Beyond the Binding Site In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development.docVIP

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Beyond the Binding Site In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development.doc

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Beyond the Binding Site In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

BeyondtheBindingSite:InVivoIdentificationoftbx2, smarca5andwnt5basMolecularTargetsofCNBPduring EmbryonicDevelopment PabloArmas1.,EzequielMargarit1.,ValeriaS.Mouguelar1,MiguelL.Allende2,NoraB.Calcaterra1* 1InstitutodeBiolog?′aMolecularyCelulardeRosario(IBR),ConsejoNacionaldeInvestigacionesCient?′ficasyTe′cnicas(CONICET)-FacultaddeCienciasBioqu?′micasy Farmace′uticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, (S2000FHQ) Rosario, Argentina, 2FONDAP Center for Genome Regulation, Facultad de Ciencias,UniversidaddeChile,Santiago,Chile Abstract CNBPisanucleicacidchaperoneimplicatedinvertebratecraniofacialdevelopment,aswellasinmyotonicdystrophytype2 (DM2)andsporadicinclusionbodymyositis(sIBM)humanmusclediseases.CNBPishighlyconservedamongvertebrates andhasbeenimplicatedintranscriptionalregulation;however,itsDNAbindingsitesandmoleculartargetsremainelusive. ThemaingoalofthisworkwastoidentifyCNBPDNAbindingsitesthatmightrevealtargetgenesinvolvedinvertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound invivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequencyofputativeG-quadruplexDNAsecondarystructure.Moreover,aninsilicoapproachenabledustoestablishthe CNBPDNA-bindingsiteandtopredictCNBPputativetargetsbasedongeneontologytermsandsynexpressionwithCNBP. ThedirectinteractionbetweenCNBPandcandidategeneswasprovedbyEMSAandChIPassays.Besides,theroleofCNBP upontheidentifiedgeneswasvalidatedinloss-of-functionexperimentsindevelopingzebrafish.Wesuccessfullyconfirmed thatCNBPup-regulatestbx2bandsmarca5,anddown-regulateswnt5bgeneexpression.Thehighlystringentstrategyused in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonicdevelopment.Furthermore,itrepresentsanovelapproachtowardunderstandingthebiologicalfunctionand regulatorynetworksinvolvingCNBPinthebiologyofvertebrates. Cit