MQP‐BIO‐JBD‐0011

CharacterizationofSenescenceRegulationbySmurf2andNotch3invitroandinvivo

AMajorQualifyingProjectReport

SubmittedtotheFacultyofthe

WORCESTERPOLYTECHNICINSTITUTE

inpartialfulfillmentoftherequirementsforthe

DegreeofBachelorofScience

in

BiologyandBiotechnology

by

___________________________________LauraFineman

April26,2012

APPROVED:___________________________________ ___________________________________HongZhang,Ph.D. JosephB.Duffy,Ph.D.CellBiology BiologyandBiotechnologyUMassMedicalSchool WPIProjectAdvisorMajorAdvisor

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ABSTRACT

Oneofthemainfunctionsofatumorsuppressoristorepresscelldivision,

sometimesbyinducingcellularsenescence.Smurf2andNotch3havepreviously

beenidentifiedinourlaboratoryastumorsuppressorsinvitro.Itisnotwell

understoodhowthesegenesregulatesenescence.Thepurposeofthisprojectisto

betterunderstandthemechanismsunderlyingthefunctionsofthesegenesin

senescence.AgeneticscreenusingshorthairpinRNAs(shRNAs)wascarriedoutto

identifygenesdownstreamofSmurf2orNotch3inthesenescencepathways.Five

candidategeneswerefurtheranalyzedinfibroblasts.Tocharacterizethefunctionof

Smurf2insenescenceinvivo,aCre‐LoxPsystemwasusedtostudytheconsequence

ofSmurf2overexpressioninmice.IfoundthatSmurf2impactsgrowthand

regulatesp21inyoungmice.

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ACKNOWLEDGEMENTS

IwouldliketothankProfessorHongZhangforgivingmetheopportunityto

joinhislab,andforhissupportandguidancethroughouttheexperimentaland

writingprocess.IwouldliketothankHangCuifordonatinghertimeinhelpingme

becomeproficientatthetechniquesandassaysnecessaryforthecompletionofthis

project.IwouldalsoliketothankYahuiKong,CharusheilaRamkumar,andIvan

Lebedevforteachingmeadditionalskillsthatwereusefulformyproject.Finally,I

wouldliketothankProfessorJosephDuffyforhisguidanceinchoosingagoodlabto

workin,hisassistancewithMQPediting,andhissupportasmyacademicadvisor

forthepastfouryears.

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TABLEOFCONTENTS

SignaturePage…………………………………..……………………………………………………………….1

Abstract……………………………………………………………………………………………………………..2

Acknowledgements…………………………………………………………………………………………….3

TableOfContents……………………………………………………………………………………………….4

Background………………………………………………………………………………………………………..5

MaterialsAndMethods……………………………………………………………………………………..14

Results…………………………………………………………………………………………………….……….22

Discussion………………………………………………………………………………………………………..34

References……………………………………………………………………………………………….………38

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BACKGROUND

ReplicativeSenescence

Normalcells,includingfibroblastcells,inculturereplicateafinitenumberof

timesbeforeenteringsenescence(HayflickandMoorhead,1961).Whenentering

senescence,cellsarepermanentlyarrestedintheG1phaseofthecellcycle,meaning

theydonotenterthereplicativeSphase(Sherwoodetal.,1988).Thisisnottobe

confusedwithquiescentcells,whicharetemporarilyarrestedintheG0phasedueto

ahighcelldensity(Zhang,2007).Senescentcellshaveadistinctmorphology;they

usuallyarelargerthanyoungcells(Bayreutheretal.,1988).Senescentcellsalso

expressanacidicbeta‐galatosidase.WhenstainedatapHof6,thisenzyme

producesabluecoloringthatbecomesdetectableafterabout16hours(Dimrietal.,

1995)

TelomereShortening

Telomeresarestructuresattheendsofchromosomesthatfunctionin

chromosomalprotection(Kimetal.,1994).Theenzymetelomeraseisawayof

maintenanceofthesechromosomalends;however,itisonlyfoundinimmortal

cancercells(Kimetal.,1994).Themajorityofsomaticcellsdonotexpressthis

enzyme(Kimetal.,1994).Asthesesomaticcellsdivide,theamountoftelomeric

DNAdecreases;thislossandeventualdeletionofnecessarysequencesinthe

genomecontributestothefinitereplicationobserved(Harleyetal.,1990).

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SenescencePathways

Telomereshortening,orDNAdamageandcellstressarethetwobigreasons

whycellswillsenesce.Eachsignalinducesaparallelpathway:itisfoundthatDNA

damageinducesthep53‐p21pathway(Herbigetal.,2004)andcellstressinduces

thep16‐RBpathway(BenantiandGalloway,2004).

p53‐p21Pathway

ATM(ataxia‐telangiectasiamutated)andATR(Rad3‐related)areprimary

PI3K‐likeproteinkinasesintheDNAdamagepathway(Herbigetal.,2004).ATM

respondstodouble‐strandbreaksandATRisaprimarymediatorinultravioletlight

damageandstalledreplicationforks(Herbigetal.,2004).Thesekinaseshavebeen

showntophosphorylateandactivatep53,whichinducesreplicativesenescence

(Herbigetal.,2004).

p53affectscellularreplicationbyit’sabilitytobindtoDNA;increased

bindingabilityleadstoreplicativesenescence(Vazirietal.,1997).Disruptionofthe

p53proteinhasbeenshowntoextendproliferationofhumanfibroblastcellsand

p53levelsareshownincreaseinagingfibroblastcells(Vazirietal.,1997).The

increaseofp53isduetoitsphosphorylationbyATMandATRfollowingDNA

damagefromshrinkingtelomeres(Vazirietal.,1997).Increasedp53binding

increasesthetranscriptionofthep21gene,acellcycleregulatorwhoseexpression

hasbeenseentoincreaseinsenescingcells(Herbigetal.,2003).

