Genome-wide RNAi analysis

ofJAK/STATsignalingcomponentsinDrosophila

Gyeong-HunBaeg,1,2RuiZhou,1,2andNorbertPerrimon1,31DepartmentofGenetics,HowardHughesMedicalInstitute,HarvardMedicalSchool,Boston,MA02115,USA

Thecytokine-activatedJanuskinase(JAK)/signaltransducerandactivatoroftranscription(STAT)pathwayplaysanimportantroleinthecontrolofawidevarietyofbiologicalprocesses.Whenmisregulated,JAK/STATsignalingisassociatedwithvarioushumandiseases,suchasimmunedisordersandtumorigenesis.TogaininsightsintothemechanismsbywhichJAK/STATsignalingparticipatesinthesediversebiologicalresponses,wecarriedoutagenome-wideRNAinterference(RNAi)screeninculturedDrosophilacells.Weidentified121geneswhosedouble-strandedRNA(dsRNA)-mediatedknockdownsaffectedSTAT92Eactivity.Ofthe29

positiveregulators,13arerequiredforthetyrosinephosphorylationofSTAT92E.Furthermore,wefoundthattheDrosophilahomologsofRanBP3andRanBP10arenegativeregulatorsofJAK/STATsignalingthroughtheircontrolofnucleocytoplasmictransportofSTAT92E.Inaddition,weidentifiedakeynegativeregulatorofDrosophilaJAK/STATsignaling,proteintyrosinephosphatasePTP61F,andshowedthatitisa

transcriptionaltargetofJAK/STATsignaling,thusrevealinganovelnegativefeedbackloop.OurstudyhasuncoveredmanyuncharacterizedgenesrequiredfordifferentstepsoftheJAK/STATsignalingpathway.[Keywords:JAK/STATsignaltransductionpathway;Drosophila;RNAinterference]Supplementalmaterialisavailableathttp://genesdev.org.

ReceivedApril1,2005;revisedversionacceptedJune20,2005.

TheevolutionarilyconservedJanuskinase(JAK)/signaltransducerandactivatoroftranscription(STAT)cascadeplaysakeyroleinawidevarietyofbiologicalprocessessuchastheimmuneresponse,tumorigenesis,anddevel-opment.Thispathway,regulatedbyalargenumberofcytokinesandgrowthfactors,hasemergedasanessen-tialfacetofvertebratesignaling.Criticalrolesofthere-ceptor-associatedJAKsandtheirsubstratetranscriptionfactorsSTATshavebeendemonstratedthroughthegen-erationofgeneknockoutmice.JAK1-deficientmicedieperinatallyandareunabletonurse(Rodigetal.1998),whileJAK2mutantmicedisplayembryoniclethalityduetoanemia(Neubaueretal.1998;Parganasetal.1998).MicelackingJAK3haveprofoundreductionsinthymocytes,BandTcells,asobservedinthecaseofautosomalseverecombinedimmunedeficiency(SCID)mice(Nosakaetal.1995;Parketal.1995;Thomisetal.1995).Similarly,STAT-deficientmiceareeitherim-munocompromisedordisplayhematopoieticdefects(Durbinetal.1996;Merazetal.1996).Ontheotherhand,constitutiveactivationofJAKsand/orSTATsiscorrelatedwithtumorigenesisthroughtheirintimate

23Theseauthorscontributedequallytothiswork.Correspondingauthor.

E-MAILperrimon@receptor.med.harvard.edu;FAX(617)432-7688.

Articlepublishedonlineaheadofprint.Articleandpublicationdateareathttp://www.genesdev.org/cgi/doi/10.1101/gad.1320705.

connectiontogrowthfactorsignaling,apoptosis,andan-giogenesis(YuandJove2004).Furthermore,studiesinmodelgeneticsystems,suchasDrosophila,Xenopus,andzebrafishhaveshownthattheJAK/STATpathwayparticipatesinanunusuallybroadsetofdevelopmentaldecisionsthatincludecellfatedetermination,cellmi-gration,planarcellpolarity,convergentextension,andstemcellmaintenance(Houetal.2002).Althoughmuchworkhasbeendoneonthispathway,manyquestionsremaintobeaddressed.Inparticular,theexactmolecu-larmechanismsbywhichJAK/STATsignalinginte-gratesandtransducescuesfromnumerousextracellularsignalingmoleculestotriggerspecificgeneticprogramsinvivoremaintobeelucidated.Inaddition,STATshavebeenshowntobeactivatedbyatleastfourdistinctmechanismsinmammals(Bromberg2001),andmanyaspectsofthisregulationremainonlypartiallyunder-stood.

Inmammals,geneticapproachestoidentifyandchar-acterizecomponentsoftheJAK/STATpathwayhavebeenpredominantlydependentoncell-line-basedgenet-icsorgenetargeting,whichislabor-intensiveandoftentime-consuming(Velazquezetal.1992).Moreover,in-terpretationofmammaliangeneticstudiesisfurthercomplicatedbytheredundancywithinindividualcom-ponentsoftheJAK/STATpathway.Incontrast,Dro-sophilamelanogasterishighlyamenabletogeneticma-nipulationsandhasservedasanexcellentmodelorgan-

GENES&DEVELOPMENT19:1861–1870©2005byColdSpringHarborLaboratoryPressISSN00-9369/05;www.genesdev.org1861

Baegetal.

ismtostudytheJAK/STATpathway.GeneticstudiesinDrosophilahaveidentifiedseveralcanonicalcompo-nentsoftheJAK/STATpathway,includingcytokine-likemoleculesUnpaired(Upd);Domeless/MasterofMarelle(Dome/Mom),theUpdreceptordistantlyrelatedtothemammaliangp130subfamily;Hopscotch(Hop),theDrosophilahomologofvertebrateJAK;STAT92E,theSTATprotein;andSOCS36E,anegativeregulatoroftheJAK/STATpathway(Houetal.2002).However,theinherentlimitationsofforwardgeneticapproachesmakeitlikelythatmanygenesremainunidentified.Recently,thedevelopmentofhigh-throughputgenome-wideRNAi-basedtechnologyinculturedDrosophilacellsof-fersarapid,systematic,andcomplementarymethodol-ogyfordissectinggenefunctions(Boutrosetal.2004;Dasguptaetal.2005).Thisquantitativecell-basedRNAianalysisalsoofferstheadvantageofuncoveringgenefunctionassociatedwithsubtlephenotypesand/orre-dundancythatmightnotbereadilyidentifiablethroughgeneticstudies,includingthoseinsensitizedgeneticbackgrounds(Bachetal.2003).Furthermore,withabun-dantgenetictoolsreadilyavailable,Drosophilaisasu-periorgeneticallytractablesystemfortheinvivovali-dationofcandidategenes.

