
DEGs(top10categories;p-values<0.05)betweenNCCITvs.EB,NCCITvs.EB+EtOHandEBvs.EB+EtOH,respectively.Numbersinthecharts
representtherelativegenesenriched.
doi:10.1371/journal.pone.0149976.g002
mRNAbyshRNAincreasesdensityofdendriticspines[48].DAB1proteinisanimportant
candidateduringneuraldevelopment.ItwasreportedthathumanDAB1proteinaffectsforma-
tiontoneuriteinculturedchickenretinalcells[49].Additionally,mouseDab1isinvolvedin
developmentofdendrites[50]anditdecreasedaxonogenesisofaxons[51],sameasratThy1
[52].Furthermore,Yaguchietal.[53]reportedthatsuppressionofmousePrkcshmRNAby
shRNAincreasedneuritogenesisofN1E-115cellsincellculture.NF-kappaBsignalingpro-
motesbothcellsurvivalandneuriteprocessformationinnervegrowthfactor-stimulatedPC12
cells[54].Wehavepreparedafunctionalnetworkforthese11genesonlywheretheirproposed
rolesinneuronaldevelopmentwereplotted(Fig3B).Wevalidatedallofthe11genesby
qRT-PCR.Weobservedthatallofselectedgenesweresignificantlyalteredtoactivateneuronal
developmentasIPAnetworkclaimedexceptELK1(Fig3C).Thus,weassumedthatthere
wouldbeanassistanceeffectexertedbyethanolitselfduringthedevelopmentprocess.
ASetofTranscriptionFactor-encodingGenesareAlteredbyEthanol
Atthisstagewewerepromptedtoprofileethanol-responsiveTFsavailableinourdatasets.To
verifythegroupofethanol-targetedTFs,wecomparedourDEGswithalistofhumanTFspro-
videdbyVaquerizasetal.,2009[55].WesearchedforTFsinouralldatasetsandidentifieda
hugenumberofTFsthatareethanolresponsive.Wehavelistedatotalof42TFsinNCCITvs.
EB,atotalof15TFsinNCCITvs.EB+EtOHandatotalof35TFsinEBvs.EB+EtOHdataset.
ThenumberofalteredTF-encodinggenesshowedaclearviewofthedeteriorativeeffectof
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ProfilingofEthanol-ResponsiveTranscriptionFactors
Fig3.Activationoftheneuronaldevelopmentprocessbyethanol.A)Networkforthecategory
“developmentofneuron”thatwasadoptedfromIPA.Therelationshipismentionedatthebottom.B)A
functionalnetworkanalysisofthe11DEGsthatwerepredictedtoactivateneuronaldevelopment.Boxes
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ProfilingofEthanol-ResponsiveTranscriptionFactors
representproposedrolesinneuronaldevelopmentinaspectofexpressionpatternsfoundinEBvs.EB
+EtOHdataset.C)qRT-PCRanalysisfortherelativemRNAexpressionofthe11genesresponsibleforthe
activationofneuronaldevelopment.TheexpressionvaluewasnormalizedtotheGAPDHexpressionlevel.
ValuesarerepresentedasaveragemRNAexpression±SEMbars,n=3replicates.Asterisksindicate
statisticallysignificantchangesbasedonadjustedp-values<0.05.
doi:10.1371/journal.pone.0149976.g003
ethanolduringearlydevelopment.AllidentifiedTFsarelistedinTable2.Toobserveaclear
comparisonbetweenthese3datasetswehavedrawnaVenndiagramwhereintersectional
comparisonswererepresentedina2by2comparisonmanner(Fig4A).Basedonthe1.0log
2
fold-changecriteriaforminingthebiologicaldata,heatmapswereconstructedwherethe
expressionchangesweredetectedveryeasily(Fig4B,4Cand4D).Wealsodrewheatmapsfor
commonTF-encodinggenesbetweenNCCITvs.EBandEBvs.EB+EtOHdatasets(Fig4E).
WehavedividedalldifferentiallyexpressedTFsinto4clusterswhereCluster1,2,3and4rep-
resentup-up,up-down,down-downanddown-uprelationshipsbetweenthecompareddata-
sets,respectively.Unfortunately,wedidnotgetanygenesundercluster1and3where6and10
geneswerelistedundercluster2and4,respectively(Fig4E).Wealsodrewlinegraphtogener-
alizetheexpressionpatternsofthecommondifferentiallyexpressedTFsamongthesedatasets
(Fig4F).Therelativeexpressiondifferenceswereclearlynoticeableinthosefigures.
WealsoperformedenrichmentanalysisofknownTFmotifsusingknownTFmotifsinthe
JASPERdatabasetoidentifymotifsenrichedinthesedifferentiallyexpressedTF-encoding
genes.Wefoundthattherewereatotalof80TFsinNCCITvs.EB,atotalof28TFsinNCCIT
vs.EB+EtOHandatotalof13TFsinEBvs.EB+EtOH,whosebindingsitesweresignificantly
overrepresentedinthepromoterregionofdifferentiallyexpressedTF-encodinggenes(p
<0.05).Top5enrichedmotifsarepresentedinFig4G,4Hand4I.Ouraimwastoprofileetha-
nol-responsiveTFsthatwillhelpotherresearcherstofindmoremolecularmechanismcon-
ductedbyethanol.So,weemphasizedtheTFsfoundinEBvs.EB+EtOHdatasetandchose
severalgenesrandomlytovalidatetheirexpressions.Expressiongraphshowedtheconsistent
resultswithRNA-seqanalysis(Fig5A).ToobservetheexpressionofselectedTF-encoding
genesinresponsewithethanolwealsostudieddifferentconcentrationsofethanol(15mM,30
mMand50mM)whichmimiclow,mediumandhighexposureunits.qRT-PCRanalysis
showedthatlowandhighdosesweremoreeffectualtodevelopingEBs(Fig5B).
Discussion
Alcoholanddevelopmentaldisorderisawell-establishedphenomenonbutthemannerin
whichalcoholexertsitstoxiceffectisstillnotwellunderstood.Here,wehavetriedtoexplore
genomicalterationsmediatedbyethanolduringearlydevelopmenttoprofileethanol-targeted
geneexpressions.Inthisstudy,weusedNCCITcellsandappliedRNAsequencingtoprofile
ethanol-targetedgenesandtriedtocategorizethegeneticalterationsaccordingtomolecular
andcellularfunctions.WehavelistedagroupofTFsthatwereregulatedbyethanolduring
earlydevelopmentand,toourknowledge,wearethefirstgrouptoprofileasetofethanol-
responsiveTFs.Thereisasimilarstudypreviouslypublished[56]byourgroupwherewetried
tofindproteomicchangesbyethanolusingMALTI-TOFMS.Intheformerstudywetreated
NCCITcellsbyethanolfromtheverybeginningwithoutwaitingforEBformation.Insimple
word,theformationofEBwasunderethanolexposureitself.Butintherecentstudywedid
formEBatfirstandthenexposedtoethanol.Thelateroneismuchclosertomimicembryonic
stateexposedbyethanol.
FromtheRNAsequencingdata,weobtainedalargenumberofgenesthatweredifferen-
tiallyexpressed.Thenumberofalteredgeneshighlightedaverysimpleanalysisthatethanolis
PLOSONE|DOI:10.1371/journal.pone.0149976 March1,2016 10/19