protein isdrasticallydownregulated inhuman inflammatorybowel pathophysiology, consistentwithitsbroadtissuedistribution. This reports have indicatedabroaderroleforCYLDinmammalian activity (Ikeda andDikic,2006;Massoumietal.,2006).Recent consequence, atleastinpart,ofincreasedBcl3-dependentNF- skin cancerthanwild-typemiceandtheirsusceptibility is a Cyld Nomenclature Database(HUGO)].Interestingly, micewithboth and/or NEMO[alsoknown asIKBKGandIKK ability ofCYLDtoinducedeubiquitylationTRAF2,TRAF6 negative regulation ofthispathway appearstobemediatedbythe et al.,2003;Regamey etal.,2003;Trompouki etal.,2003).The factor receptorsuperfamily (Brummelkampetal.,2003;Kovalenko that theproteinproductof adult animals,islocalizedinthefatbody, ovaries,testes,digestivetractandspecificareasofthenervoussystem.We demo these issuesbycharacterizinganorthologof of CYLDinvivoarepoorlyunderstood.We establishedageneticallytractableanimalmodeltoinitiatesystematicinvestigati that isanegativeregulatoroftheNF- [email protected]) *Authors forcorrespondence (e-mail:[email protected]; 54124 Thessaloniki,Greece. Petros Ligoxygakis regulator ofNF- et al.,2003;Trompouki etal.,2003).CYLD isalsoanegative its tumor-suppressing function(Borodovsky etal.,2002;Kovalenko deubiquitylating activity, whichhasbeenassociatedgeneticallywith codes fora956aminoacidcytoplasmic protein(CYLD) with inactivated, indicatingthat Tumors develop inindividuals whohave bothcopiesofthegene single gene,named cases ofcylindromatosis arecausallyassociatedwith defects ina 2000). Thisfamilial diseaseandthemajority(57%)ofsporadic to tumorsofskinappendagescalledcylindromas (Bignell etal., Familial cylindromatosis isanautosomal-dominantpredisposition INTRODUCTION Oxford OX13QU,UK. Accepted 11April 2007 1 KEY WORDS:Cylindromatosis, The cylindromatosis( Theodore Tsichritzis antibacterial defense suppressor generegulatestriglyceridecontentand A (2007)doi:10.1242/dev.02859 Development 134,2605-2614 CYLD CYLD inabroadrangeoffunctionsassociatedwithfathomeostasisandhostdefence infections. Allmutantphenotypesdescribedwerereversibleuponconditionalexpressionof under starvationconditions.Furthermore,flieswithcompromised Greece. Biomedical SciencesResearch CenterAl.Fleming,34FlemingStreet, 16672Vari, Institute ofImmunologyand Drosophila alleles inactivated aremoresusceptible tochemicallyinduced expression isassociatedwithalteredfatbodymorphologyinadultflies,increasedtriglyceridelevelsandsurvival 3 Department ofBiochemistry, UniversityofOxford, SouthParksRoad, ␬ 4 B activation bymembersofthetumornecrosis Department ofBiology, AristotleUniversityofThessaloniki, CYLD CYLD 3, 2 Institute ofMolecularBiologyandGenetics, 1 * andGeorgeMosialos , PeerC.Gaentzsch CYLD (Biggs etal.,1996;Biggs1995). ) geneismutatedinhumantumorsofskinappendages.Itencodesadeubiquitylatingenzyme(CYLD) Drosophila CYLD ortholog ofthehumancylindromatosistumor Drosophila is atumorsuppressorgene. ␬ B andJNKsignalingpathways,invitro.However, thetissue-specificfunctionandregulation , Fatbody, Hostdefense,NF-kappaB CYLD (CYLD), likeitsmammaliancounterpart,isadeubiquitylatingenzyme.Impairmentof ␥ 3 , StylianosKosmidis – HumanGene in 1,4, Drosophila * CYLD ␬ B . Drosophila CYLD CYLD al., 2001).IntheTNFR1pathway, RIP1isessentialfor NF- mammalian TNFreceptorinteracting protein1(RIP1)(Georgel et IMD, acentralplayerofthe pathway andahomologofthe LC andPGRP-LE,thesignalistransducedfromcellsurface to the Naitza andLigoxygakis,2004).Following microbial recognitionvia pathway (Kaneko andSilverman, 2005; TNFRSF1A –HUGO) cascade broadlyresemblingthemammalianTNFR1(alsoknown as organisms, including orthologs of CYLD issignificantly conserved acrossevolution, becauseputative lymphocytes inmice(Costelloetal.,2005;Reiley etal.,2006). disease anditisrequiredfortheproperdevelopment ofT known asBG4–FlyBase)ordTAK1 (alsoknown asTAK1 – involves similarcomponents.Fliesdeficient in IMD,dFADD (also Stanger etal.,1995).In mitogen activated kinase(MAPK)activation (Kelliher etal.,1998; mediated innateimmunity. analyzed thepotentialinvolvement of Drosophila CYLD we soughttovalidate themodelbyinvestigating theinvolvement of (Bignell etal.,2000).Basedonprevious findings usinghumancells, CYLD andthetwo proteinshave asimilardomainorganization CYLD (FlyBasegeneID:CG5603)is45%homologoustohuman analysis andcontainsaputative orthologofCYLD. is well-characterized,offers enormousadvantages forgenetic model system. characterize asimpler, yetappropriatelyrepresentative, animal functional characterizationofCYLD,wesoughttoestablishand complexity ofthebiologicalrolesthisprotein.To facilitate the multiple tissuesareaffected whenthegeneismutatedhighlight complex andhighlyregulated signalingpathways, andthefact that elegans In The broadtissuedistribution ofCYLD,itsimplicationintwo Drosophila 2 expression exhibitedreducedresistancetobacterial Drosophila , AnthonyE.Brown (Bignell etal.,2000). CYLD Drosophila peptidoglycan recognitionproteins(PGRPs)PGRP- is broadlyexpressedduringdevelopmentand,in , IMD(forimmunedeficiency) defines asignaling in NF- Drosophila Drosophila melanogaster can bereadilyidentified inavariety of Drosophila CYLD ␬ was chosenforthisanalysis,becauseit B/IKK signaling.Inthiscontext, we 3 , EfthimiosM.Skoulakis transgenes. Ourresultsimplicate . RESEARCH ARTICLE , IMDtransducesasignalthat Drosophila CYLD and Caenorhabditis Drosophila nstrate in IMD- ␬ on of 2 B and 2605 ,