ThereareanumberofstepsinvolvedinsignalingthroughtheJAK/STATpathway,includingphosphory-lationandnucleocytoplasmicshuttlingofSTAT92E.Wehopedtoidentifynewmembersofthiscanonicalpath-wayaswellasproteinsthatmightfunctionasmodula-torsbyregulatingdifferentstepsofthispathway.Tothisend,weperformedagenome-wideRNAiscreenincul-turedDrosophilacellsandidentified121genesthatrep-resent29potentialpositiveand92negativeregulatorsoftheJAK/STATpathway.Importantly,amongthesewerefivecanonicalcomponentsoftheJAK/STATpathway,includingUpd2,Dome,Hop,STAT92E,andSOCS36E,indicatingtherobustnessandvalidityofthisapproach.The29positiveregulatorswerefurtheranalyzedbyex-aminingtheeffectoftheirdouble-strandedRNA(dsRNA)-mediatedknockdownonSTAT92Etyrosinephosphorylation.WealsodemonstratethatDrosophilahomologsofRanBP3andRanBP10areinvolvedinSTAT92Enucleocytoplasmictransport.Finally,wechar-acterizedthefirstproteintyrosinephosphatase,PTP61F,thatnegativelyregulatestheDrosophilaJAK/STATpathway.Together,thesefindingsunderscoretherobust-nessofgenome-wideRNAiscreeningapproachestoun-covernovelregulatorsinvolvedindifferentstepsinsig-nalingpathways.Results

GeneratingaJAK/STATreportergeneinDrosophilaSOCS36E(Drosophilahomologofsuppressorofcyto-kinesignalinggenefamily)encodesanegativeregulatoroftheJAK/STATsignalingpathwayinDrosophila,andhasbeenshowntobetranscriptionallyactivatedbyJAK/STATsignaling(CallusandMathey-Prevot2002;Karstenetal.2002).UponcloseexaminationoftheSOCS36Egenomicregion,weidentifieda441-bpfrag-

1862GENES&DEVELOPMENT

mentintheenhanceroftheSOCS36EgenethatcontainstwopotentialSTAT92E-bindingsites.TogenerateaJAK/STATreporter,weplacedfivetandemrepeatsofthisgenomicfragmentupstreamofaminimalheat-shockpromoter-drivencDNAencodingthefireflylucif-erasegene(Fig.1A),hereinreferredtoas10XSTAT92E–luciferase.Toconfirmthatthisreportergenewasre-sponsivetoJAK/STATsignalingandtoselectaDrosophilacelllinethatwouldallowfortheidentifica-tionofbothpositiveandnegativeregulatorsofSTAT92Eactivity,wefirsttransfectedvariousDrosophilacelllineswith10XSTAT92E–luciferaseandanActinpro-moter-drivenRenillaluciferaseexpressionvector(Act-Renilla)togetherwithvariousdsRNAs.WetesteddsRNAsagainstknownJAK/STATcomponentsandquantifiedtheactivityofJAK/STATsignalingbymea-suringrelativeluciferaseunits(RLU),whichequaledtheratiooftheabsoluteactivityoffireflyluciferasetoRenillaluciferase.ADrosophilaSchneidercelllinede-rivative(S2-NP)exhibitedrobustendogenousJAK/STATactivity,andthisactivitywassensitivetoRNAima-nipulations.TheadditionofdsRNAsagainstpositiveregulators,suchasSTAT92E,Hop,andDome,ledtoa12-to24-folddecreaseinthereporteractivity,whereasdsRNAagainstanegativeregulator,SOCS36E,increaseditsactivitybythreefold(Fig.1B).ThusthereportergenefaithfullyreflectedJAK/STATsignalinginS2-NPcells.Acell-basedgenome-wideRNAiscreenanddataanalysis

ToidentifyadditionalmodulatorsoftheJAK/STATpathwaywhoseloss-of-functionaffectsSTAT92Eactiv-

Figure1.GeneratingaJAK/STATreporterconstruct.(A)Sche-maticrepresentationofthe10XSTAT92E–luciferasereporterconstruct.FivecopiesofagenomicfragmentfromtheSOCS36EintronicregioncontainingtwoSTAT92E-bindingsiteswereplacedupstreamofahspminimalpromoter-drivenfireflylu-ciferasegene.(B)DrosophilaS2-NPcellsweretransfectedwith10XSTAT92E–luciferaseandAct-RenillatogetherwithdsRNAsagainstvariouscanonicalcomponentsoftheJAK/STATpathway.Luciferaseassaywasperformed4dlater,andthereporteractivitywasnormalizedastheratiooffirefly/Re-nilla.Notethatthecontrolvaluewassetto1.Theresultswerefromtwoindependentexperiments.

Genome-widestudyofJAK/STATsignaling

ity,weperformedagenome-wideRNAiscreenusingcul-turedDrosophilaS2-NPcellsin384-wellplates.Weusedalibraryof∼21,300dsRNAs(Boutrosetal.2004)thattarget>95%oftheannotatedgenesintheDrosophilagenome(Hildetal.2003).Luciferasevalueswereana-lyzedandpotentialcandidategeneswereassignedonthebasisoftheirdeviationfromtheplateaverageforeachgivenplate(seeMaterialsandmethods).Intheprimaryscreen,weidentified474candidategenesthatinclude259genesthatreducedJAK/STATsignalingbymorethantwostandarddeviations(SD)and215genesthatincreasedsignalingbymorethanthreeSDwhenknockeddownbyRNAi(Fig.2A).Importantly,amongthesegenesweindependentlyidentifiedfivecanonicalcomponentsoftheJAK/STATpathway:Upd2,Dome,Hop,STAT92E,andSOCS36E,confirmingtherobust-nessofthescreen(Fig.2B).

Ofthese474candidategenes,188representsequencesnotannotatedbytheBerkeleyDrosophilaGenomeProject(whichwerebasedonaninclusivealgorithmfordeterminingORFsintheflygenome)(Hildetal.2003),ribosomalproteins,andproteinsinvolvedinRNApro-cessingandtranslation(datanotshown).Thesegeneswerenotpursuedfurther.Wenextrepeatedthesameassaywiththeremaining286genesinduplicate.Two-hundred-twocandidategenes(71%)identifiedinthepri-maryscreenwereverified.Intheprimaryscreen,weusedAct-Renillafornormalizingthetransfectioneffi-ciency.BecausethecandidategenesfromtheprimaryscreenweredeterminedbycalculatingtheFirefly/Re-nillaratio,itisconceivablethatsomeofthemmightarisefromtheeffectofcertaindsRNAsontheActinpromoter.Thus,toremovecandidategenesthatmightaffecttheActinpromoterandnottheJAK/STATrespon-siveelement,werepeatedourreporterassayusingaPolIIIpromoter-drivenRenillaluciferaseexpressionvector(polIII-Renilla).Wefoundthat81candidates(40%)fell

intothiscategoryandwerenotpursuedfurther(datanotshown).Thusthisscreenidentified121candidategenesthatspecificallymodulateJAK/STATsignalinginS2-NPcells(SupplementaryTable1).Importantly,Upd2,acy-tokine-likemolecule,isamongthepositiveregulatorsidentifiedinthescreen.Thus,theendogenouslyex-pressedUpd2isresponsibleforbasallevelsofJAK/STATsignalinginS2-NPcells.

These121geneswereassignedtocategoriesbasedontheirpredictedmolecularfunctions,proteindomains,andreportsfromtheliteraturetohelpustopredictfunc-tionsandgeneratetestablehypothesesforfurtherchar-acterization(Fig.2B).Thesecategoriesinclude(1)ca-nonicalJAK/STATsignalingpathwaycomponent,(2)ki-nase/phosphatase,(3)chromatinremodeling,(4)proteintrafficking,(5)celladhesion,(6)structuralmolecule,(7)transcriptionfactor,and(8)miscellaneous.