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p16‐RbPathway

Althoughtelomereerosionisamainpartofthereplicativesenescence

model,otherfactorsalsocontributetothesenescentphenotype(Hütteretal.,

2002).Oxidativedamageisamajorcauseofagingaswell;theoncogeneRASis

activatedafteroxidativedamageoccursandcaninduceprematuresenescence

(Hütteretal.,2002).

ActivatedRASup‐regulatesthecyclin‐dependentkinaseinhibitorp16

(BenantiandGalloway,2004),whoseexpressionisindependentoftelomere

degradationandp53expression(Herbigetal.,2004).p53isabletoinhibitcyclin‐

dependentkinase(CDK)4andCDK6,whoinactivateRb(retinoblastoma)(Ruasand

Peters,1998).Soincreasedlevelsofp16leadtoincreasedlevelsofRb(Ruas,1998).

However,Rb‐inducedsenescenceisnotcompletelyp16dependent;p21hasbeen

showntoup‐regulateRbinthesamewayasp16(Beauséjouretal.,2003).Rb

activationinducessenescencebyrepressionofE2F‐dependentpromoters,leading

toG1cellcyclearrest(Benantietal.,2004).

NotchSignaling

TheNotchsignalingpathwayisnotfullyunderstood,butitconsistsofthe

Notchreceptors,ligands,modifiers,andtranscriptionfactors(Allenspachetal.,

2002).Notch3isoneoffourmammalianNotchreceptors;itdiffersfromNotch4in

thatitcontainscytokineresponse(NCR)regionsanddiffersfromNotch1and

Notch2inthatitlacksC‐terminaltranscriptionalactivationdomains(TAD)

(Allenspachetal.,2002).

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NotchSignalingPathway

Notchsignalingstartswithbindingtotheligand;thisreleasestheactive

intracellulardomainofNotch(ICN)intothecytoplasm(Allenspachetal.,2002).

Fromthecytoplasm,itrelocatestothenucleus,whereitinducestranscriptionof

severalbasic‐helix‐loop‐helix‐orangeproteinsthatrepressgeneexpression

(Allenspachetal.,2002).Oneofthemajorplayersintranscriptionalactivationisthe

Mastermind‐like(MAML)protein;itaidsinthestabilizationoftheDNA‐binding

complexthatincludesICN(Oyamaetal.,2007).

Notch3andPathways

IthasbeenshownthatNotch3depletionofthecelldecreasedthelevelsof

p21;thiswasconsistentwiththedecreasedlevelsoftheICNtarget,HES1

(Giovanninietal.,2009).HES1increasehasbeenpreviouslylinkedwithup‐

regulationofp21(Giovanninietal.,2009).

Smurf2RegulatesSenescence

Smurf2,orSMADspecificE3ubiquitinproteinligase,encodesa748amino

acidhumanproteinthatisresponsibleforubiquitinatingSmad2,whichleadstoits

degradation;SmadsareimportantregulatorsforTGF‐β(transforminggrowth

factor‐beta)(Linetal.,2000).

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Smurf2andSenescence

Microarrayanalysisfromourlabhasshownthattelomereshortening

increasesSmurf2levels;furthermore,overexpressionofthegeneisalsosufficientto

inducereplicativesenescence(ZhangandCohen,2004).Twoformsofthisprotein

areusedinthisproject:theabove‐mentionedwildtypeformandacystineto

alaninemutationatposition716intheHECTdomainoftheprotein(Linetal.,

2000).AlthoughtheC716AmutantisunabletoubiquitinateSmad2,itstilloperates

underasimilarmechanismtothewildtype(Zhangetal.,2008).

Smurf2inthePathway

Smurf2hasbeenshowntoaffectboththep53/p21andthep16/Rb

pathways;however,itrequiresatleastoneofthepathwaystobefunctionalinorder

toinducesenescence(ZhangandCohen,2004).Smurf2entersthepathwayat

eitherp21orp16(Figure1).IncreaseinSmurf2hasbeenshowntocorrelatewith

increasedexpressionofp21(Zhangetal.,2008).Smurf2doesnotdirectlyinteract

withp16,Id1(inhibitorofdifferentiationofDNAbinding1)actsasamediator

(Kongetal.,2011).Id1repressesp16expression,sotheubiquitinationand

degradationofId1bySmurf2allowsp16tobeexpressedandinducesenescence

(Zhengetal.,2004).

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Figure1:Notch3’sandSmurf2’sInteractionwiththep53‐p21andthep16‐RBSenescencePathways.Notch3isshowntoup‐regulatep21levels.Smurf2hasbeenshowntobothup‐regulatep21levelsandup‐regulatep16levelsthroughrepressionofId.

IdentificationofGenesDownstreamofNotch3UsingshRNAScreening

TheinvitropartofthisMQPconsistedofdeterminingwhatgeneswere

downstreamofSmurf2andNotch3.Smurf2screeningpreviouslydonebyourlab

hadalreadygeneratedagroupofclonesthatneededtobevalidated,soI

concentratedonNotch3screening.Theideawasifagenethatwasinfact

downstreamofeitherSmurf2orNotch3wasknockeddown,thesenescence

Id

SenescenceSignals

ATM/ATR

p53

p16

p21

RB

E2F

Notch3Smurf2

SenescencePhenotype

RAS

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pathwaywouldbeinterruptedandfibroblastcellswouldbeabletoescapeSmurf2

orNotch3‐inducedsenescence.

TheshRNAlibrariesusedforthescreeningweretheExpressionArrestpGIPZ

Lenti‐viralshRNAmirLibraryfromOpenBiosystems,asshowninFigure2.The

librarywasdividedintopools,eachpoolcontaining5,000shRNAs.Thelenti‐viral

vectoriscompromisedoftwocomplementary22bpsequencesthatwouldpairinto

ahairpinsequencewhentranslatedintomRNA.

