PeaksdefiningwhereDNAwas
boundtothehistonemarksof
interest(H3K4me3andH3K9ac)were
identifiedandqualityscored.Using
theUCSCgenomebrowseritwas
observedthattreatmentBandA+B
hadverysimilarpeakshifts,which
differedfromthecontrolgroupin
severalimportantgenicregions.The
KLF4genedescribedaboveis
providedasanexampleforH3K4me3
(top).Importantly,manygenes
identifiedthroughdifferential
analysiswithChIP-seqdidnotshow
upintheRNAexpressionanalysis.
Forexample,theepithelial-specific
factorEHFscoredhighlyinChIP-seq
(bottom),butnotintheRNA-seq.
Conversely,itwasnotedthatmany
differentiallyregulatedgenesfrom
theRNA-seqdidnothaveChIP-seq
peaksofsignificantqualityfor
comparison.
Promoter/GenicRegion
Ch
IP
Pea
k
INTRODUCTION
GENEANDEPIGENETICEXPRESSIONPATTERNSOFSAME-GENOME
SKINEQUIVALENTCONSTRUCTSEXPOSEDTOSINGLEAND
COMBINATIONSOFCOSMETICACTIVEINGREDIENTS
BretStephens1,JinNamkoong2,DaleKern2,RemonaGopaul2,HelenE.Knaggs2
1WasatchScientificServices,SaltLakeCity,UnitedStates
2CenterforAnti-AgingResearch,NuSkinEnterprises,Inc.,Provo,UnitedStates
?Oryzasativa,NarcissustazettabulbandSchizandra
chinensisfruitextractsarecurrentlyusedinskincare
formulations,howeverthesub-cellulareffectsofthese
extractsonskincellsispoorlyunderstood.
?RNA-seqandChIP-seqrepresentcontemporarymethods
toobtainasnapshotofgeneexpressionandepigeneticstate.
?Skinequivalentmodelsconsistingofadultfemalematched
donorkeratinocytesandfibroblastsshouldmoreclosely
replicateskinbiologyofadultfemalescomparedto
monolayerculturemodelsortypicalcommercially
constructedtissuescontainingcellsfromdifferentdonors.
OBJECTIVE
Investigategeneandepigeneticexpressionpatternsofsame-
genomekeratinocyte/fibroblasttissueconstructsexposedto
Oryzasativa,NarcissustazettabulbandSchizandrachinensis
fruitextractsindividuallyandincombination.
METHODS
RESULTS
I.RNA-seqidentifiesmodulationofgenesexpression
andpathwaysinvolvedinskindifferentiation.
TableA.Overalltherewere394,1484,and1940genes
differentiallyregulatedatalevelofgreaterthan2-fold
inductionorrepressionfortreatmentsA,B,andA+B,
respectively.TreatmentBhadamoredramaticeffecton
transcriptionalchangesthantreatmentA.Interestingly,itwas
observedthatthereweresignificantlymoredown-regulated
genesthanup-regulatedgenesineachtreatment.Alsoof
note,thegenesdifferentiallyexpressedintreatmentA+B
overlappedmorecloselywithtreatmentBthantreatmentA,
suggestingthatthecontributionoftreatmentBismore
dominantinthecombinatorialtreatment.
CONCLUSIONS
Toourknowledge,thisisthefirstcomprehensivesignaling
studyevaluatingtheeffectsoftheseextractsinamodelsystem
intendedtobestreplicatethemostcommonusersofcosmetic
productsbyutilizingmatchedadultfemalekeratinocytesand
fibroblasts.TheRNA-seqresultssuggestthattheseextracts
(moreparticularlyN.tazettaandS.chinensis)reduceexpression
ofmanyskin-specificfactorsthatplayaroleinterminal
differentiation,leadingtoanexpressionprofilethatmimics
youthfulskin.Asexpected,ChIP-seqanalysisprovedusefulin
validatingRNA-seqfindings.Moreimportantly,theChIP-seq
resultshaveexposedadditionalpatternsatpotentiallykeygenic
regionsthatwerenotevidentintheRNA-seqdata.Conversely,
ChIP-seqwasnotabletoidentifyalldifferentiallyregulated
transcripts.Therefore,usingbothplatformsinconcertcan
provideadditionallayersofunderstandingcomparedtojusta
singlemethod.Furtherminingofthisdata,combinedwith
follow-upstudiesareplanned.
REFERENCES
1Huangetal.NatureProtoc.2009;4(1):44-57
2Henryetal.FrontBiosci.2012;1(17):1517-32
ConflictofInterest
JN,DK,RGandHKareemployeesofNuSkinEnterprises,Inc.
BSreceivedfundingfromNuSkinEnterprises,Inc.
RNAIsolation
LibraryGeneration
Crosslink
Chromatin
Bioinformatics
Gen
eE
xp
res
sio
n
TreatmentGroups
Discard
LibraryGeneration
Sequencing
Bioinformatics
Compare
LabelTreatmentConditions
CUntreatedcontrol
AOryzasativaextract
BNarcissustazettabulbextract,Schizandrachinensisfruitextract
A+BCombinationofAandB
H3K4me3
Selection
GenesExpressed≥2-fold≤2-fold
TreatmentA81313
TreatmentB4571027
TreatmentA+B7211219
Top5GeneOntology(GO)TermBiologicalProcesses
TreatmentATreatmentB
1EpidermisdevelopmentEpidermisdevelopment
2KeratinizationEctodermdevelopment
3EctodermdevelopmentEpidermalcelldifferentiation
4ResponsetowoundingKeratinization
5KeratinocytedifferentiationKeratinocytedifferentiation
TableB.Genesidentifiedasup-ordown-regulatedwerefed
intothegeneontologyprogramDAVID1(version6.7).Pathways
ofhighestprobabilityarelistedinorder.Itwasnotedthat
treatmentBandtreatmentA+Bdramaticallyreducedthe
expressionofgeneslocatedintheepidermaldifferentiation
complex(EDC).TheEDCisenrichedforgenesassociatedwith
epidermalterminaldifferentiation:S100genes,smallproline-
richregion(SPRR)genes,andlatecornifiedenvelope(LCE)
genes2.Ofnote,KLF4(amasterstemcellregulatory
transcriptionfactor)controlsexpressionofEDCgenesandalso
wasfounddown-regulatedinoursamples.
II.ChIP-seqfindingsenhanceexpressionresults.
KLF4
UntreatedC
TreatmentA
TreatmentB
TreatmentA+B
EHFAltStart
UntreatedC
TreatmentA
TreatmentB
TreatmentA+B
Contact:DaleKern,dkern@nuskin.com
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