Next,weassayedthe29positiveregulatorsincellsstimulatedwithexogenousUpd,awell-characterizedli-gandforJAK/STATsignaling.Wefoundthat27geneswerevalidatedinthisassay,stronglysuggestingthatthescreenhasidentifiedbonafidecomponentsoftheJAK/STATsignalingpathway(SupplementaryTable1).ThisassayrevealedthatUpd2andCG17836arenotrequiredforUpd-inducedJAK/STATsignaling.SinceUpd2isanendogenouslyexpressedcytokineresponsibleforbasallevelsofJAK/STATsignaling,itisnotexpectedtoberequiredfortheJAK/STATsignalelicitedbyexogenousUpd.

IdentificationofgenesthataffecttyrosinephosphorylationofSTAT92E

Tomoreclearlyelucidatetherolesofpositiveregulators,weassayedtheirrequirementforthephosphorylationofSTAT92E.TyrosinephosphorylationisakeystepinSTATactivationuponcytokine/receptorstimulation.

Figure2.DataanalysisfortheJAK/STATscreen.(A)Scatterplotforthreerepresenta-tivescreenplates.Cutoffsweresetas2SDbelowthemeanor3SDabovethemeanRLU.Notethatallthree“spikedin”con-troldsRNAsagainstSTAT92Ewereidenti-fied.(B)PiechartdepictingcategoriesofgenesidentifiedintheJAK/STATscreen.

GENES&DEVELOPMENT1863

Baegetal.

Thus,monitoringsteady-statelevelsofphosphorylatedSTATindsRNA-treatedcellswouldprovideinsightintothemolecularfunctionsofourcandidategenes.Asex-pected,wefoundthatUpdstimulationofS2-NPcellsleadstoadramaticincreaseintyrosine-phosphorylatedSTAT92E,asshownbyWesternblotanalysis(Fig.3A).Next,wetestedtheeffectofdsRNAsagainstthe29posi-tiveregulatorsonUpd-inducedSTAT92Ephosphoryla-tion.Thirteengenes(besidesSTAT92E)werefoundtoberequiredforUpd-inducedSTAT92Ephosphorylation(Fig.3B,C;SupplementaryTable1).Asexpected,thesegenesincludedthecanonicalcomponentsDomeandhop.Incontrasttotheinitialassayintheprimaryscreen,hereweusedexogenousUpdtoactivateSTAT92Ephos-phorylation,andthuswewereunabletoidentifygenesthatactupstreamofthereceptor,suchasUpd2.Notably,twoofthe13genes(CG16790andCG4329)thatregulateSTAT92Ephosphorylationhavenopredictedfunction,yetclearlyhavehumanorthologs;furtherinvestigationoftheirmolecularfunctionsinJAK/STATsignalinginDrosophilamayadvanceourunderstandingofthemam-malianpathway.

Interestingly,thisassayrevealedthatRNAiknock-downofthecyclin-dependentkinase2gene(cdc2)re-sultedinadecreaseinSTAT92Etyrosinephosphoryla-tion(Fig.3B),suggestingthatcdc2modulatesJAK/STATsignalingbyaffectingtyrosinephosphorylationofSTAT92E.Consistentwiththisobservation,Warts/Lats,

Figure3.IdentificationofgenesrequiredforUpd-inducedty-rosinephosphorylationofSTAT92E.(A)Act-STAT92E-HAwastransfectedintoS2-NPcellstogetherwithdsRNAagainstlacZ.Cellsweresplitintotwodishes3.5daftertransfection.HalfofthecellswerecoculturedwithS2-NPcellstransfectedwithAct-Upd∼12hpriortoharvestandtheotherhalfremaineduntreated.Cellextractsweresubjectedtoimmunoprecipitationusinganti-HAantibodiesandtheimmunoprecipitateswereanalyzedbyimmunoblottingusinganti-phospho-Tyr-STAT92EandHAantibodies.NotethatUpd-inductionleadstoadramaticincreaseinSTAT92Ephosphorylation.(B)Act-STAT92E-HAwastransfectedintoS2-NPcellstogetherwithvariousdsRNAstargetingpositiveregulatorsidentifiedinthescreen.CellswerestimulatedwithUpd∼12hpriortoharvest.CellextractsweresubjectedtoWesternblotanalysisusinganti-phospho-Tyr-STAT92EandHAantibodies.dsRNAsagainstLacZandlilliserveascontrol.(C)ListofgenesrequiredforUpd-inducedSTAT92Ephosphorylation.

18GENES&DEVELOPMENT

whichhasbeenshownbothbiochemicallyandgeneti-callytointeractwithcdc2andtonegativelyregulateitskinaseactivity(Taoetal.1999),wasidentifiedinourscreenasapotentialnegativeregulatorofJAK/STATsig-naling.TheseresultssuggestthatSTAT92Eplaysanim-portantroleinWarts/Lats-mediatedinhibitionofcellproliferation.

Wealsoidentifiedechinoid(ed)asapositiveregulatorrequiredforUpd-dependentSTAT92Etyrosinephos-phorylation.edencodesacelladhesionmoleculeandhasbeenshowntobeanegativeregulatoroftheEGFRsig-nalingpathwayduringDrosophilaeyedevelopment(Baietal.2001;Islametal.2003).PreviousexperimentshaveshownbothpositiveandnegativeinteractionsbetweentheJAK/STATpathwayandtheEGFRpathway.Forex-ample,STAT92EmutantsphenocopymutantsintheEGFRpathway(Yanetal.1996).Furthermore,studiesusingmammaliantissueculturesystemshavedemon-stratedthatEGFRsignalingactivatesbothJAK1andSTAT1(Quelleetal.1995;Leamanetal.1996).Inaddi-tion,EGFR-inducedcellmigrationismediatedpredomi-nantlybytheJAK/STATpathwayinprimaryesophagealkeratinocytes(Andletal.2004).Similarly,edhasbeenshowntoberesponsiblefordefectivecellmigrationinCaenorhabditiselegans(VogelandHedgecock2001).ThereforestudyingtheroleofedinJAK/STATsignalingindifferentcontextsmayfacilitateourunderstandingofthegeneticandbiochemicalmodeofSTATactivationbyEGFRsignaling,andprovideinsightsintothemecha-nismsgoverningcancercellmetastasisinhumans.IdentificationofgenesthataffectnucleartranslocationofSTAT92E

AnotherstepintheactivationoftheJAK/STATsignal-ingpathwayisthetranslocationofSTATsintothenucleus.Inrestingcells,STATsresidemainlyinthecytoplasm.Uponcytokinestimulation,theyarephos-phorylatedonkeytyrosineresiduesandrapidlytranslo-catetothenucleus,wheretheytrans-activatetargetgenes.PreviousstudieshaveshownthatImportin␣5andRanarerequiredforthenuclearimportofphosphory-lated(activated)STATs(Sekimotoetal.1997).Toresetthecellsafterstimulation,STATsareexportedoutofthenucleusintothecytoplasminpreparationforthenextroundofsignalingusinganExportin-1/CRM-1-depen-dentmechanism(McBrideetal.2000).Theseobserva-tionssuggestthatdefectivenucleocytoplasmicshuttlingofSTATscandisruptsteady-statedistributionofSTATsandinduceaberrantbiologicalresponses.Amongall121candidates,weidentifiedsevengenesthatarepoten-tiallyinvolvedinproteintraffickingbasedontheirpre-dictedmolecularfunctionsandproteindomains(Fig.2B;SupplementaryTable1).TheseincludeRab26,Ran,CG10225,whichencodestheDrosophilahomologofRan-bindingprotein3(RanBP3),CG11763,whichen-codestheDrosophilahomologofRan-bindingprotein10(RanBP10),andtheDrosophilahomologofCellularAp-optosisSusceptibilitygeneproduct(CAS)thatwasini-tiallyidentifiedasaRan-bindingprotein.Inaddition,we

alsoidentifiedDrosophilahomologsofTransportin1andNucleoporin196,whichhavebeenimplicatedinproteinimportand/orexportinmammals.WethereforeexaminedthesubcellularlocalizationofphosphorylatedSTAT92Eunderconditionswhereeachofthesevencan-didateswasdepletedbyRNAiexceptRad26.Asacontrolwefoundthatunderrestingconditionstyrosinephos-phorylatedSTAT92Ewasdetectedpredominantlyinthecytoplasm(Fig.4A–C).Moreover,weobservedasignifi-cantreductioninphosphorylatedSTAT92ElevelsinthecytoplasmwhencellsweretreatedwithdsRNAagainst