Figure2:DesignoftheshRNAmirvector.TheshRNAmirsystemincorporatestwo22bpsequencesthatarecapableofbindingandformingahairpininalenti‐viralconstruct.(AdaptedfromOpenBiosystems)

Thehairpinthatformsactivatesthecellulardrosha/dicerresponse,resulting

inexpressionarrestforthecorrespondingtranscript(Figure3).Droshaanddicer

processthehairpinshRNAmirtoasinglestrand.TheRISCcomplexbindstothe

singlestrandofshRNAmirandthetranscript,allowingdegradationandsilencingof

thegene.

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Figure3:shRNAmirExpressionArrestMechanism.CellsinfectedwithasingleviruscontainingashRNAmirconstructleadstotheexpressionarrestofatranscript.(AdaptedfromOpenBiosystems)

FunctionofSmurf2InVivo

Smurf2ubiquitousexpressionhasnotbeenstudiedmuch;previousinvivo

studiesstudiedtissuespecificexpressionforosteoblastfunction.Inthisproject,we

useamousemodeltostudytheconsequenceofSmurf2overexpression.

Knockout

Previously,theSmurf2knockoutmousemodelwasfirstcreatedbytheZhang

groupattheNationalCancerInstitute.Thismouseshowsgenomeinstabilitythat

leadstoincreasedsusceptibilitytovariouscancers(Blanketal.,2012).Ourlabalso

generatedknockoutmicewithadifferentstrategy,whichhaveasimilarphenotype

(inpress).

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Overexpress

OurlabhascreatedaSmurf2knock‐inmouseusingaCre‐LoxPsystem.A

Rosa26‐LoxP‐STOP‐LoxP‐Smurf2mousewasbackcrossedtoaC57BL/6

background.TheRosa26isastrongpromoterthatiscapableofubiquitous

expressionattheearlyembryonicstage(Soriano,1999).TheLoxPflankingsystem

allowsexpressionofthegeneofinterestuntilrepressedwithaCre‐LoxPsystem

(Rajewskyetal.,1996).Inthiscase,theSTOPisflankedbytheLoxP,andrepresses

theexpressionofSmurf2.ToactivatetheSmurf2gene,themicewerecrossedwith

Sox2‐CremicethatwereabletodisrupttheSTOPsiteandallowexpressionof

Smurf2startingatanearlyembryonicstage.Figure4showsthemechanismthat

occursinSmurf2knock‐inmicewhencrossedwiththeSox2‐Cremice.

Figure4:MechanismofSox2‐Cre‐LoxPsysteminSmurf2knock‐inmice.Smurf2geneandLoxP‐STOP‐LoxPinsertionintoRosa26locusinhibitsexpressionofSmurf2.AdditionofSox2‐CreexcisesSTOPandallowsexpressionofSmurf2.

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MATERIALSANDMETHODS

DNACloning

Cloningwasusedinthisprojectobtainandamplifyplasmidconstructs

neededforDNAtransfection.TheseplasmidsincludedpMD2‐VSVG,pCMVdR8.74,

dsRED,fourdifferentconstructsofCDC42BPA,andfourdifferentconstructsof

AHCYL1.

BacterialCulture

AllplasmidswereobtainedbyXL‐2E.colistockcontainingtheconstruct.The

plasmiddsREDwasculturedunderkanamycinselection;asmallamountofbacteria

wasculturedin6mLofLBmediumcontainingkanamycinforamplificationand

collectedviaminiprep.Allotherconstructswereculturedunderampicillin

selection;asmallamountofbacteriawasstreakedoutontoampicillinplates.Single

colonieswerepickedandculturedin6mLofLBmediumcontainingampicillin.The

CDC42BPAandAHCYL1plasmidswerecollectedviaminiprep.ThepMD2‐VSVGand

pCMVdR8.74constructswereculturedfurtherinpreparationofcollectionvia

midiprep.

PlasmidIsolation

PlasmidDNAsinthisprojectwereisolatedusingQuiagen’sQIAprep®Spin

MiniprepKitandQuiagen’sHiSpeed®PlasmidPurificationKit.

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DNATransfectionin293T

Thisprojectrequiredexpressionofplasmidconstructsincelllines.TheDNA

wasdeliveredinalenti‐viralconstructtointegratetheircontentsintotheinfected

hostcell.Virusespackagedwerelenticontrolvector,Notch3,Smurf2WT,and

Smurf2C716A.Humanembryonickidney293Tcellsfromculturewereplatedata

densityof4.5x106cells/p100tissueculturedishesinDMEMmediumwith10%

fetalbovineserum.Transfectionoccurredthenextdaywith2MCaCl2and2XHBSat

acellconfluenceofabout70%.

TheDNAwasmixedina15mLtube;6.5ugofpackagingvectorpCMVdR8.74,

3.5ugofenvelopevectorpMD2‐VSVG,0.2ugofreportervectordsRED,and10uLof

theDNAofinterest.Thevolumewasraisedto437.5uLwith0.1%TEinH20.Themix

wasvortexedatahighspeed,followedby62.5uLofCaCl2and500uLofHBS,both

addeddropwise.Thefinalsolutionwasaddeddropwiseontothe293Tcells.

Twenty‐fourhourslater,themediumwaschangedto9mLoffreshDMEMwith10%

FBS.Forty‐eighthourspost‐transfection,thecellswereobservedunder

fluorescencemicroscopyforthepresenceofdsRED.Supernatantsofplateswith

morethan70%ofcellsexpressingdsREDwerecollectedandstoredat‐80°Ctokill

anylivingcellscollectedalongwiththevirus.

shRNAScreening

GeneknockdownwithshRNAplasmidsallowedforidentificationofpossible

genesinvolvedinNotch3inducedsenescence.