DEVELOPMENT MATERIALS ANDMETHODS fat homeostasis. functionally associatedwiththeIMDpathway andthatitregulates demonstrate that reversed uponrestorationof expression aresensitive tobacterial infections,aphenotypethatis expressed inembryosandadults.Flieswithimpaired Drosophila antimicrobial peptidegenes. 2003). Active Relishregulates directlythetranscriptionof of Relishalongwiththe DREDD, acaspase-typeprotease,isinvolved inthefullactivation family memberthatcanbeactivated byproteolyticcleavage. phosphorylates theprecursorformofRelishprotein,anNF- et al.,2001;Zhou2005).The which dependsonthemammalianproteins(Dengetal.,2000;Wang UBE2V1 –HUGO),similarlytotheactivation ofmammalianIKK, (also known asUBE2N–HUGO)andUEV1 theflyhomologsofubiquitin-conjugatingenzymesUBC13 on Silverman etal.,2000).Activation oftheIKKcomplex depends human NEMOprotein(Luetal.,2001;Rutschmann2000b; latter sharessignificant homologytotheregulatory subunit ofthe The formercodesfortheIKK (5 pCDNA3FLAGDMCYLD asthetemplateandprimersDMCYLDF3 by threePCRreactions.Thefirst PCRwas performedwith (Trompouki etal.,2003).pCDNA3FLAGDMCYLD(C284S) was generated frame withtheFLAG epitopetogeneratepCDNA3FLAGDMCYLD Not TTATATCTG-3 DMCYLDR4 (5 (5 melanogaster Full-length Plasmid construction contains two subunits identified bythe downstream ofdTAK1 intheactivation ofNF- (AMPs) (Leulieretal.,2002;NaitzaVidal etal.,2001). infections andfail toactivate therelevant antimicrobialpeptides FlyBase) areextremely sensitive toGram-negative bacterial 2606 Eco respectively. GST-DmCYLD(1-317) was generated bycloningintothe GST-DmCYLD(C284S), glutathione-S-transferase (GST)-DmCYLD or 3 (Amersham-PharmaciaBiotech)digested with YLD(C284S) bydigestionwith produced frompCDNA3FLAGDMCYLD orpCDNA3FLAGDM C same manneraspUAST.1DMCYLD. The full-lengthDMCYLDfragment - of theplasmidpUAST.1. pUAST.1DMCYLD(C284S) was generatedinthe with pUAST.1DMCYLD was generatedbydigestingpCDNA3FLAGDMCYLD pCDNA3FLADDMCYLD togeneratepCDNA3FLAGDMCYLD(C284S). Nde DMCYLDR6 (seeabove). TheresultingPCR product was digestedwith previous two PCRsastemplateandtheprimersDMCYLDF3 AGC TTTGC-3 CCAGCTACCTTGAT-3 the sametemplateandprimersDMCYLDF4(5 AAGGTAGCTG GAAT TATGG-3 c-MYC-tagged CTTGTGGGC-3 DMCYLDR2 (5 pCDNA3FLAGDMCYLD asatemplateandthe primersDMCYLDF1and Ј Ј In thisarticle,weshow that,like itsvertebrate counterpart, The -GGCTCGATAC CACCATTACAAA-3 -GCTCTAGAATGAATTCCAAATCCGATTATGAAGC-3 I, andwas clonedintothecorresponding sitesofpCDNA3FLAG in RI and I and Kpn RESEARCH ARTICLE Drosophila I and Hin Not D. melanogaster CYLD isadeubiquitylatingenzymethatbroadly Xba complementary (c)DNA usingtheprimers:DMCYLDF1 Drosophila Ј dIII andclonedintothe Ј I sitesofpGEX5X-3aPCRfragment generatedusing ). ThethirdPCRwas performedwiththeproductsof Ј ). TheresultingPCRproductwas digested with Ј -CTGACCTACGCCGGCGGCTCTAATGGTACATCA - Ј ) digestedwith I andsubcloningthefragmentintocorrespondingsites -GACTGGATGCGGC CGCCTGTAATTTCGAAT AT - I Drosophila ␬␤ Ј ) andDMCYLDR6(5 CYLD, the iae(K)cmlx(seebelow) acts kinase (IKK)complex Drosophila CYLD was amplified byPCRfrom Eco ␤ EcoR Ј CYLD homolog, whereastheproductof ). ThesecondPCRwas performedwith RI and CYLD isbiochemicallyand Drosophila I and IKK complex (Stoven etal., ird5 expression. Inaddition,we Not Ј ) andDMCYLDR5(5 Not Drosophila Nde I was clonedintopGEX5X- and Eco I. To generate6 and I YDR openreading CYLD-RC Ј RI and -CCGATGAGTGAA - kenny ␬ ␣ B. Thiscomplex Ј -CACCATA ATT - (also known as Hin IKK complex Not ( key dIII sitesof I togenerate Ј ϫ ) mutants. Eco ) and HIS- and Ј CYLD RI and -ATC - ␬ D. B A vector. eyrto ihBT[1 rehydration withBBT formaldehyde and58.8%heptane,rinsedwithmethanol.After Triton X-100,pH7.4)containing5%NGS. ATCGTTAACG-3 TCATTC-3 primers: AcKEY-F, 5 LD18356 ( ( as anantigen. following annealedphosphorylatedoligosinto pAc5.1/V5-HIS A(Invitrogen) withac-MYCepitopebyligatingthe MYC-HIS Awas generatedbyreplacingtheV5epitopeofplasmid CCCCTCGAGATGGTACATCATTA TATCTGTGC-3 AAATGATCTTAAACAACAA AAG TAAAAC-3 using thefollowing PCRprimers:AcCYLD-F:5 8 mMHEPESpH6.9,4MgSO 180 Embryos fromeachdevelopmental stageweredechorionated,fixed in Immunohistochemical analysisof Biotechnology usingaGSTfusionof The rabbitanti-CYLDantibodywas generatedbySantaCruz Generation ofanti-CYLDantibody frame (ORF)was clonedintothe mercaptoethanol) andboiledfor5minutes.Extractswere subjectedtoSDS- cracking buffer (10mMphosphatepH7.4,8Murea,1%SDS, b- expression ofGSTfusion proteins.Cells(1.5ml)werelysedin150 incubated for4hoursinthepresence of0.1mMIPTGtoinducethe of thetransformantswerediluted tenfold,grown for1hour andthen CYLD(541-956), GST-CYLD orGST-CYLD(C284S). Overnight cultures galactosidase fusionweretransformed withplasmidsexpressing GST, GST- MC1061 bacteria( Deubiquitylation assay for microscopy. PBS andmountedwithDAKO mountingmedium(DAKO Corporation) minutes atroomtemperature,andthenthey wererinsedthree timeswith were placedinNileredsolution(0.5mg/mlacetone) for 5 rinsed for10minutesatroomtemperatureinPBS.Subsequently, they sections. Thesectionswereplacedongelatin-covered glassslidesand in OCT(SakuraFinetekEurope)andwerecryosectioned8 in PBScontaining25%sucrose.Thefollowing day, theflieswereplaced for 10minutesatroomtemperatureandwereincubatedovernight at4°C containing 4%paraformaldehyde.Subsequently, they wererinsedinPBS Female fliesupto5daysoldwerefixed for5hoursat4°CinPBS Staining withNilered antibodies weredilutedinPBHT(20mMNaH were mountedwithDAKO mountingmedium(DAKO Corporation).All 5 2001; Skoulakis andDavis, 1996).Theprimaryantibodywas usedat processed forimmunohistochemistryasdescribedpreviously (Philipetal., Sagittal paraffin sections(8 Immunofluorescence analysis containing 2%NGS. Canada Balsam(Sigma).AllantibodiesweredilutedinBBT-250 ial,teebyswr isdwt B (1 Finally, theembryoswererinsedwithPBT secondary antibody(1:2000dilution)for2hoursatroomtemperature. overnight at4°C,rinsedthefollowing daywithBBT-250 andstainedwith 2 hours,stainedwiththeappropriateprimaryantibody(400ng/ml) 250 (BBT, 250mMNaCl)containing2%normalgoatserum(NGS)for 20, 1%bovine serumalbumin (BSA)],embryoswereblocked withBBT- GAAATAAGTTTTTGTTCTT-3 AGAAGATCTGA-3 pAc5.1/V5-HISA: cMyc-S,5 DAB solution(1 mlPBT, 1mgDAB, 0.1%H stained withABCreagent(Vectastain