Figure4.DrosophilahomologsofRanBP3(CG11763)andRanBP10(CG10225)areinvolvedinphosphorylatedSTAT92Enucleocytoplasmicshuttling.CellsweretreatedwithvariousdsRNAsandthentransfectedwithAct-Upd4dlater(G–O)orremaineduntransfected(A–F).Immunostainingwasperformedusinganti-phospho-Tyr-STAT92Eantibody(green).DAPIstain-ingwasemployedtovisualizethenuclei(red).Notethatasig-nificantaccumulationofphosphorylatedSTAT92Einthenu-cleiofcellstreatedwithdsRNAagainsteitherCG11763orCG10225wasdetecteduponUpdinduction,comparedwithcellstreatedwithdsRNAforlacZ.

Genome-widestudyofJAK/STATsignaling

thereceptordome(Fig.4D–F).UponstimulationwithUpd,STAT92Eaccumulatesinthenucleiof27.2%(n=162)ofcells(Fig.4,cf.G–IandA–C).Theseresultsillustratethespecificityandsensitivityofourassay.In-terestingly,wefoundthatcellstreatedwithdsRNAsagainstCG11763orCG10225displayedasignificantin-creaseinphospho-STAT92EnuclearaccumulationuponUpdstimulation(Fig.4,cf.J–L,M–OandG–I,80%,n=200and59%,n=200,respectively).ThiswasnotduetochangesinthetotalphosphorylationlevelsofSTAT92E(datanotshown).Wecouldnotdetectsignifi-canteffectsofdsRNA-mediatedknockdownofCasorTrnonSTAT92Etranslocation(datanotshown).Ontheotherhand,theroleofRanandNup98inSTAT92EtranslocationcouldnotbeassessedinthisassayduetodifficultiesinintroducingtheUpdexpressionvectorintocellsuponRNAiknockdownofthesetwogenes(datanotshown).Takentogether,theseresultsstronglysug-gestthattheDrosophilahomologsofRanBP3andRanBP10arenovelregulatorsofJAK/STATsignalingthataffectsignal-dependentSTAT92Enucleartransport.Theroleofproteintyrosinephosphatase61F(PTP61F)intheJAK/STATpathway

AnotherimportantstepintheJAK/STATsignaltrans-ductionpathwayisthedephosphorylationofthesignal-ingmoleculesJAKsandSTATs.Inmammals,severalPTPshavebeenimplicatedinthedephosphorylationofJAKand/orSTATproteinsbothinthecytoplasmandinthenucleus(ShuaiandLiu2003).Incontrast,noPTPshavebeenidentifiedthatregulateJAK/STATsignalinginDrosophila.PTP61Fwasidentifiedasastrongnega-tiveregulatorinourscreen.KnockdownofPTP61FbyRNAiresultedinamorethanfourfoldincreaseinSTAT92E-dependentreporteractivity(Fig.5A).PTP61FencodestheDrosophilahomologofmammalianPTP-1B,whichhasbeenshowntoattenuateinsulin,PDGF,EGF,andIGF-Isignalingbydephosphorylatingtyrosineresi-duesofJAKsand/orSTATsinmammaliantissueculture(AokiandMatsuda2000;Myersetal.2001).Wethere-foretestedthehypothesisthatPTP61FmightserveasthetyrosinephosphataseforHop.Weobservedadra-maticincreaseintyrosinephosphorylationofHopuponRNAiknockdownofPTP61F(Fig.5B),suggestingthatHopmaybeasubstrateofPTP61F.WealsodetectedasignificantincreaseinSTAT92EphosphorylationincellstreatedwithdsRNAagainstPTP61F(Fig.5C).ThisisconsistentwiththenotionthatSTAT92Eisadown-streamtargetofHop,althoughwecannotruleoutthepossibilitythatbothHopandSTAT92EmaybetargetsofPTP61F.

InbothmammalsandDrosophila,SOCS,anegativeregulatoroftheJAK/STATpathway,hasbeenshowntobetranscriptionallyactivatedbyJAK/STATsignaling,thusgeneratinganegativefeedbackloop.ThispromptedustoexaminetheexpressionpatternofPTP61FandwhetheritsexpressionisresponsivetoJAK/STATsig-nalinginvivo.WefoundPTP61Fisexpressedinastripedpattern,reminiscentoftheSTAT92Eexpressionpattern

GENES&DEVELOPMENT1865

Baegetal.

Figure5.PTP61FnegativelyregulatestheJAK/STATpathwayinDrosophila.(A)KnockdownofPTP61FbyRNAiactivatestheJAK/STATreporteractivity.DrosophilaS2-NPcellsweretransfectedwith10XSTAT92E–luciferaseandAct-RenillatogetherwithdsRNAsagainstlacZorPTP61F.Luciferaseassaywasperformed4dlaterandthereporteractivitywasnormalizedastheratiooffireflytoRenilla.Thecontrolvaluewassetas1.Theresultswerefromtwoindependentexperiments.(B)Act-Myc-HopwastransfectedintoS2-NPcellstogetherwithdsRNAsagainstlacZorPTP61F.Cellswereharvestedandcelllysateswereimmunoprecipitatedwithanti-Mycantibody.Immunoprecipitateswereanalyzedbyimmunoblottingusinganti-phospho-Tyroranti-Mycantibodies.Notethatanincreaseinphospho-HoplevelswasdetecteduponRNAiknockdownofPTP61F.(C)Act-STAT92E-HAwastransfectedintoS2-NPcellstogetherwithdsRNAsagainstlacZorPTP61F.Cellswereharvestedandcelllysateswereimmunoprecipitatedwithanti-HAantibody.Immunoprecipitateswereanalyzedbyimmunoblottingusinganti-phospho-Tyr-STAT92Eoranti-HAantibodies.Anin-creaseinphospho-STAT92ElevelswasdetecteduponRNAiknockdownofPTP61F.(D)RNAinsituhybridizationusingSTAT92EorPTP61Fprobeswasperformedonwild-typestage9–10embryos(panelsa,b),embryosoverexpressingUpdunderthecontrolofpaired-Gal4(panelc),andhopGLCembryos(paneld).NotethatPTP61Ftranscriptlevelsaredramaticallyincreasedinthepaireddomain(panelc)anddecreasedinembryolackinghopactivity(paneld).(E)GeneticinteractionsbetweenUpdandPTP61F.Over-expressionofUpdintheeyeunderthecontrolofGMR-Gal4resultsinadramaticovergrowthanddeformationintheadulteye(cf.panelsaandb).RemovingonecopyofPTP61Ffurtherenhancesthisphenotype(panelc),whereasintroductionofaPTP61Ftransgenerescuesthisphenotype(paneld).