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CellInfectionwithshRNAVirusandPuromycinSelection

WS1humanfibroblastcellswereplatedat1.0x105cells/p100.Eachp100

platewasinfectedwith5uLofshRNAlibraryinmediumcontaining1ug/uLof

polybrene.Theplateswerechangedwithfreshmediumthefollowingday.The

shRNAplasmidscontainpuromycinresistance;twodayspostinfection,medium

containing1ug/mLofpuromycinwasadded.Ap60platecontainingWS1cellsas

wellwasusedasaselectioncontrol;theplatewasemptyafterabout3daysof

puromycinexposure.Secondroundinfectionoccurredafterselection.

CellInfectionwithNotch3VirusandBSDSelection

WS1cellsthatremainedviableafterpuromycinexposureweretheninfected

withNotch3virusinmediumcontaining1ug/uLofpolybrene.Theplateswere

changedwithfreshmediumthefollowingday.TheNotch3plasmidcontainsBSD

resistance;twodayspostinfection,mediumcontaining1ug/mLofpuromycinand

2ug/mLofBSDwasadded.Ap60containingWS1cellswasagainusedasaselection

control;theplatewasemptyafter4‐5daysofBSDexposure.Thecellswerekept

underpuromycinandBSDselectionfor12days,whencoloniesstartedformingon

theplates.

IsolationofGenomicDNA

GenomicDNAwasthenisolatedfromthecoloniesofthescreenusingspot

trypsinization.Eachcolonywasculturedinanindividualwellona24‐wellplate

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untilalmostreachingconfluence;genomicDNAwasisolatedusingthefollowing

protocol.CellswerewashedwithPBS,andthen200uLofalysisbuffer/5Xprotease

Ksolutionwasadded.Thecellswereincubatedat37°Cfor2‐3hours,andthe

supernatantwascollectedtoa1.5mLEppendorftube.Next,200uLofisopropanol

wasmixedinandthetubewasincubatedfor10‐15minutesatroomtemperature

beforebeingspundownfor15minutesat14,000rpm.Thesupernatantwas

aspiratedandthepelletwaswashedwith70%ethanol;thetubewasspundown

againfor7minutesat14,000rpmandthenairdried.Afterthepelletwasdry,50uL

ofH20wasaddedandthetubewaslefttoincubateovernightat37°C.

PolymeraseChainReaction

TheGenomicDNAextractedfromeachcolonywasamplifiedusing

PolymeraseChainReactionandwasfacilitatedwithTaqDNAPolymerasepurchased

fromNewEnglandBiolabs.TheamplifiedregionincludedtheshRNAmirsequence;

theamplificationproductwassenttobesequenced.

Sequencing

TheDNAPCRproductwaspurifiedandsenttoGenewizInc.forsequencing.

shRNAConformation

ThreeshRNAs(PIAS4,AHCYL1,andCDC42BPA)werevalidatedbytesting

knockdownefficiencyandgrowthcurves.

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RNAIsolation

RNAwasisolatedfromthecellspriortothesecondaryinfectionofNotch3or

Smurf2toidentifythelevelofknockdowneachshRNAinduces.RNAwasisolated

usingaTrizol‐basedprotocol,andthenrunwithgelelectrophoresistodetermine

theintergrityoftheRNA.

RT‐PCR

Invitrogen’sSuperscript®IIReverseTranscriptasewasusedtotranscribe

theextractedRNAintocDNA.

Real‐timeqPCR

ThereversetranscriptaseproductwasrunusingaBio‐RadiQ5machineand

softwarewiththeaidofSYBR®Green.

GrowthCurves

Cellswereplatedin6‐wellplatesataround2x104cells/wellwithtwowells

foreachsampletimepoint.Eachsetofwellswascountedonday1,3,5,and7with

day1beinglessthan20hoursaftercellswereplated.AZ1Coulter®Particle

Counterwasusedtocounteachsamplethreetimes;theaveragewastaken.

Genotyping

Miceweregenotypedandmarkedonthetoeatage7‐10days.

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GenomicDNAExtraction

Approximately2mmoftailwassnippedandplacedinaPCRtubewith75uL

ofNaOHandEDTAlysisbufferandincubatedat95°Cfor30minutesandthen

cooledto4°Cfor15minutes.Then75uLofTris‐HClbufferwasaddedandmixedto

neutralizethesolution.

PolymeraseChainReaction

TheextractedgenomicDNAwasamplifiedusingPolymeraseChainReaction

usingprimersthatsurroundedtheregionofinterest,Smurf2WT,Smurf2C716A,or

Cre.AmplificationwasaidedbyLambdaBiotech’sTaqPolymerase.ThePCRproduct

wasrunona1%agarosegelwithgelelectrophoresistodeterminethegenotypes.

OBSERVATIONOFCONSEQUENCEOFSMURF2INVIVO

ObservationoftheSmurf2overexpressedmicewascarriedoutbyabi‐

weeklyandweeklyrecordofweightaswellasdeterminationofgeneexpressionby

Westernandreal‐timePCR.

GrowthCurve

Growthwasmeasuredfromshortlyafterbirthuntildeath.Weightwas

measuredingramsbyplacingthemouseinabeakerthatwaszeroedonabalance.

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OrganExtraction:RNAandLysateExtraction

Eightorgans;liver,heart,lung,kidney,pancreas,spleen,colon,andskinwere

harvestedandweighed.Eachorganwasfrozeninliquidnitrogenandcrushedinto

powderforsubsequentuseforlysateandRNAextraction.

RNAwasextractedfromthespleenusingtheabovementionedTrizolbased

procedure.TheRNAwasreverse‐transcribedandanalyzedwithreal‐timePCR.