EliteHRPkit),developed with key ␮ g/ml andthesecondaryantibodyat1:2000.Afterwashing, thesections ), thefull-length Drosophila Ј and AcKEY-R, 5 Ј , andclonedinto Ј Escherichia coli key Ј and cMyc-AS,5 Genomics ResourceCenter)usingthefollowing -CCCGAATTCCAAAAT GTCGGACGAAGAG - ϫ ORF, was PCRamplified fromthecDNA clone phosphate-buffered saline(PBS),0.1%Tween- ␮ Ј m) ofentireadultflieswereobtainedand Ј -CGAAGAAC AAAAACTTATTTC TGA- -CCCCTCGAG GTTTTTATCCAAAC A- Ј Eco . V5and6 Eco RI- Drosophila Drosophila ) expressing aubiquitin-Met- RI- Xho Ј -CCG GTCAGATCTTCT TCA- Xho ϫ ieo A51cMCHSA I siteofpAc5.1/c-MYC-HIS 4 HIS C-terminal-tagged I sitesofthepAc5.1/V5-HIS and 2mMEGTA in4% 2 2 O PO ϫ Development 134(14) CYLD aminoacids1-317 2 PBS, 0.1%Tween-20), Ј ) andmountedinliquid embryos 4 , 500mMNaCl,0.2% and AcCYLD-R:5 Bst Ј -CCCGAATTCC A- Ј . PlasmidpAc5.1/c- BI– Age I-digested kenny ␮ ␮ l of ␤ m Ј - -

DEVELOPMENT ATCTCCTTG-3 0.1% SDS,50mM Tris, pH8.0)sixtimes,eachfor 10minutes,followed by buffer (900mMNaCl,1.0%Triton X-100,0.5%sodiumdeoxycholate, rocking atroomtemperature.Immunoprecipitates werewashed withCoIP c-MYC agaroseaffinity gel(Sigma-Aldrich)for1.5hourswithagitation GATCGTGAAGAAGCGCAC-3 according totheprotocolsoutlinedinHybridHunterhandbook(Invitrogen). 30°C anddiploidswereselected.Activation ofthe transformed intothe cDNAs intotheB42activation-domain vector pYESTrp2 (Invitrogen) and request). Eachpathway memberwas preparedbycloningtheirfull-length pathway (afulllistofgenesandPCR primersequencesisavailable upon as baitinamating-basedtwo-hybrid screen againstothermembersoftheIMD LYS::(4lex the manufacturer’s protocol.Briefly, 7.5 was performedusingtheEffectene transfectionreagent(Qiagen)accordingto CYLD regulates fatcontentandhostdefense tablets (RocheAppliedScience).Lysates were incubatedwith100 Aldrich), supplementedwithComplete MiniProteaseInhibitorCocktail 0630, 0.5%sodiumdeoxycholate,0.1% SDS,50mMTris, pH8.0)(Sigma- pAcKEY/V5 alone,in100 with 2 werepreparedfrom5mlculturesoftransientlytransfectedcells Cell lysates Co-immunoprecipitation blotanalysis andwestern ml Drosophila Drosophila S2 cellculture andtransfection A cDNA correspondingtothefull-lengthORFof Yeast two-hybridscreening protein was measuredusingtheBio-Rad D commercial kit(Biosis)accordingtomanufacturer’s instructions.Total for 3minutes.SupernatantswereanalyzedTAG contentusinga centrifugation at5000rpm(6150 Tween-20, incubatedat70°C for 5minutesandclearedtwiceby genotype, approximately36hoursold,werehomogenizedin100 For triglyceride(TAG)-content analysis,threegroupsofeightmalefliesper Triglyceride contentandstarvationassay TGGCTGGTAC-3 CGGAGGAACCTTTAC-3 analysis was performedusingtheprimersDMCYLDF7(5 synthesis kit(Fermentas),accordingtomanufacturer’s instructions.PCR manufacturer’s instructions.cDNA was preparedusingtheRevert AidcDNA corresponding bodypartsusingtheRNA-Wiz kit(Ambion),accordingto RNA was extracted fromeithereightmaleorfive femalefliesorfromthe Analysis of and westernblottingusinganti-GSTantibody. Expression ofGSTfusionproteinswas detectedwithSDS-PAGE (8.5%gel) PAGE (6%gel)andwesternblottingwitharabbitanti- cerevisiae The resultingplasmid,pHyb-CYLD,was transformedintothe cloned intotheLexA DNA-binding domainvector pHybLex/Zeo (Invitrogen). response in12-hourintervals (Gronke etal.,2003). rate was estimatedbycountingthenumber offliesunabletoexhibit asit-up starved infood-freevialswithunlimitedwater supplyat25°C.Thedeath groups of25malefliespergenotype,approximately36hoursold,were according tomanufacturer’s instructions.For thestarvation assay, three plates 24hoursbeforetransfection.DNA (2 with 1 Mating was performedbyoverlaying 1 abovementioned plasmidsandyeaststrainswas employed asapositive control. JRA –FlyBase)andFOS(alsoknown asKAY –FlyBase)usingthe 6lexAop-LEU2 post-transfection andincubatedat25°Cfor48hours. using a1:10Effectene ratio.Cellswerewashed oncein1 –1 penicillin G,and50 ␮ l ofEGY48onaYPAD plate.Plateswereincubated for1-2daysat ␮ g eachofpAcCYLD/c-MycandpAcKEY/V5or2 Aop- medium supplementedwith10%heat-inactivated FBS,50units strain L40[ S2 cells(Invitrogen) weremaintainedat25°CinSchneider’s Drosophila ; Invitrogen). An interactionbetweenJUN(bait;alsoknown as HIS3 Ј ) forloadingcontrol. Ј ); DMCYLDF3andDMCYLDR6;rp49F1(5 ) URA3::(8) S. cerevisiae ␮ g/ml streptomycinsulfate (Invitrogen). Transfection CYLD expression byRT-PCR ␮ MATa his3 Ј l RIPA buffer (150mMNaCl,1.0%IgepalCA- ) andDMCYLDR9(5 lexAop g Ј ) andrp49R2(5 ) for1minuteand13,000rpm(16,000 strain EGY48( ␮ ϫ ⌬ -lac l ofanovernight cultureofL40flies 10 200 trp1-901leu2-3112ade2 ␮ 5 Z) g) was usedforeachtransfection C cells ml Protein Assaykit(Bio-Rad) GAL4 lacZ Drosophila –1 reporter genewas tested MAT ] (Invitrogen) and used were platedin60mm Ј ␣ -CGCTCGACA - ϫ ␤ Ј ura3 trp1his3 PBS at8hours Saccharomyces -GCATCTGT - -gal antibody. Ј -TAGAGC - -TAGAGC CYLD was ␮ ␮ l ofanti- l 0.05% ␮ g of g Ј - ) The expression patternof been identified bygenomicapproaches(Fig.1A,www.ensembl.org). blotting was performedaspreviously described(Rutschmannetal.,2000a). Bacterialimmunechallengeandnorthern and werefoundnottobeinfected. analyzed byPCRforthepresenceofGram-negative bacteria at 37°Cfor1hourfollowed byrecovery at29°Cfor1hour. Allstrainswere containing strain(Georgel etal.,2001).To rescuethe These were made available totheflycommunityviaBloomingtonStockCentre. insertions fromtheinsertionalmutagenesisstocksgeneratedbyExelixis and phenotypes, strain (1:5000 dilution;Invitrogen). transferred toPVDFmembranes.Blotswereprobedwithanti-V5antibody type), The following strainsweremadeandusedinthisstudy: analysis Drosophila boiling in2 a final wash withRIPA buffer alone.Immunoprecipitateswereelutedby suppressor gene Drosophila adult stagesof CYLD isbroadly expressed duringembryonic and RESULTS primers usedinB(DCYF3andDCYR6) are shown.