(Fig.5D,panelsa,b).Inaddition,overexpressionofUpdunderthecontrolofprd-Gal4resultedinadramaticin-creaseinPTP61Ftranscriptlevelsinthepaireddomain(Fig.5D,panelc).Furthermore,levelsofthePTP61FtranscriptweregreatlyreducedinembryoslackingHopactivity(Fig.5D,paneld),suggestingthatPTP61Ftran-scriptionisdependentonactiveJAK/STATsignaling.Takentogether,theseresultsdemonstratethatPTP61FexpressionrespondstoJAK/STATsignalinginvivo.ThesedatasuggestedthatlossofPTP61FwouldresultinanincreaseinJAK/STATsignaling.Thus,wenextexaminedthegeneticinteractionbetweenPTP61FandcanonicalcomponentsoftheJAK/STATpathway,usingDf(3)ED4238,adeficiencyuncoveringthePTP61Fgene.WetestedtheinteractionintheDrosophilaeyefollow-ingoverexpressionofUpdusingGMR-Gal4driver,whichcausesadramaticovergrowthanddeformationoftheadulteye(Fig.5E,panelb)(Bachetal.2003;Chenetal.2003).TheseverityofthisphenotypeisproportionaltothestrengthoftheJAK/STAT-mediatedsignal,asre-movingonecopyofSTAT92EsignificantlysuppressestheGMR-Updeyephenotype(Bachetal.2003;datanotshown).ConsistentwithPTP61Fbeinganegativeregu-latoroftheJAK/STATsignalingpathway,flieshetero-zygousforDf(3)ED4238showedanenhanceddeformedeyephenotype(Fig.5E,panelc).APTP61FtransgenerescuesthisenhanceddeformedeyephenotypeinfliesheterozygousforDf(3)ED4238(Fig.5E,paneld).Inaddition,thePTP61FtransgenealsorescueslethalityinfliescarryingUAS-UpdGMR-Gal4/+;Df(3)ED4238/+,presumablycausedbyleakyexpressionofUAS-UpdinconjunctionwithPTP61Fdeficiency(SupplementaryTable2).

Nextweexaminedthegeneticinteractionbetween

1866GENES&DEVELOPMENT

PTP61FandHop.FliescarryingadominanthyperactiveHopallele(HopTum-l)displaydecreasedviabilityandtheformationofmelanotictumors(Harrisonetal.1995;Dearolf1998).Thistumorformationphenotypeissen-sitivetogenedosage.Previousstudieshaveshownthatreducingthelevelsofpositiveregulators,suchasSTAT92E,Cdk4,andCycE,increasestheviabilityand/ordecreasestumorformation(Houetal.1996;Chenetal.2003).WethereforemonitoredbothviabilityandmelanotictumorformationinfemalesheterozygousforHopTum-landcomparedtheseresultstofemaleshetero-zygousforbothHopTum-landDf(3)ED4238.RemovingonecopyofPTP61FinHopTum-lheterozygousfemalesleadstoasignificantdecreaseinsurvivalrateandadra-maticenhancementintheformationofmelanotictu-mors(Table1).Altogether,theseresultsdemonstratethatPTP61FisabonafidenegativeregulatoroftheJAK/STATpathwayinDrosophila.Discussion

Here,wereportthefirstgenome-wideRNAiscreenfornovelcomponentsoftheJAK/STATsignaltransductionpathway.Thisscreenhasuncovered116novelgenesthatregulateJAK/STATsignalinginDrosophila,inad-ditiontofivepreviouslycharacterizedcanonicalJAK/STATcomponents.ThisdemonstratesthatourscreenwassuccessfulinidentifyinggeneswithspecificrolesintheJAK/STATpathway.Wefurthershowedthat13ofthe29positiveregulatorsarerequiredforUpd-inducedSTAT92Ephosphorylation.Inaddition,wefoundtwonovelregulatorsofSTAT92Enucleartranslocation.Fi-nally,weidentifiedPTP61FasthefirstproteintyrosinephosphatasethatnegativelyregulatesJAK/STATsignal-inginDrosophilabothinvitroandinvivo,anddemon-stratedthatPTP61FisatranscriptionaltargetofJAK/STATsignaling.

Amongtheidentifiedgenes,40(32.8%)hadnopre-dictedmolecularfunctionand/orrecognizableproteindomain,suggestingthatthescreenidentifiedmanyun-characterizedgeneswithessentialrolesinJAK/STATsignaling(Fig.2B;SupplementaryTable1).Notably,“Re-ciprocal-Best-Blast”(RBB)analysisrevealedthat88genes(72.7%)identifiedinthescreenhavehumanor-thologs,suggestingaconservedroleinthemammalianJAK/STATsignalingpathway(SupplementaryTable1).Thelistofcandidategenesidentifiedinthisscreen

Table1.LossoffunctionofPTP61FenhancesTum-lphenotypesGenotype

Viable%withtumors

Tum-l/+;TM3,Sb/+

35019.43Tum-l/+;Df(3)ED4238/+

177

.83

Resultsfromthreeindependentexperiments.

FemalefliesheterozygousforbothDf(3)ED4238andhopTum-ldisplayedasignificantdecreaseinviabilityandadramaticin-creaseinmelanotictumorformationcomparedtohopTum-lfe-malefliesheterozygousforonlyhopTum-l.

Genome-widestudyofJAK/STATsignaling

onlyminimallyoverlapswiththatgeneratedfromsimi-largenome-wideRNAistudiesontheWntandHedge-hogsignalingpathways(Dasguptaetal.2005;K.Nybak-ken,pers.comm.),indicatingthatwehaveidentifiedmanygenesthathaveaspecificroleintheJAK/STATsignalingpathway.Moreover,∼73%ofourcandidategeneshavewell-conservedhumanorthologs,suggestingthatcell-basedassaysinDrosophilacanserveasasimpleassaysystemtorapidlyidentifyandcharacterizegenesthatmayplaysimilarrolesinthemammalianJAK/STATpathway.

Clearlytheresultsfromthescreenpresentedherewillprovidethefoundationformanyfollow-upinvestiga-tions,aseachofthenewlyidentifiedgeneswillneedtobecarefullyvalidatedinvivofortheirrolesinJAK/STATsignaling.Validationintheflysystem,aswellasinothermodelsystemsforthoseevolutionarilyconservedfac-tors,willprovidefurtherinsightsintoourglobalunder-standingofJAK/STATsignaling.Materialsandmethods