LysatewasextractedfromalltheorgansusingRIPAbufferwith20Xprotease

inhibitor.Concentrationwasmeasuredat562nanometers.Lysateswererunona

WesternblotandprobedforSmurf2,p21,p16,andtubulin.

BoneMarrowExtraction

Theentirebonemarrowextractionprocesswasperformedonice.Micewere

double‐killedusingIsofluraneandcervicaldislocation.Hindlimbswereseveredat

hipjointandforelimbsattheshoulderjoint.Limbswereplacedinap60plate

containingastaining(Biotin‐,Flavin‐,Pheno‐reddeficientRPMI1640)medium.Skin

andmusclewasscrapedawaytothebonewitharazorbladeandthekneeand

elbowjointsweredisarticulatedtoharvestthefemur,tibia,andhumorousbones.

Theendsoftheboneswerecuttoexposethemarrowcavities;boneswere

completelyflushedwithstainingsolutionusinga5mLsyringeanda25Gneedle.An

18Gneedleissubsequentlyusedtobreakthelargechunksofmarrowintosmall

pieces.Cellsarefilteredwitha70umnylonmeshintoa15uLtubeandthefinal

volumeofcellsisbroughtupto10mLwiththestainingsolution.

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A10Xdilutionofcellsiscountedusingahemocytometer.Thebonemarrowwas

extractedforRNAusingthemethodmentionedaboveinprepforrtPCR.

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RESULTS

ProductionofSmurf2andNotch3Viruses

HEK‐293Tcellswereusedtopackagethevirusesusedinthisprojectvia

transfection.AplasmidexpressingaredfluorescentproteindsREDwasusedin

transfectiontodeterminetheefficiencyofthetransfections;Figure5shows

representativeimagesof293Tcellsaftertransfection.StrongerdsREDfluorescence

correlateswithabetterefficiency;efficiencieslowerthat80%werenotused.

Figure5:HEK‐293TTransfectionEfficiency.TransfectionefficiencyisshownbydsREDfluorescence:regularlight(left)andgreenfluorescentlight(right)showstheusualpercentageofHEK‐239Tinfectedbythevirus.

shRNAScreenforGenesInvolvedinNotch3‐InducedSenescence

OurlabhadpreviouslyscreenedseveralcandidatesusingSmurf2,soI

concentratedonscreeningwithNotch3.Sevengeneswereidentifiedfromthe

Notch3screen;Table1showsthegenesidentifiedalongwiththechromosome

locationandthesenseshRNAsequence.CDC42bindingproteinkinasealpha

(CDC41BPA)ishighlightedinthetable;thisgenewasstudiedfurtherinattemptto

confirmthatitoperatesdownstreamofNotch3.BetweenthepreviousSmurf2

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screeningandthecurrentNotch3screen,fivegeneswereanalyzedfurther:SUMO2,

Catalase,PIAS4,AHCYL1,andCDC42BPA.

Pool# GeneName Identity

MatchChromosomeLocation SenseshRNAsequence5’‐>3’

8 Inter‐genicregion 21/22 8 CCGTCTCCAGACATTTAAAGAT

9 Inter‐genicregion 21/22 1 CTGAAGATGTACTGGAACTAAA

9

Glutamatereceptor,ionotropic,kainite3

21/22 1p34‐p33 CCCTTGGTTTCTCCTATGAGAT

10

CDC42bindingprotein

kinasealpha

21/22 1q42.11 CAAGCTGGAAGTTCATACAGAA

11TranscriptionfactorAP‐2

beta21/22 6p12 ACCTCCCTTCCTCACATTGTTA

13 Inter‐genicregion 21/22 14 AGCATGTCACTTGTTCTGTTAA

13 Inter‐genicregion 21/22 20 AGCCTTAAGTTCCACCACACTA

Table1:SevenCandidateGenesThatMayActDownstreamofNotch3.AsummaryofthesequencingdataobtainedfromtheNotch3screening.TheshRNAtargetedgenesareshown,aswellasthenucleotidematchbetweenthegeneandsiRNAandchromosomelocation.CDC42bindingproteinkinasealphawaschosentoresearchfurtherasapossiblecandidate.

TestofKnockdownofCandidateGenes

TotalRNAwasextractedfromcellsinfectedwithlenti‐virusexpressing

shRNAtargetingSUMO2,Catalase,PIAS4,AHCYL1,andCDC42BPA.Afterreverse

transcription,RT‐qPCRwasusedtodetermineiftheshRNAswereabletoinducea

sufficientknockdowninthecells.

SUMO2andCATALASEshRNAwasunabletoproduceanadequate

knockdown(datanotshown),soconfirmationofthesegeneswasdiscontinued.

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BothshRNAstestedinducedmorethan50%knockdownofPIAS4inWS1

fibroblastcells(Figure6)wheninfectedwith200uLofshRNAlenti‐virus.

Figure6:PIAS4_7andPIAS4_9shRNAsProduceaMoreThanFiftyPercentKnockdowninWS1cells.RNAwascollectedfromWS1cellsandreversetranscribedtocDNAforrealtimePCRfromWS1cellstransfectedwith200uLnon‐silencingshRNA(blue),PIAS4_7(red),andPIAS4_9(green).Non‐silencingshRNAwasnormalizedto1.

FourdifferentshRNAsweretestedforAHCYL1,givingvariousknockdowns

fora100uLvirusinfectioninWS1fibroblastcells(datanotshown).ThebestshRNA

wereretestedinLF1cellswith250uLofvirus,givingmorethanan80%knockdown

(Figure7).