( have beendescribed,andare shownhere. ThepositionsoftheCYLD Drosophila melanogaster Fig. 1.Reversetranscriptase-PCRanalysisof Gram-positive bacterialinfections,respectively, andthe (Rutschmann etal.,2000a)aspositive controlsforGram-negative and Additional strainsusedwere of different PelementandpiggyBacinsertions(Thibaultetal.,2004). isogenic strainwas usedasabackgroundbyExelixis ingeneratingaseries used intheRT-PCR reaction (lowerpanel). known as body compartmentsofadult theindicated isolated attheindicateddevelopmental stagesandin primers DMCYLDF3(DCYF3)and DMCYLDR6 (DCYR6),andRNAswere expression wasdetectedby reverse usingthe transcriptase(RT)-PCR used a Df(2L)j27/CyO flieswereobtainedfromtheBloomingtonStockCenter. We UAS white RpL32 - CYLD/+; CYLD isogenic strainasawild-typecontrolinallexperiments. A ϫ stocks, infection experiments and northern blot stocks, infectionexperimentsandnorthern contains aputative orthologofthemammaliantumor SDS samplebuffer, separatedby13%SDSPAGE, and f00814 RNAwasusedasa control fortheamountof cDNA ) UAS-CYLD/+; CYLD CYLD Drosophila ( . Five transcriptsof CYLD f00814 . ( w1118 Drosophila CYLD A relish f00814 ) Fivetranscriptsof ;hs-Gal4/MKRS ), flies (upperpanel). (Hedengren etal.,1999)and f00814 development RESEARCH ARTICLE f02494 ;hs-Gal4/MKRS , Drosophila CYLD (CYLD rescue)andfour CYLD d10472 was investigated by Drosophila CYLD B ) CYLD CYLD yolkGAL4 UAS-CYLD expression in RP49 was incubated and f00814 ( DmCYLD Wolbachia (also f00135 -driver- mutant 2607 white (wild have dif ) .

DEVELOPMENT nerve cord was notablystrongerthan thatofearlierstages,while the embryonic stage14,accumulation ofCYLDinthebrainandventral relatively abundant inthebrainandventral nerve cord(Fig.2B).By 12, CYLDcontinuedtoaccumulate inthedeveloping midgutandwas of theanteriorandposteriormidgut (Fig.2A).Duringembryonicstage protein was detectedmainly inmesodermanddeveloping segments throughout stage1-5embryos(data notshown) but, bystage7,the reactivity totheantibody(Fig.2D). D1 chromosomalareainwhich homozygous fortheDf(2L)J21deficiency, whichdeletesthe31C7- antibody to developed (seeMaterialsandmethods). Thespecificity ofthis pattern was theninvestigated usingananti-CYLDantibodythatwe three majoradultbodysegments (Fig.1B).Theproteindistribution found inalldevelopmental stages(embryo, larva andpupa)inall reverse transcriptase(RT)-PCR, and posterior midgutrudiment;vnc,ventralnervecord. rudiment; br, brain;hg,hindgut;mg,midgut;ms,mesoderm;pmg, (H), intheantenna(I)andeye(J).amg,anteriormidgut (8 polyclonal anti-CYLDantibodyonadult settings asinE,endogenousCYLDlevelswere detectedwitharabbit antibody andthesamesecondaryusedinF-J.Using using anon-specificrabbitpolyclonalantibody(anti-GFP)asprimary CYLD inadulttissuesbyimmunofluorescence. (E)Negativecontrol locus, didnotstainwiththeanti-CYLDantibody. ( homozygous fortheDf(2L)J21deficiency, whichdeletesthe cord (vnc),themidgut(mg)andhindgut(hg).(D)Stage12embryos Atstage14,CYLDisdetectedatthebrain(br),ventralnerve (C) forming midgut(amgandpmg)intheventralnervecord (vnc). stage 12,CYLDislocalizedinthedevelopingbrainarea (br),inthe the posteriormidgutrudiment(pmg)andinmesoderm(ms).(B)At stage 7,CYLDisexpressed intheanteriormidgutrudiment(amg), ( Fig. 2.DistributionofCYLDin 2608 A-D ␮ m). CYLDwasdetectedinovaries(F),testes(G),thefatbody ) DetectionofCYLDinembryosbyimmunohistochemistry. (A)At RESEARCH ARTICLE Drosophila CYLD was confirmed becauseembryos Drosophila CYLD Drosophila CYLD w1118 resides, didnotshow any -specific transcriptswere embryos andadults. sagittal paraffin sections E-J CYLD was present ) Detectionof CYLD antibody. lysates ofanequalnumberbacteriawithpolyclonalanti-GST expressed atsimilarlevels,asdetectedbyimmunoblottingofwhole-cell ( bacteria were analyzedbyimmunoblottingwithapolyclonalanti- proteins, wholeextractsfrom anapproximately equalnumberof galactosidase antibody. ThepositionsofUb- Fig. 3. transformed withtheindicatedexpression constructsforg expressing aubiquitin- terminal region of protease domainofhumanCYLDandthecorresponding C- The highdegree ofsimilaritybetweentheubiquitin-specific Drosophila the antibody(datanotshown). eliminated withtheadditionofexcess antigenthatwas usedtoraise adult tissuestotheanti-CYLDantibodycouldbealmostcompletely of thebrain,suchasopticlobe(datanotshown). Thereactivity of (Fig. 2J),whereastheproteinwas weaklydetectableinvarious areas accumulated stronglyintheproboscis,antenna(Fig.2I)andeye not shown) andinthefat body(Fig.2H). Inaddition,CYLD protein accumulationwas detectedinintestinalepithelialcells(data the developing oocyte (Fig.2F),and inthetestes(Fig.2G).Weaker expression was detectedinthecytoplasm ofnurseandfolliclecellsin nervous system. or functionofthemidgutandmesodermalderivatives, aswellinthe 2C). Together, thesedatasuggestaroleforCYLDinthedevelopment protein remainedabundant inthemidgutandposteriorintestine(Fig. transferase DmCYLD(C284S). ( catalytic domain,was unable tocleave ubiquitinfromtheUb- containing aserineinsteadof conserved cysteine intheputative 1 and3).Bycontrast,amutated formof bacteria expressing aubiquitin- enzyme. For thispurpose, into theabilityof CYLD was alsocapableofcleaving theUb- fusion (Fig.3,comparelanes 1and4).Asexpected, human cleaved ubiquitin fromtheUb- fusion proteinwas tested. Wild-type and itsabilitytocleave theubiquitinmoietyfromUb- ␤ -gal) are indicated.( nautfis CYLDwas detectedinseveral tissues.Strong In adultflies, Drosophila (GST): GST-HCYLD(538-956), GST-DmCYLD orGST- CYLD hasdeubiquitylatingactivity A (Dm)CYLD isadeubiquitylatingenzyme. Drosophila ) AfterinductionoftheGST-domain-containing Drosophila B ␤ ) TheGST-domain-containing proteins were -galactosidase (Ub- Drosophila CYLD toactasadeubiquitylating ␤ CYLD promptedaninvestigation ␤ gal fusion(Fig.3,comparelanes -galactosidase (Ub- CYLD was introducedinto Drosophila ␤ ␤ gal) fusionwere gal and Development 134(14) ␤ Drosophila gal fusion(Fig. 3, ␤ -galactosidase CYLD readily ␤ lutathione-S- gal) fusion, CYLD, E. coli ␤ ␤ ␤ - gal gal