JAK/STATreportergene

A441-bpgenomicfragmentintheenhancerofSOCS36Econ-tainingtwopotentialSTAT92E-bindingsiteswasamplifiedbyPCR,usingfivedifferentsetsofoligos:(1)CTGCAGGAACCACTCAGAGTGCCTGCGTGT(PstI),GAATTCATACAAAACTGTCTTAGGTGTTTA(EcoRI);(2)CTGCAGGAACCACTCAGAGTGCCTGCGTGT(PstI),CTGCAGATACAAAACTGTCTTAGGTGTTTA(PstI);(3)GAATTCGAACCACTCAGAGTGCCTGCGTGT(EcoRI),GAATTCATACAAAACTGTCTTAGGTGTTTA(EcoRI);(4)AGATCTGAACCACTCAGAGTGCCTGCGTGT(BglII),AGATCTATACAAAACTGTCTTAGGTGTTTA(BglII);(5)GCGGCCGCGAACCACTCAGAGTGCCTGCGTGT(NotI),GCGGCCGCATACAAAACTGTCTTAGGTGTTTA(NotI).EachamplifiedgenomicfragmentcontainingdifferentrestrictionenzymesiteswassequentiallysubclonedintopP[UAST](PhelpsandBrand1998).ThegenomicfragmentamplifiedusingthefirstsetofoligoswassubclonedintothePstI/EcoRIsitesofpP[UAST]togenerate2XSTAT92E.ThegenomicfragmentamplifiedusingthesecondsetofoligoswassubclonedintothePstIsiteof2XSTAT92Etogenerate4XSTAT92E.ThegenomicfragmentamplifiedusingthethirdsetofoligoswassubclonedintotheEcoRIsiteof4XSTAT92Etogenerate6XSTAT92E.ThegenomicfragmentamplifiedusingthefourthsetofoligoswassubclonedintotheBglIIsiteof6XSTAT92Etogenerate8XSTAT92E.Next,thehsp70minimalpromoterelementwasamplifiedfrompP[UAST]byPCRusingoligosGCGGCCGCAGCGGAGACTCTAGCGAGCG(NotI)andCTCGAGAATTCCCTATTCAGAGTTCT(XhoI).Thishsp70minimalpromoterwassubclonedintotheNotI/XhoIsitesof8XSTAT92Etogenerate8XSTAT92E–hsp70.Again,thegenomicfragmentamplifiedusingthefifthsetofoligoswassubclonedintotheNotIsiteof8XSTAT92E–hsp70vectortogenerate10XSTAT92E–hsp70.Finally,anXhoI/XbaIfragmentcontainingthefireflyluciferasegenefromthepGL3–luciferasevector(Promega)wassubclonedintotheXhoI/XbaIsitesof10XSTAT92E–hsp70togenerate10XSTAT92E–luciferase.

TogenerateareporterconstructcontainingsixSTATconsen-sussites,twopairsofoligos—(1)TTCTGGGAAACCGTTTATACGCTGCGTTCGCGGAAACCGTTTATACGCTGCGTTCTGGGAAACCGTTTATAC,AACGGTTTCCCAGAACGCAG

GENES&DEVELOPMENT1867

Baegetal.

CGTATAAACGGTTTCCGCGAACGCAGCGTATAAACGGTTTCCCAGAATGCAand(2)GCTGCGTTCGCGGAAACCGTTTATACGCTGCGTTCTGGGAAACCGTTTATACGCTGCGTTCGCGGAA,AATTTTCCGCGAACGCAGCGTATAAACGGTTTCCCAGAACGCAGCGTATAAACGGTTTCCGCGAACGCAGCGTATA—wereannealed,respectively,andclonedto-getherintopP[UAST]usingPstIandEcoRIsites.Subsequently,anXhoI/XbaIfragmentcontainingthefireflyluciferasegenefromthepGL3–luciferasevector(Promega)andthehsp70mini-malpromoterweresubclonedintotheresultingvectorusingXhoI/XbaIandNotI/XhoIsites,respectively,togeneratethe6XSTAT–synthetic-lucconstruct.Celllines

ThecelllinethatweusedisaderivativeofS2cellsandwasoriginallyaPeptidoglycan-responsivecellline.Duringthecourseofmaintenanceinourlab,however,wehavenoticedsubtlemorphologicalchangesinthesecells.Mostimportantly,theyarenolongerresponsivetoPeptidoglycantreatment.Thus,thesecellshaveevolvedtoanewS2cellderivative,andwerethusreferredtoas“S2-NP.”Acell-basedRNAiscreen

Alibraryof∼21,300dsRNAsrepresentingtheDrosophilage-nomewasaliquottedinto384-wellplates(∼80ngdsRNA/well).Foreachwell,0.5ng10XSTAT92E–luciferase,10ngAct-Re-nilla,and110ngpAc-PL(servingascarrierDNA)weremixedwith0.96µLEnhancerin15µLEC(Qiagen)andincubatedatroomtemperaturefor5min.Then0.42µLofEffectenereagentwereaddedandthemixturewasimmediatelydispensedintoeachwellcontainingdsRNA.Afterincubationatroomtem-peraturefor10min,40µLS2-NPcells(106cells/mL)weredis-pensedintothewell.Luciferaseassayswereperformed96haftertransfectionusingDualGloreagents(Promega).Foreachwell,thereporteractivity,referredtoasrelativeluciferaseunits(RLU),wascalculatedastheratiooffireflyluciferasetoRenillaluciferase.Foreachplate,themeanandSDofRLUwerecalcu-lated.dsRNAsthatcausedaRLUvaluetobeeithertwoSDormorebelowthemeanorthreeSDormoreabovethemeanwereselectedascandidategenes.Theassaywasconductedindupli-catetoreducetherateoffalsepositivesandtoincreasethereproducibilityofindividualcandidates.Forthesecondaryscreen,286dsRNAswereresynthesizedusingtheMegaScriptkit(Ambion)andaliquottedinto384-wellplates(80ngdsRNA/well).Transfectionandluciferaseassaywereperformedasde-scribedabove.InexperimentsinvolvingpolIII-Renilla,thesameamountofpolIII-RenillawasusedaswithAct-Renilla.ToassaycandidategenesincellsstimulatedwithUpd,20pg10XSTAT92E–luciferase,5ngAct-Renilla,105ngpAc-PL,and1ngAct-UpdweretransfectedtoS2-NPcellstogetherwith80ngdsRNA.Inexperimentsinvolving6XSTAT–synthetic-luc,2ng6XSTAT–synthetic-luc,20ngpolIII-Renilla,80ngpAc-PL,and20ngAct-UpdweretransfectedtoS2-NPcellstogetherwith80ngdsRNAperwell.Inalltheabove-mentionedexperi-ments,luciferaseassayswereperformed96haftertransfection.ImmunoprecipitationandWesternblotanalysis

ForanalyzingUpd-inducedSTAT92Ephosphorylation,anex-pressionplasmidforHA-taggedSTAT92E(Act-STAT92E-HA)wastransfectedintoS2-NPcellstogetherwithdsRNAagainstLacZ.Cellsweresplitintotwodishes3.5daftertransfection.HalfofthecellswerecoculturedwithS2-NPcellstransfectedwithAct-Upd∼12hpriortoharvest(Harrisonetal.1998)and

1868GENES&DEVELOPMENT

theotherhalfremaineduntreatedascontrol.Cellextractsweresubjectedtoimmunoprecipitationusinganti-HAantibodies,andtheimmunoprecipitateswereanalyzedbyimmunoblottinganalysisusinganti-phospho-Tyr-STAT92Eandanti-HAanti-bodies.TheeffectofRNAiknockdownof29potentialpositiveregulatorsonSTAT92Etyrosinephosphorylationwasinvesti-gatedbyWesternblotanalysis.S2-NPcellsweretransfectedwithAct-STAT92E-HAtogetherwithdsRNAtargetingeachofthepositiveregulators.Fourdaysaftertransfection,cellswerecoculturedwithS2-NPcellstransfectedwithAct-Updfor∼12hpriortoharvest.ThecelllysateswereresolvedbySDS-PAGE,transferredtoPVDFmembrane,andsubjectedtoimmunoblot-tinganalysisusinganti-phospho-Tyr-STAT92Eantibody(CellSignaling).Themembranewasthenstrippedandreprobedwithanti-HAantibody(Upstate)todetectSTAT92E-HAasaloadingcontrol.ToexaminetheeffectofdsRNA-mediatedknockdownofPTP61FonthephosphorylationstatusofHopandSTAT92E,Act-Myc-HoporAct-STAT92E-HAwastransfectedintoS2-NPcellstogetherwithdsRNAsagainstlacZorPTP61F.Cellswereharvestedandcelllysateswereimmunoprecipitatedwithanti-Mycoranti-HAantibodies,respectively.Immunoprecipitateswereanalyzedbyimmunoblottingusinganti-phospho-Tyroranti-phospho-Tyr-STAT92Eantibodies,respectively.Themem-braneswerestrippedandreprobedwithanti-Mycoranti-HAantibodies,respectively.Immunohistochemistry