‐0.2

0

0.2

0.4

0.6

0.8

1

1.2

KnockdownofPIAS4

NS

PIAS4_7

PIAS4_9

25

Figure7:AHCYL1_4shRNAProducesaMoreThanEightyPercentKnockdowninLF1cells.RNAwascollectedfromLF1cellsandreversetranscribedtocDNAforrealtimePCRfromLF1cellstransfectedwith250uLofnon‐silencingshRNA(blue)andAHCYL1_4(green).Non‐silencingshRNAwasnormalizedto1.

FourdifferentshRNAswerealsotestedforCDC42BPA;thesealsogave

variousknockdownsfora100uLvirusinfectioninWS1fibroblastcells(datanot

shown).ThebesttwoshRNAswereretestedinLF1cellswith250uLofvirus,giving

morethana40%knockdown(Figure8).

0

0.2

0.4

0.6

0.8

1

1.2

KnockdownofAHCYL1

NS

AHCYL1

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Figure8:CDC42BPA(1,7)shRNAProduceaMoreThanFortyPercentKnockdowninLF1cells.RNAwascollectedfromLF1cellsandreversetranscribedtocDNAforrealtimePCRfromWS1cellstransfectedwith250uLofnon‐silencingshRNA(blue),CDC42BPA_1(red),andCDC42BPA_7(green).Non‐silencingshRNAwasnormalizedto1.

ValidationofPIAS4,AHCYL1,andCDC42BPA

Inordertoensurethatthecandidatesthatwerescreenedwerenot

background,growthcurveswereused.WS1orLF1fibroblastcellswithnon‐

silencing,PIAS4,AHCYL1,andCDC42BPAshRNAwereinfectedwithlenti‐virus

controlvectorandeitherSmurf2WTaswellasSmurf2C716AorNotch3challenge

viruses.Thecellswereplatedfourdayspost‐infection(Notch3)orfivedayspost‐

infection(Smurf2WTandC716A).

Figure9belowshowsthegrowthcurvedatafromPIAS4_7andnon‐silencing

knockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2C716A

challengeinWS1cells.ThedatashowsthatPIAS4/lenticontrolgrowthrateisabout

thesameasnon‐silencing/lenticontrol.ThedataalsoshowsthatPIAS4knockdown

(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2C716A,does

0

0.2

0.4

0.6

0.8

1

1.2

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Knockd

ownofCDC

42BP

A

NS

CDC42BPA_1

CDC42BPA_7

27

notleadtoasignificantrescueofsenescencewhencomparedtonon‐silencing

knockdown(solidblueandred).

Figure9:PIAS4_7KnockdownDoesNotLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),PIAS4/lenti‐viruscontrolvector(dashedgreen),PIAS4/Smurf2WT(dashedblue),andPIAS4/Smurf2C716A(dashedred).DatashowsthatPIAS4cellschallengedwitheitherSmurf2WTorSmurf2C716Aarenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.

Figure10belowshowsthegrowthcurvedatafromAHCYL1andnon‐

silencingknockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2

C716AchallengeinLF11cells.ThedatashowsthatAHCYL1/lenticontrolgrowth

rateisslowerthannon‐silencing/lenticontrol.ThedataalsoshowsthatAHCYL1

knockdown(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2

C716A,doesnotleadtoasignificantrescueofsenescencewhencomparedtonon‐

silencingknockdown(solidblueandred).

0

100000

200000

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500000

0 1 2 3 4 5 6 7 8

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ber

DaysinCulture

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NS+C

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28

Figure10:AHCYL1KnockdownDoesNotLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),AHCYL1/lenti‐viruscontrolvector(dashedgreen),AHCYL1/Smurf2WT(dashedblue),andAHCYL1/Smurf2C716A(dashedred).DatashowsthatAHCYL1cellschallengedwitheitherSmurf2WTorSmurf2C716Aarenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.

Figure11belowshowsthegrowthcurvedatafromCDC42BPA_1andnon‐

silencingknockdowncellswithalenti‐viruscontrolvectororNotch3challengein

LF1cells.ThedatashowsthatCDC42BPA/lenticontrolgrowthrateisfasterthan

non‐silencing/lenticontrol.ThedataalsoshowsthatCDC42BPAknockdown

(dashedblue),whenchallengedwithNotch3,doesnotleadtoasignificantrescueof

senescencewhencomparedtonon‐silencingknockdown(solidblue).

0

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ber

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NS+S

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A4+V

A4+S

A4+C

29

Figure11:CDC42BPA_1KnockdownDoesNotLeadtoSignificantRescueofNotch3InducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti(solidgreen),non‐silencingshRNA/Notch3(solidblue),CDC42BPA/lenti(dashedgreen),andCDC42BPA/Notch3(dashedblue).DatashowsthatCDC42BPAcellschallengedwithNotch3arenotrescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Notch3.

Figure12belowshowsthegrowthcurvedatafromCDC42BPA_7andnon‐

silencingknockdowncellswithalenti‐viruscontrolvector,Smurf2WT,orSmurf2

C716AchallengeinLF11cells.ThedatashowsthatCDC42BPA/lenticontrolgrowth

rateisslowerthannon‐silencing/lenticontrol.ThedataalsoshowsthatCDC42BPA

knockdown(dashedblueandred),whenchallengedwithSmurf2WTandSmurf2

C716A,doesleadtoasignificantrescueofsenescencewhencomparedtonon‐

silencingknockdown(solidblueandred).