DEVELOPMENT RT-PCR reaction (lowerpanel). RpL32 were compared withwild-typeflies(w1118). analyzed byreverse for transcriptase(RT)-PCR element-insertion lines transcripts of (DCYF7) andDMCYLDR9(DCYR9)]are shownrelative tothefive were used[DMCYLDF3(DCYF3),DMCYLDR6(DCYR6),DMCYLDF7 allele ofthegene,henceforthcalled (data notshown). Therefore, thisstrainrepresentsabonafide mutant embryos withanti-CYLDantibodyfailed todetectCYLD protein the RT-PCR analysis,stainingof In ordertostudythebiologicalroleofCYLDin fat bodymorphology Defective expression of integrity ofitsconserved C-terminalcatalyticdomain. has autonomousdeubiquitylatingactivity thatdependsonthe lane 2).Therefore,like itsvertebrate ortholog, CYLD regulates fatcontentandhostdefense it isfunctionally redundantwithanothergene inthegenome. function iseitherdispensablefor properembryonicdevelopment, or morphological anomalies.This indicatesthat surprisingly viableanddid notexhibit obvious external ( Fig. 4.MolecularcharacterizationofP-elementinsertionlines. distribution indeveloping embryos, mRNAs inthe disrupt ordisruptedincompletelytheexpression of insertions inintron2of possible transcriptsfromthegene(Fig.4B).Bycontrast, the strong chosen forfurtheranalysisbecausethisallelecanbeconsidered a which containsaP-elementinsertioninthefirst intron (Fig.4A),was transposons wereverified byPCR(datanotshown). Strain obtained fromtheExelixis collection.Thegenomiclocationsofall insertionsinthe P-element strains containing A The positionsofP-elementinsertionsanalyzedandtheprimersthat ) ) RNAwasusedasacontrol fortheamountofcDNAusedin CYLD Drosophila CYLD f00135 hypomorph lackingdetectableexpression ofall f00135 , f02494 Drosophila CYLD , that havebeenidentified.( f00814 and Drosophila CYLD d10472 CYLD , CYLD f00814 f02492 rp49 f00814 CYLD f00814 strains. Consistentwith either didnotappearto and homozygous-mutant (also knownas Drosophila homozygotes were Given theprotein . Drosophila CYLD CYLD mRNA. Theresults d10472 CYLD Drosophila B alters ) TheP- locus were -associated were f00814 CYLD , four , lipid droplets incryosectionsfrom adultwild-typeflies(G)or ( hematoxylin andEosinstainingofparaffin sections(8 CYLD However, histologicalanalysisrevealed thatthemajorityof eoyeW % leain()alteration (%) alteration(%) w1118 In2/30(6.67%) severely altered fatbodyisshown(seealsoResultsandFig.5). of thefatbody. Thepercentage offlieswithnormal(WT),moderatelyaltered or WT(%) A totalof30adultfemaleflieseachgenotype were analyzedfor themorphology CYLD w1118 Genotype mutant flies Table 1.Fatbodymorphologyinwild-typeand homozygotes (H). CYLD morphology. Fig. 5.Impaired and CYLD-rescue (F)flies.( staining showed thatfat bodycellsin 5D), in othertissuesandorgans [compare theoocyte fromwild-type(Fig. absence ofCYLDdidnotprecipitateapparentmorphologicaldefects as theCYLD-rescuestrainfromthispointon)(Fig.5C,Table 1).The the controlofheat-shockpromoterin was reversed uponinducibleexpression ofa (Fig.5B,Table 1).Thisphenotype abnormal sizeandmorphology background ( YDrsu 00 00 – 10.00 90.00 CYLD rescue retained adegree offunctionalitydespitetheirmorphological were abletoaccumulatelipids.Thisinturnindicatedthat they D-F Oocyte morphologyinwild-type(D), ) f00814 f00814 f00814 le,themorphologyoffatbodycould notbedetermined. flies, f00814 homozygotes (B)andCYLD-rescue (C)flieswere analyzedby adult homozygotesexhibited fat bodycellsofan (Fig. 5E)andCYLD-rescue5F)flies].Nilered UAS-CYLD/+;CYLD ( A-C Drosophila CYLD ) Adultfemaleabdomensfrom wild-type(A), 33 – – 93.33 66 66 46.67 26.67 26.66 G , H ) Nilered stainingforthepresence of Fat bodymorphology expression altersfatbody f00814 RESEARCH ARTICLE CYLD oeaeSevere Moderate ;hs-Gal4/MKRS CYLD f00814 CYLD f00814 CYLD adult homozygotes homozygotes (E) ␮ CYLD transgene under m). f00814 , referredto f00814 CYLD mutant 2609 f00814

DEVELOPMENT shown. Representative datafrom atleastthree independent experimentsare type strainw1118(3.08±0.20lipiddropletscm lipid droplets(5.77±0.26cm CYLD alterations (Fig.5G,H).Ameasurementoflipiddropletsrevealed that ( values (±s.d.)from atleastthree independentexperimentsare shown. were notsubjectedtoheat-shock[DmCYLDrescue(NO HS)].Mean shock inductionof ( triglyceride contentandincreased survivalunderstarvation. Fig. 6.Impaired 2610 analyzed: wildtype(WT) 2-3-day-old malefliesafterhatching.Thefollowingstrainswere TAGs wereincreasedby79.8%in level of Total TAG contentwas foundtobeinversely proportional tothe the characteristicmorphologyof thefat body(Gronke etal.,2005). andthereforecontributing to cytoplasm andnucleus to oneside, usually occupy alarge portionofthecell,thusrestricting the phospholipid layerthatsurroundstheTAGs. They areopaqueand structures calledlipiddroplets.Theseconsistofasingle primarily intheformoftriglycerides(TAGs) incharacteristic fat bodyisthemajorsiteoffat storagein into thefat contentofflieswithdefective The alteredmorphologyofthefat bodypromptedaninvestigation content associated withincreased totaltriglyceride Defective expression of compared with controlanimals(Fig.6A).However, UAS-CYLD/Y;CYLD B A A B 100 ) Survivalunderstarvationconditionsof2-3-day-oldmaleflies. % survival µg TAG / mg protein Triglyceride (TAG) content(expressed as ) 10 20 30 40 50 60 70 80 90 100 200 300 400 500 600 700 800 0 0 12243648607284961081201321440 f00814 RESEARCH ARTICLE Drosophila CYLD T(11)f01 mYDrsu DmCYLD rescue DmCYLD f00814 (w1118) WT DmCYLD rescue f00814 WT (w1118) adult homozygoteshadsignificantly morebut smaller Drosophila CYLD f00814 UAS-CYLD ;hs-Gal4/MKRS w1118 transcription andCYLD-rescue fliesthat expression. Morespecifically, total * , CYLD Drosophila CYLD ie(hr) time expression results inelevated CYLD f00814 (DmCYLD rescue) afterheat- ␮ homozygotes (f00814), Drosophila f00814 –2 g TAG per CYLD ) comparedtothewild- –2 adult homozygotes ). expression. The ␮ . Fat isstored g protein) of eceN HS) rescue(NO is CYLD f00814 morphology ofthefat bodyin homeostasis, afunctionthatismostlikely relatedtothe altered lysates of blotofanti-c-Myc-immunoprecipitatedV5 western materialfrom Drosophila DREDD, IRD5andKenny).CYLDinteractsspecificallywithKenny, the the IMDpathway(PGRP-LC,PGRP-LE,PGRP-LA,IMD,dTAK1, dFADD, with freeaccesstowater. 