Forimageanalysis,cellswerebathedwithvariousdsRNAsfor4dandthentransfectedwithAct-Updorleftuntreated.Twenty-fourhoursaftertransfection,cellswerefixedandstan-dardimmunohistochemistrywasperformedusinganantibodyagainstphospho-Tyr-STAT92E.DAPIstainingwasemployedtovisualizethenuclei.AccumulationofphosphorylatedSTAT92Einthenucleiwasanalyzedandimagesacquiredundertheconfocalmicroscope.

Flystocksandgeneticinteraction

Flylinesweremaintainedaccordingtostandardprocedure.Thefollowingflylineswereused:hopC111/FM7(BinariandPerrimon1994),hopTum-l/FM7(adominanttemperature-sensitiveallele)(HanrattyandDearolf1993),paired-Gal4(BrandandPerrimon1993),UAS-Upd(Harrisonetal.1998),UAS-UpdGMR-Gal4/CyO(thisstudy),UAS-PTP61F(thisstudy),andUAS-PTP61FDf(3)ED4238/TM3(thisstudy).FemalescarryinggermlineclonesofhopC111weregeneratedusingtheFLP-DFStechnique(ChouandPerrimon1996).VirginfemalesofthegenotypehopC111FRT101/FM7werematedwithmalesofthegenotypeovoD1FRT101/Y;FLP38.Theresultinglarvaewereheat-shockedfor2hat37°C.hopC111FRT101/ovoD1FRT101femaleswerecrossedwithFM7/Ymales.ForPTP61Fgeneticinteractionstud-ies,theeyephenotypeofUAS-UpdGMR-Gal4/+adultflieswascomparedtothatofUAS-UpdGMR-Gal4/+;Df(3)ED4238/+adultflies.hopTum-l/+;TM3,Sb/+andhopTum-l/+;Df(3)ED4238/+femalesweregeneratedbycrossinghopTum-l/YmaleswithDf(3)ED4238/TM3,Sbfemales,weremaintainedat29°C,andwerescoredforviabilityandthepresenceofmelanotictumors.

Acknowledgments

WethankDr.KentNybakkenforkindlyprovidingtheAct-RenillaandpolIII-Renillaplasmids.WethankmembersoftheDrosophilaRNAiScreeningCenterforreagentsandtechnicalassistance.WethankRamanujDasguptaandKentNybakken

forcommunicatingdatapriortopublication.SpecialthanksgotoBernardMathey-Provot,SaraCherry,RamanujDasgupta,PamelaBradley,andRichardBinariforcriticallyreadingthemanuscript.N.P.isaHowardHughesMedicalInstituteinves-tigator.G.-H.B.wassupportedbyTheMedicalFoundation/CharlesA.KingTrustpost-doctoralfellowship.R.Z.isaLeu-kemiaandLymphomaSocietyfellow.

Noteaddedinproof

SupplementaryTable1liststheinformationonthedsRNAs.PleasenotethattheresultsobtainedwithdsRNAswithpoten-tialoff-targeteffectswillneedfurthervalidationwithnewlysynthesizedindependentdsRNAs.

References

Andl,C.D.,Mizushima,T.,Oyama,K.,Bowser,M.,Nakagawa,H.,andRustgi,A.K.2004.EGFR-inducedcellmigrationismediatedpredominantlybytheJAK–STATpathwayinpri-maryesophagealkeratinocytes.Am.J.Physiol.Gastrointest.LiverPhysiol.287:G1227–G1237.

Aoki,N.andMatsuda,T.2000.Acytosolicprotein-tyrosinephosphatasePTP1Bspecificallydephosphorylatesanddeac-tivatesprolactin-activatedSTAT5aandSTAT5b.J.Biol.Chem.275:39718–39726.

Bach,E.A.,Vincent,S.,Zeidler,M.P.,andPerrimon,N.2003.AsensitizedgeneticscreentoidentifynovelregulatorsandcomponentsoftheDrosophilajanuskinase/signaltrans-ducerandactivatoroftranscriptionpathway.Genetics165:1149–1166.

Bai,J.,Chiu,W.,Wang,J.,Tzeng,T.,Perrimon,N.,andHsu,J.2001.ThecelladhesionmoleculeEchinoiddefinesanewpathwaythatantagonizestheDrosophilaEGFreceptorsig-nalingpathway.Development128:591–601.

Binari,R.andPerrimon,N.1994.Stripe-specificregulationofpair-rulegenesbyhopscotch,aputativeJakfamilytyrosinekinaseinDrosophila.Genes&Dev.8:300–312.

Boutros,M.,Kiger,A.A.,Armknecht,S.,Kerr,K.,Hild,M.,Koch,B.,Haas,S.A.,Consortium,H.F.,Paro,R.,andPerri-mon,N.2004.Genome-wideRNAianalysisofgrowthandviabilityinDrosophilacells.Science303:832–835.

Brand,A.H.andPerrimon,N.1993.Targetedgeneexpressionasameansofalteringcellfatesandgeneratingdominantphe-notypes.Development118:401–415.

Bromberg,J.F.2001.ActivationofSTATproteinsandgrowth

control.Bioessays23:161–169.

Callus,B.A.andMathey-Prevot,B.2002.SOCS36E,anovel

DrosophilaSOCSprotein,suppressesJAK/STATandEGF-Rsignallingintheimaginalwingdisc.Oncogene21:4812–4821.

Chen,X.,Oh,S.W.,Zheng,Z.,Chen,H.W.,Shin,H.H.,and

Hou,S.X.2003.CyclinD–Cdk4andcyclinE–Cdk2regulatetheJak/STATsignaltransductionpathwayinDrosophila.Dev.Cell4:179–190.

Chou,T.B.andPerrimon,N.1996.TheautosomalFLP-DFS

techniqueforgeneratinggermlinemosaicsinDrosophilamelanogaster.Genetics144:1673–1679.

Dasgupta,R.,Kaykas,A.,Moon,R.T.,andPerrimon,N.2005.

FunctionalgenomicanalysisoftheWingless/Wntsignalingpathway.Science308:826–833.

Dearolf,C.R.1998.Fruitfly‘leukemia.’Biochim.Biophys.Acta

1377:M13–M23.

Durbin,J.E.,Hackenmiller,R.,Simon,M.C.,andLevy,D.E.

Genome-widestudyofJAK/STATsignaling

1996.TargeteddisruptionofthemouseStat1generesultsincompromisedinnateimmunitytoviraldisease.Cell84:443–450.