0

50000

100000

150000

200000

250000

300000

350000

0 1 2 3 4 5 6 7 8

Cell#

Day#

NS+V

NS+N3

C+V

C+N3

30

Figure12:CDC42BPA_7KnockdownDoesLeadtoSignificantRescueofSmurf2WTandSmurf2C716AInducedSenescence.CellgrowthdatawascollectedfromLF1cellstransfectedwithnon‐silencingshRNA/lenti‐viruscontrolvector(solidgreen),non‐silencingshRNA/Smurf2WT(solidblue),non‐silencing/Smurf2C716A(solidred),CDC42BPA/lenti‐viruscontrolvector(dashedgreen),CDC42BPA/Smurf2WT(dashedblue),andCDC42BPA/Smurf2C716A(dashedred).DatashowsthatCDC42BPAcellschallengedwitheitherSmurf2WTorSmurf2C716Aarerescuedfromthecontrolsenescencephenotypeshownbynon‐silencingshRNA/Smurf2WTandnon‐silencingshRNA/Smurf2C716A.

SenescenceRegulationbySmurf2InVivo

ToanalyzewhathappenswhenSmurf2isoverexpressedinmice,we

weighedthemiceeveryonetotwoweeksandcheckedSmurf2,p21,andp16

expressionlevelsinorgansandbonemarrow.

Thereweresevenmiceinthelitter:threewereSox2‐CreandfourwereSox2‐

Cre/Smurf2.Forthefirstmonth,themicewereweighteveryotherweek;theywere

weighedeveryweekforthesecondmonth(Figure13).Thedatashowsthatthe

Sox2‐Cre/Smurf2(blue)miceweighlessthantheSox2‐Cre(red)micefor

approximatelythefirstfortydays.

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31

Figure13:Smurf2OverexpressingMiceWeightLess.Micewereweighedeverytwoweeksandtheneveryweekfortwomonths.Thelitterwassevenmice:threewereSox2‐Cre(red)andfourwereSox2‐Cre/Smurf2(blue).

Todetermineproteinexpression,lysatewasextractedfromeightorgansand

runonaWesternblot.Theorgansextractedwereliver,kidney,spleen,pancreas,

colon,lung,heart,andskin.Themembranewasprobedwiththreeantibodies:

Smurf2,p21,andp16,butp21andp16wereundetected.AsseenwithFigure14,

Smurf2proteinisoverexpressedinSox2‐Cre/Smurf2micewhencomparedtoSox2‐

Cremice.

liverkidneyspleenpancreascolonlungheartskin‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐5858585858585858

Figure14:Smurf2ProteinisOverexpressedinSox2‐Cre/Smurf2Mice.WesternblotofSox2‐Cremouse#5andSox2‐Cre/Smurf2mouse#8organs.Organsincludeliver,kidney,spleen,pancreas,colon,lung,heart,andskin.MembranewasprobedwithSmurf2.

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60

Weight(gram

s)

DaysafterBirth

Sox2‐Cre/Smurf2

Sox2‐Cre

Smurf2tubulin

32

ThegeneexpressionofSmurf2,p21,andp16wasdeterminedusinganassay

moresensitivethanaWesternblot.RNAwasextractedfromspleenandanalyzed

usingreal‐timePCR.AsseeninFigure15,Smurf2andp21RNAisoverexpressedin

Sox2‐Cre/Smurf2(blue)micewhencomparedtoSox‐Cre(red)mice.Thedataalso

showsthatp16isnotoverexpressedinSox2‐Cre/Smurf2mice.

Figure15:Smurf2andp21RNAisOverexpressedinSox2‐Cre/Smurf2mice.RNAwasextractedfromspleeninSox2‐Cre(blue)andSox2‐Cre/Smurf2(red)mice.ExpressionofSmurf2,p21,andp16wasanalyzed.Sox2‐Cregeneexpressionwasnormalizedto1.

ThegeneexpressionofSmurf2,p21,andp16RNAwasalsodeterminedwith

bonemarrowandanalyzedusingreal‐timePCR.AsseeninFigure16,Smurf2RNAis

overexpressedinSox2‐Cre/Smurf2(blue)micewhencomparedtoSox‐Cre(red)

mice.Thedataalsoshowsthatp21isnotsignificantlyoverexpressedinSox2‐

Cre/Smurf2miceandthatp16expressionisundetectable.

0

1

2

3

4

5

6

7

RelativeGeneExpression

Smurf2p21p16

Sox2‐Cre

Sox2‐Cre/Smurf2

33

Figure16:Smurf2RNAisOverexpressedinSox2‐Cre/Smurf2mice.RNAwasextractedfrombonemarrowinSox2‐Cre(blue)andSox2‐Cre/Smurf2(red)mice.ExpressionofSmurf2,p21,andp16wasanalyzed.Sox2‐Cregeneexpressionwasnormalizedto1.

0

0.5

1

1.5

2

2.5

RelativeGeneExpression

Smurf2p21p16

Sox2‐Cre

Sox2‐Cre/Smurf2

34

DISCUSSION

Discussion

ThisprojectwassuccessfulinusingshRNAmirgeneknockdownscreeningto

identifypotentialgenesdownstreamofNotch3.Sevencandidategeneswere

identifiedfromthisscreeningprocess,oneofwhichwasfurtherinvestigatedwith

fivegenesintheSmurf2senescencepathway.Mydatasuggeststhatknockdownof

CDC42BPAallowspartialescapeofSmurf2inducedsenescence,whileknockdown

ofCDC42BPAdoesnotallowsignificantrescuefromNotch3‐inducedsenescence

andknockdownofPIAS4andAHCYL1doesnotallowsignificantrescuefrom

Smurf2‐inducedsenescence.

Thisprojectwasalsosuccessfulindeterminingpreliminarycharacterization

ofthefunctionofSmurf2insenescenceregulationinvivo.Smurf2overexpressionis

abletoimpactgrowthandregulatep21inyoungmice.

PIAS4,AHCYL1,andCDC42BPA

PIAS4,AHCLY1,andCDC42BPAwereidentifiedascandidategeneswhose

down‐regulationallowstheescapeofSmurf2‐orNotch3‐inducedsenescencein

WS1andLF1cells.