36hoursandwerethen placedinvialswithoutfoodbut for upto this purpose,newly emerged adultswereallowed tofeednormally survive forlongerperiodsoftimeunderstarvation conditions.For proteins indicatedonthetopofpanel. ( Fig. 7. hc didnotsignificantly altertheresistance tostarvation ofcontrol shock homozygotes remainedalive attheendofthistimeperiod.Heat- data suggestthat homozygotes [Fig.6A,CYLDrescue(NOHS)].Therefore, our expression of the for 6A). Significantly, intheabsenceofheat-shockrequired contained almostwild-typelevels ofTAGs (CYLDrescue,Fig. homozygotes thatexpressed transgenic that was notsignificantly different fromthatof expression suggestedthat The increasedlevels ofTAGs inflieswithimpaired starvation associated withincreased resistance tonutrient Defective expression of dead after4days,whereas more than40%of were controls (Fig.6B).Controlanimals andCYLD-rescueflies significantly longerwithout asourceofnutrientsincomparisonwith CYLD/Y;CYLD A ) Quantitativeyeasttwo-hybridassaywithallofthecomponents Drosophila Drosophila ortholog ofthemammalianprotein NEMO(IKK f00814 YD(mYD neat ihKny(dIKK CYLD (DmCYLD)interactswithKenny S2 cellstransfectedwithplasmidsexpressing the ;hs-Gal4/MKRS Drosophila CYLD Drosophila CYLD CYLD CYLD f00814 rspiaCYLD Drosophila CYLD isrequiredforTAG f00814 flies possessedaTAG content homozygotes mightbeableto f00814 homozygotes survived homozygotes. Development 134(14) Drosophila CYLD transgenes, ␥ is CYLD CYLD ). ( B ) Anti- CYLD UAS- f00814 f00814 ␥ ).

DEVELOPMENT a ratioof type w1118flies(w)andin CYLD is themostlikely causeoftheobserved resistancetostarvation of levels ofCYLDresultinanelevation inthelevels ofTAGs, which animals (datanotshown). Collectively, thedatasuggestthatreduced CYLD regulates fatcontentandhostdefense (±s.d.) from three independentexperimentsare shown. (DmCYLD/yolk) before andafter type w1118(w)andfliesoverexpressing expression (expressed asaratioof determined before andafterinfectionwith Drosophila CYLD Fig. 8.Antimicrobial peptideexpression inflieswithimpaired above, andtheknown principalroleofthisorgan in Because ofthedeficits infat bodystructureand functiondescribed infections associated withsusceptibilitytobacterial Defective expression of 2005). Inhuman cells,CYLDinteractswithNEMO andregulates homolog ofmammalianTNFR signaling(Kaneko andSilverman, Gram-negative sepsisdependsontheIMDpathway, the bacterial responsesarecontrolled bytheToll signaling cascade, Ligoxygakis, 2004).Whereasanti-fungal andanti-Gram-positive depends largely onthetypeofinvading pathogen(Naitzaand these responseshave broadspecificity, theirpatternofexpression synthesis andsecretionbythefat bodyofpotentAMPs.Although 2006; Trompouki etal.,2003). (Brummelkamp etal.,2003;Kovalenko etal.,2003;Reiley etal., ␬ consistent withthegiven connectionofmammalian CYLDwithNF- altered susceptibilitytoinfections.Furthermore,thiswould be defence, wewanted todeterminewhetherlossof A B B/IKK signalingandwiththefunctionofimmunesystem Dipt/RP49 normalised The hallmarkofthe

Dipt/RP49 normalised 2 0. 4 0. 6 0. 8 0. 2 1. 4 1. 1. 8 1. 6 0 1 .2 0. 4 0. 6 0. 8 0. 1. f00814 0 1 2 Diptericin homozygotes. un w wf00 in itrcnepeso hatrinfection 6hafter expression Diptericin fected unin expression. : rp49 mYDoverexpression DmCYLD fe cted Drosophila

(also knownas DmCYLD/yolk CYLD 81 ( E. coli A 4w f00814 Drosophila CYLD Diptericin ) w Diptericin systemic immuneresponseisthe E.coli homozygotes (f00814)was infection. (A,B)Meanvalues CYLD RpL32 E. coli : rp49

expression [expressed as DmCYLD/yolk in thefatbody ) mRNAlevels]inwild- . ( mRNA levels)inwild- B Drosophila CYLD ) E. coli Diptericin Drosophila is Drosophila f00814 host type; relish/Dif, positivecontrols; f00814: independent experimentsare shownineachpanel.WT, w1118wild was monitored for7days.Representative datafrom oneoutoffour negative ( bacteria. susceptible toinfectionbyGram-negativeandGram-positive regulate theToll pathway. (data notshown), suggestingthat without infectionorfollowing Gram-positive bacterialchallenge expression was detected betweenmutantandwild-typeflies the expression oftheAMPgenes Toll andIMDsignaling withandwithoutinfection,bymeasuring investigated theeffect of impaired Next, we immunoprecipitation inSchneider-2 (S2)cells(Fig. 7B). interaction betweenCYLDandKENNYwas confirmed viaco- other proteinsimplicatedintheIMDpathway (Fig.7A). The counterpart ofthemammalianNEMO(IKK CYLD was abletointeractwithKENNY, whichisthe DREDD, dTAK1, IRD5andKENNY).We foundthat pathway in Drosophila quantitative yeasttwo-hybrid assaytoinvestigate theabilityof We useda Kovalenko etal.,2003;Trompouki etal.,2003). TNFR-associatedpathways (Brummelkampetal.,2003; of the activation ofNF- Fig. 9.A Escherichia coli Drosomycin respectively. Inuninfectedflies, CYLD whereas nosignificant difference was observed between homozygotes was significantly higherthanin wild-typeflies, B % flies surviving % flies surviving A 100 120 100 120 20 40 60 80 f00814 20 40 60 80 0 0 Drosophila E. coli ( A a a a a a a a Day7 Day6 Day5 Day4 Day3 Day2 Day1 Day 0 a a Day2 Day1 Day 0 , homozygotes andwild-typeflies uponinfectionwith B CYLD tointeractwithknown membersoftheIMD Drosophila A totalof25flieswere infectedwitheitherGram- ) ( ; A)orGram-positive( Drs ) asread-outsforIMDandToll signaling, (Fig. 8A).Nosignificant difference in strain withimpaired ␬ B bydeubiquitylatingsignalingmolecules E. faecalis (PGRP-LC, PGRP-LE,IMD,dFADD, E. coli a Day4 Day 3 Time Time Drosophila CYLD E. faecalis RESEARCH ARTICLE Diptericin Drosophila Dipt CYLD CYLD a a Day7 Day6 Day 5 expression inmutant f00814 ␥ ; B)bacteria.Survival ) protein,but notwith expression is ( homozygotes. Dipt CYLD doesnot WT DmCYLD rescue Dif WT f00814 relish f00814 DmCYLD rescue expression on ; Drosophila Drosophila DptB ) and 2611 Drs