Hanratty,W.P.andDearolf,C.R.1993.TheDrosophilaTumor-ous-lethalhematopoieticoncogeneisadominantmutationinthehopscotchlocus.Mol.Gen.Genet.238:33–37.

Harrison,D.A.,Binari,R.,Nahreini,T.S.,Gilman,M.,andPer-rimon,N.1995.ActivationofaDrosophilaJanuskinase(JAK)causeshematopoieticneoplasiaanddevelopmentalde-fects.EMBOJ.14:2857–2865.

Harrison,D.A.,McCoon,P.E.,Binari,R.,Gilman,M.,andPer-rimon,N.1998.Drosophilaunpairedencodesasecretedpro-teinthatactivatestheJAKsignalingpathway.Genes&Dev.12:3252–3263.

Hild,M.,Beckmann,B.,Haas,S.A.,Koch,B.,Solovyev,V.,Bu-sold,C.,Fellenberg,K.,Boutros,M.,Vingron,M.,Sauer,F.,etal.2003.Anintegratedgeneannotationandtranscrip-tionalprofilingapproachtowardsthefullgenecontentoftheDrosophilagenome.GenomeBiol.5:R3.

Hou,X.S.,Melnick,M.B.,andPerrimon,N.1996.Marelleacts

downstreamoftheDrosophilaHOP/JAKkinaseandencodesaproteinsimilartothemammalianSTATs.Cell84:411–419.

Hou,S.X.,Zheng,Z.,Chen,X.,andPerrimon,N.2002.TheJak/STATpathwayinmodelorganisms:Emergingrolesincellmovement.Dev.Cell3:765–778.

Islam,R.,Wei,S.Y.,Chiu,W.H.,Hortsch,M.,andHsu,J.C.2003.NeuroglianactivatesEchinoidtoantagonizetheDro-sophilaEGFreceptorsignalingpathway.Development130:2051–2059.

Karsten,P.,Hader,S.,andZeidler,M.P.2002.Cloningandex-pressionofDrosophilaSOCS36EanditspotentialregulationbytheJAK/STATpathway.Mech.Dev.117:343–346.

Leaman,D.W.,Pisharody,S.,Flickinger,T.W.,Commane,

M.A.,Schlessinger,J.,Kerr,I.M.,Levy,D.E.,andStark,G.R.1996.RolesofJAKsinactivationofSTATsandstimulationofc-fosgeneexpressionbyepidermalgrowthfactor.Mol.Cell.Biol.16:369–375.

McBride,K.M.,McDonald,C.,andReich,N.C.2000.NuclearexportsignallocatedwithintheDNA-bindingdomainoftheSTAT1transcriptionfactor.EMBOJ.19:6196–6206.

Meraz,M.A.,White,J.M.,Sheehan,K.C.,Bach,E.A.,Rodig,S.J.,

Dighe,A.S.,Kaplan,D.H.,Riley,J.K.,Greenlund,A.C.,Campbell,D.,etal.1996.TargeteddisruptionoftheStat1geneinmicerevealsunexpectedphysiologicspecificityintheJAK–STATsignalingpathway.Cell84:431–442.

Myers,M.P.,Andersen,J.N.,Cheng,A.,Tremblay,M.L.,Hor-vath,C.M.,Parisien,J.P.,Salmeen,A.,Barford,D.,andTonks,N.K.2001.TYK2andJAK2aresubstratesofprotein-tyrosinephosphatase1B.J.Biol.Chem.276:47771–47774.Neubauer,H.,Cumano,A.,Muller,M.,Wu,H.,Huffstadt,U.,

andPfeffer,K.1998.Jak2deficiencydefinesanessentialde-velopmentalcheckpointindefinitivehematopoiesis.Cell93:397–409.

Nosaka,T.,vanDeursen,J.M.,Tripp,R.A.,Thierfelder,W.E.,

Witthuhn,B.A.,McMickle,A.P.,Doherty,P.C.,Grosveld,G.C.,andIhle,J.N.1995.DefectivelymphoiddevelopmentinmicelackingJak3.Science270:800–802.

Parganas,E.,Wang,D.,Stravopodis,D.,Topham,D.J.,Marine,

J.C.,Teglund,S.,Vanin,E.F.,Bodner,S.,Colamonici,O.R.,vanDeursen,J.M.,etal.1998.Jak2isessentialforsignalingthroughavarietyofcytokinereceptors.Cell93:385–395.Park,S.Y.,Saijo,K.,Takahashi,T.,Osawa,M.,Arase,H.,Hi-rayama,N.,Miyake,K.,Nakauchi,H.,Shirasawa,T.,andSaito,T.1995.DevelopmentaldefectsoflymphoidcellsinJak3kinase-deficientmice.Immunity3:771–782.

GENES&DEVELOPMENT1869

Baegetal.

Phelps,C.B.andBrand,A.H.1998.EctopicgeneexpressioninDrosophilausingGAL4system.Methods14:367–379.

Quelle,F.W.,Thierfelder,W.,Witthuhn,B.A.,Tang,B.,Cohen,S.,andIhle,J.N.1995.PhosphorylationandactivationoftheDNAbindingactivityofpurifiedStat1bytheJanusprotein-tyrosinekinasesandtheepidermalgrowthfactorreceptor.J.Biol.Chem.270:20775–20780.

Rodig,S.J.,Meraz,M.A.,White,J.M.,Lampe,P.A.,Riley,J.K.,Arthur,C.D.,King,K.L.,Sheehan,K.C.,Yin,L.,Pennica,D.,etal.1998.DisruptionoftheJak1genedemonstratesobliga-toryandnonredundantrolesoftheJaksincytokine-inducedbiologicresponses.Cell93:373–383.

Sekimoto,T.,Imamoto,N.,Nakajima,K.,Hirano,T.,andYo-neda,Y.1997.Extracellularsignal-dependentnuclearimportofStat1ismediatedbynuclearpore-targetingcomplexfor-mationwithNPI-1,butnotRch1.EMBOJ.16:7067–7077.Shuai,K.andLiu,B.2003.RegulationofJAK–STATsignallingintheimmunesystem.Nat.Rev.Immunol.3:900–911.Tao,W.,Zhang,S.,Turenchalk,G.S.,Stewart,R.A.,StJohn,M.A.,Chen,W.,andXu,T.1999.HumanhomologueoftheDrosophilamelanogasterlatstumoursuppressormodulatesCDC2activity.Nat.Genet.21:177–181.

Thomis,D.C.,Gurniak,C.B.,Tivol,E.,Sharpe,A.H.,andBerg,L.J.1995.DefectsinBlymphocytematurationandTlym-phocyteactivationinmicelackingJak3.Science270:794–797.

Velazquez,L.,Fellous,M.,Stark,G.R.,andPellegrini,S.1992.Aproteintyrosinekinaseintheinterferon␣/␤signalingpath-way.Cell70:313–322.

Vogel,B.E.andHedgecock,E.M.2001.Hemicentin,aconservedextracellularmemberoftheimmunoglobulinsuperfamily,organizesepithelialandothercellattachmentsintoorientedline-shapedjunctions.Development128:883–4.

Yan,R.,Luo,H.,DarnellJr.,J.E.,andDearolf,C.R.1996.AJAK–STATpathwayregulateswingveinformationinDro-sophila.Proc.Natl.Acad.Sci.93:5842–5847.

Yu,H.andJove,R.2004.TheSTATsofcancer—Newmoleculartargetscomeofage.Nat.Rev.Cancer4:97–105.

1870GENES&DEVELOPMENT