PIAS4

IthasbeensuggestedpreviouslythatPIAS4isinvolvedinsenescence

(Rytinkietal.,2009).PIAS,orproteininhibitorsofactivatedSTATs,isabletoinhibit

STATasitsnamesuggests(Desrivièresetal.,1996).AsSTATactivatestranscription,

35

PIASisabletoinhibittranscription(Desrivièresetal.,1996).PIAS4(PIASyor

gamma)isshowntoactasaSUMO‐E3ligaseforSmad3andp53(Imotoetal.,2003).

MyresultsindicatethatknockdownofPIAS4isnotsufficienttoallow

fibroblaststoescapeSmurf2‐inducedsenescence.Itispossiblethatthelevelof

knockdowninmyexperimentswasnotdepletedenoughtointerferewiththe

functionofPIAS4,orthatotherPIASproteins(PIAS1‐3)compensateforthe

knockdownofPIAS4.Inthecaseofcompensation,afunctioninterferenceofPIAS

activitywouldbemoresuitableinthetestoftheirfunctioninSmurf2‐induced

senescence.ItisalsopossiblethatknockdownofPIAS4onlycompromisesonlyone

ofthesenescencepathways,whichisnotsufficientforafullescapeofSmurf2‐

inducedsenescence.ItwillbeinterestingtoinvestigateifPIAS4collaborateswith

othergenesinregulatingSmurf2‐inducedsenescence.

AHCYL1

ItisknownthatAHCYLcanregulatebothsenescencepathwaysthroughp53

andp16(Lealetal.,2008).AHCYL1,ors‐adenosylhomocysteinehydrolase‐like1,is

involvedinthehydrolysisofS‐adenosyl‐L‐homocysteinetoL‐homocysteineand

adenosine(Lealetal.,2008).AlthoughthereislittleresearchonAHCYL1in

particular,inactivationofAHCYLingeneralinducedcellresistancetop53andp16‐

inducedreplicativearrest(Lealetal.,2008).AHCYLhasalsobeenshowntoinhibit

p53transcriptionalactivityandDNAdamage‐inducedtranscriptionofp21(Lealet

al.,2008).

36

MydatagivesevidencethatknockdownofAHCYL1isnotsufficienttoresuce

fibroblastsfromSmurf2‐inducedsenescence.AsverygoodknockdownofAHCYL1

wasachieved,itislikelythatthelevelofAHCYL1knockdowninterferedwithits

function.ApossibilityisthatAHCYL2wasabletocompensatefortheknockdownof

AHCYL1,soagain,afunctioninterferenceofAHCYLactivitymightbemoresuitable

intestingitsfunction.AnalternativereasonisthatAHCYL1knockdowncausesthe

WS1andLF1fibroblastcellstogrowveryslowly,evenwithoutthesecondary

infection;fastergrowthwasunabletobeachieved.SoalthoughAHCYL1knockdown

withSmurf2WTandSmurf2C716AchallengegrewsimilarlytoAHCYL1

knockdownwithlenti‐viruscontrolvector,allthreegrewsimilarlytonon‐silencing

knockdownwithSmurf2WTandSmurf2C716Achallenge.

CDC42BPA

CDC42BPAisknowntoregulatep53isthesenescencepathway(Sirotkinet

al.,2008).CDC42BPA(CDC42bindingproteinkinasealpha)knockdownisshownto

inhibittheexpressionofp53andPCNA,orproliferatingcellnuclearantigenin

ovariancells(Sirotkinetal.,2008).PCNApromotesDNAsynthesisduringSphase

andprovidesprotectionfromapoptosisduringG1phase(Sirotkinetal.,2008).

MyresultsindicatethatknockdownofCDC42BPAisnotsufficienttoallow

fibroblaststoescapeNotch3‐inducedsenescence;however,theknockdownwas

sufficienttopartiallyrescuecellsfromSmurf2‐inducedsenescence.Itispossible

thattheknockdownlevelofCDC42BPAinmyexperiments,whilesufficientto

interferewithitsfunctionwhiledownstreamofSmurf2,wasnotlowenoughto

37

interferewiththefunctionofCDC42BPAwhendownstreamofNotch3.Because

CDC42BPAisonlyshowntointeractwithoneofthesenescencepathways,thismay

notbesufficientforescapeofNotch3‐inducedsenescence.Itwillbeinterestingto

furtherdeterminetheinteractionofSmurf2withCDC42BPA,aswellasthe

interactionofCDC42BPAwithothergenesinthesenescencepathway.Thisresearch

maybeabletoenlightenusonifandhowCDC42BPAinteractswithNotch3.

Smurf2InVivo

Smurf2overexpressionwasabletoaffectgrowthinyoungmiceforashort

periodoftime.However,themiceseemedtohavecompensatedforthisabnormal

development,asthisstuntedgrowthlastedforlessthantwomonths.Smurf2

overexpressioninyoungmiceseemedtoadheretoasimilarsenescencepathway

thatoccursinvitro.Smurf2wasshowntoregulatep21,whichisconsistentwithin

vitrodata.However,asp16didnotseemtoberegulatedbySmurf2,thiscouldhave

occurredduetotheyoungageofthemice.p16hasbeenseentobevastlyincreased

duringsenescence(Alcorta,etal.,1996),indicatingthatp16willmostlikelynot

becomepresentinmiceuntilanolderage.

GrowthratesofSmurf2overexpressedmiceshouldberepeated,possibly

withweightsbeingmeasuredatmorefrequentintervals.Geneexpressionshould

alsobedeterminedatadditionalagestodetermineiftheexpressionchangesasthe

miceage.OurlabalsocontainsSmurf2C716Amice,itwillbeinterestingtoseeif

overexpressionofSmurf2C716AgivessimilarconsequencesasSmurf2WT

overexpression.

38

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