DEVELOPMENT Interestingly, infection, thesystemisoverloaded and likely reasonforthisisthat,whensepticinjuryusedas a meansof were elevated incomparisonwithwild-type controls.Themost significantly different fromwild-typeflies,whereasbasallevels mutants, IMD signalinginuninfectedandinfectedflies.Innull effect. Asexpected, overexpressing flies, whereasagain-of-functionconditionshouldhave theopposite negative bacteria, below thatofwildtypeininfectedflies. results suggestthat counterpart (theTNFRpathway), isactivated byubiquitylation.Our expression suggeststhattheIMDpathway, like itsmammalian Drosophila signaling in deubiquitylating enzymeimplicatedintheregulation ofNF- eliminated, any furtherincreaseisnotpossible. maximum, suchthateven whenaninhibitor ofIMDsignalingis of the (Bier, 2005). generated, whichmake themidealforsophisticatedgeneticscreens rapid lifecycle andthelarge numbersofindividuals thatcanbe systems. Flieshave several experimental advantages, includingtheir genes requiredforthefunctionofvisual,auditoryandimmune cancer, cardiovascular disease,metabolicandstoragediseases, those responsiblefordevelopmental andneurologicaldisorders, tools foranalyzingthefunctionofhumandiseasegenes,including Drosophila melanogaster DISCUSSION crucial roleofCYLDinanti-bacterialresponses 9, CYLD-rescuecurves). Taken together, theseresultsidentifya the abilityofthesefliestorespondbothkindschallenges(Fig. CYLD expression would beupregulated in signaling, itcanbehypothesizedthatIMD-mediatedAMPgene human counterpart,whichisanegative regulator ofTNF/NF- severe susceptibilitytobacterialinfection.Byinferencefromits CYLD-rescued flies.Parallel experiments withthe Enterococcus faecalis bacterium showed reducedsurvival afterinfectionbytheGram-negative of survival comparedwithwildtype(Fig.9).Fliesfromthisstrain expression was measured(Fig.8B). crossed tothefat body-specific condition for 2612 knock-out effect ofitsgenehas revealed a crucialfunctionin Mouse GenomeInformatics), has beendescribedinmice.The regulating deubiquitylating enzyme,A20(alsoknown asTnfaip3 – Kovalenko etal.,2003;Trompouki etal.,2003).AnadditionalIKK- NEMO-CYLD interactioninmammals (Brummelkampetal.,2003; switch tode-activate thepathway, ashasbeenproposedforthe activation. Itsinteractionwith of heat-killed bacteriawereused(datanotshown). Notably, restoration comparable results(datanotshown). Thiswas notthecasewhen over adeficiency uncovering the Interestingly, following infectionwithGram-positive orGram- To ourknowledge, thisarticleisthefirst todescribe a In thepresentstudy, wecharacterizedtheexpression andfunction In addition,wealsostudiedtheeffects ofagain-of-function Drosophila CYLD . Flieswithimpaired Drosophila RESEARCH ARTICLE Dipt E. coli Drosophila CYLD leadstoaconcomitantincreasein Drosophila CYLD Dipt expression was twiceashighinwild-typeflies. CYLD ortholog ofthehumantumorsuppressorgene (Fig. 9A)orbytheGram-positive bacterium levels ininfected expression inCYLD-rescuefliesalsorestored Drosophila . Furthermore,thediscovery thatthelossof f00814 (Fig. 9B),comparedwithwild-typeor is emerging asoneofthemosteffective Drosophila CYLD homozygotes exhibited areducedrate Drosophila Drosophila CYLD whereby , yolk- IKK Dipt CYLD Drosophila GAL4 driver and ␥ Dipt induction was significantly CYLD is alikely target forthis CYLD couldserve asa loss-of-function mutant UAS-CYLD expression reachesits expression showed mutants werenot CYLD suppressed CYLD Drosophila locus produced f00814 flies were Dipt allele gene Dipt . ␬ ␬ B B in function mightbetheprincipaldeterminantofimmunedeficiency deficiency andstrengthensourhypothesisthat fat bodystructureand indicates thathighlevels ofTAGs arenotsufficient tocauseimmune positive orGram-negative bacteria(datanotshown). Thisfinding a survival typicalofwild-typeflieswhenchallengedwithGram- counterparts (Gronke etal.,2005).Nevertheless, have 100%moreTAGs intheirbloodthanwild-type ( would alsobesensitive toinfection.Fliesmutantfor Following thesamelineofthinking,mutantswithhighbloodTAGs phagocytic activity, bacteriacouldproliferatefreelyandkillthehost. could presumablyaccumulateintheblood).Inabsenceof overloaded withthefunctionofinternalizingexcess lipids(that 2003), arenotabletoengageinphagocytosis because they are hemocytes, whicharemacrophage-like cells(MeisterandLagueux, remains tobeestablished.Apossiblehypothesiscouldthatfly Whether higherfat levels couldpotentiallybedetrimentalaswell, CYLD inthedevelopment ofTlymphocytes (Reiley etal.,2006). Interestingly, arecentreporthasidentified acrucialroleformurine be expected toinfluencesurvival toimmunechallenges. Drosophila of RIP1(Booneetal.,2004;Evans etal.,2004;Wertz etal.,2004). polyubiquitin chains,anditpromotesK48-linked polyubiquitylation that itis,atthesametime,adeubiquitylaseofK63-linked responses (Leeetal.,2000).More-recentexperiments have revealed Again, redundancy withproteinsperforming similarfunctionsin physiological propertiesattached toit(fat storage,hostdefence). effect weobserved was restrictedtothefat bodyand the not appeartoaffect embryonicdevelopment, but rather themajor and adulttissues.However, acompromise of during physiology. Thefat body isthemajorimmuno-responsive organ be offered bythedramaticchangeinfat bodymorphologyand observed alterationin signaling. of thenon-degradative functionofpolyubiquitinchainsinNF- deubiquitylating enzymesintheflyshouldstrengthenconcept phenotype (A.E.B.andP.L., unpublished).Systematicscreeningfor the 5 limiting inflammationbyterminatingTNF activation bycleaving Ub that itfunctionsasageneralnegative regulator ofIKKandJNK CYLD proteinsinhostdefence. a clearfunctionalrelationshipbetweenmammalianand (Reiley etal.,2006)togetherwiththeresultsofthisstudyestablish broad patternof with otherubiquitinhydrolases. Similarly, ouranalysisshowed a and stimulatoryconditions,whichpointstoafunctionalredundancy conditions, theroleofCYLDmayberestrictedtospecific celltypes (Schmidt-Ullrich etal.,2001).Therefore,underphysiological development ofepidermalappendices by activating NF- TNFR family memberectodysplasinAreceptorcontrols the display aphenotypethatisrestrictedtoepidermalbodyregions. The the distinctsensitivity ofthe for thedeathof cellular activities (Boutrosetal.,2002).Anintriguing explanation is amoregeneralphenomenoninvolving several pathways and the humoralaspectofresponse,overall survival toovert infection Although measuringAMPgeneexpression isagoodindicationof bmm De-regulation oftheIMDpathway, asexemplified bythe Even thoughtheanalysisofCYLDactivity incelllinessuggests CYLD Ј ), aTAG lipasehomologoustohumanadipocyte TAG lipase, Drosophila untranslated region (UTR)ofthisgenehave animmune f00814 contains anorthologof homozygotes. Collectively, findings byReiley etal. CYLD Drosophila CYLD systemic defenceandsignificant changeswould Dipt f00814 regulation, cannotaccount,however, for homozygotes following infectioncould Lys63 Drosophila CYLD chains, cylindromatosis patients A20 expression inbothembryonic , andP-elementinsertionsin Development 134(14) CYLD mutants toinfection. ␣ bmm -induced NF- expression did flies showed Drosophila brummer ␬ ␬ ␬ B B B

DEVELOPMENT CYLD observed inCYLD-deficient flies,mightexplain theresistanceof al., 2003),andthis,inconjunctionwithincreasedTAG levels the JNKpathway contributes tooxidative stressresistance(Wang et (Reiley etal.,2004;Wang etal.,2005).In shown tobenegatively regulated byCYLDinmammaliancells FOXO isactivated bytheJNKpathway, whichitselfwas recently signaling (GoberdhanandWilson, 2003).Ithasbeenshown that target isthetranscriptionfactor FOXO, whichisactivated byINR signaling downstream ofreceptoractivation. Onesuchpossible This finding suggeststhat is notaffected bythedecreaseinCYLDlevels (datanotshown). CYLD have foundthattheexpression of (ILPs)] homologoustohumaninsulin(Brogioloetal.,2001).We INR. INRisactivated byneuropeptides[insulin-relatedpeptides that 1999; Clancy etal.,2001;Tatar etal.,2001).Therefore,itispossible insulin receptor(INR)orChico,anINRsubstrate(Bohnietal., Increased TAG contenthasbeenobserved inflieswithmutated Bignell, G.R.,Warren, W., Seal,S.,Takahashi, M.,Rapley, E.,Barfoot,R., Biggs, P. J.,Chapman,P., J.andStratton,M.R. Lakhani,S.R.,Burn, Biggs, P. J.,Wooster, R.,Ford, D.,Chapman,P., Mangion,J.,Quirk,Y., Bier, E. References Lymphoma Society ofAmerica. Medical Research CouncilUK(toP.L.). G.M.isaScholaroftheLeukemiaand CT-2005-018704) (toG.M.)andbyaCareer EstablishmentGrantfrom the Research FrameworkProgramme oftheEuropean Union,Project INCA(LSHC- from theHoward HughesMedicalInstituteandbyfunding undertheSixth anti-CYLD antibody. scholarship Thisworkwassupportedbyaninternational Tronick (SantaCruzBiotechnology)forassistancewiththegenerationof Oxford) fortheuseofFuji-filmFLA3000phosphorimager;andSteve providing themutantstrainsusedinthisstudy;D.Sherratt(Universityof generation oftransgenicflies;thecuratorsExelixiscollectionfor Technology, Heraklion,Greece) foradviceandtechnicalassistancewiththe Molecular BiologyandBiotechnology, FoundationforResearch and The authorswouldliketothankI.LivadarasandC.Savakis(Instituteof where Nevertheless, possiblefunctionaldeficits inthenervous system, other tissuescouldbeenvisaged (ChenandFischer, 2000). CYLD regulates fatcontentandhostdefense and/or metabolismin starvation. ThisresultsuggeststhatCYLDisinvolved infat storage associated withincreasedTAG contentandresistancetonutrient demonstrated thatimpaired in thecontext ofunderstandingdisease. establishing furtherthevaluable cross-talkbetweenfliesandhumans counterpart, isalsoinvolved intheregulation oftheJNKpathway, required todeterminewhether Borodovsky, A.,Ovaa,H.,Kolli, N., Gan-Erdene, T., Wilkinson,K.D.,Ploegh, Bohni, R.,Riesgo-Escovar, J.,Oldham,S.,Brogiolo, W., Stocker, H.,Andruss, Boone, D.L.,Turer, E.E.,Lee,G.,Ahmad, R.C.,Wheeler, M.T., Tsui, C., of thefamilialcylindromatosis tumour-suppressor gene. Green, H.,Brown, C.,Biggs,P. J.,Lakhani, S.R.etal. 1375-1377. suppressor geneinvolvedin the developmentofcylindromas. The cylindromatosis gene(cyld1) onchromosome 16qmaybetheonlytumour for itsrole asatumoursuppressor gene. (turban tumoursyndrome) gene localisedtochromosome 16q12-q13:evidence Easton, D.F., J.andStratton,M.R. Burn, Nat. Rev. Genet. organ sizebyCHICO,aDrosophila homologofvertebrateIRS1-4. B. F., Beckingham,K.andHafen,E. 165. responses. modifying enzymeA20isrequired forterminationofToll-like receptor Hurley, P., Chien, M.,Chai,S.,Hitotsumatsu,O.etal. 875. Drosophila f00814 f00814 (2005). Drosophila, thegolden bug,emergesasatoolforhumangenetics. Drosophila Nat. Immunol. animals, but willbethefocusoffutureexperiments. We homozygotes tonutrientstarvation. Furtheranalysisis 6 CYLD isrequiredforpropersignaltransductionby , 9-23. CYLD accumulates,werenotinvestigated in 5 , 1052-1060. Drosophila Drosophila Drosophila CYLD Drosophila CYLD (1999). Autonomouscontrol ofcelland Drosophila ILP2 Nat. Genet. , andpossiblyinmammals. (1995). Familialcylindromatosis CYLD mightregulate INR Drosophila 11 , 441-443. Nat. Genet. (2000). Identification (2004). 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