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ADENOVIRUSINDUCEDADENO-ASSOCIATEDVIRUSGENEEXPRESSIONIS NOTDEPENDENTONAAVNON-STRUCTURALREPPROTEIN By DANIELFRANCISLACKNER ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2002 Copyright2002 by DanielFrancisLackner Thisworkisdedicatedtobothofmyparents,JohnandSallyLackner. ACKNOWLEDGMENTS Inmylab,Iwouldliketothankagreatnumberofpeople. First,Iwouldliketo thankBillMcDonald. Hewastheunfortunatepostdocthathadtositnexttomeasafirst yeargraduatestudent. HeansweredeverystupidquestionIeveraskedandtaughtmeto formthereallyimportantones. TwootherimportantpostdocswereChristianand Corrina. Dr.TeshendorfintroducedmetothewondersandpeculiarityoftheEuropean lifestylefromtheGermanperspective. HewillbethemeasuretowhichIcompareall futuremedicaldoctors. CorrinahasexposedmetotheSpanishversionoftheEuropean lifestyleandcreatedmyentireknowledgeoftheneurosciencefield. IhaveenjoyedthelargenumberofChinesepostdocsinthelab,whichintroduced metotheChineseculture. Wei-jin,Pei,Wu,Yan,andLong-Gonhavetaughtmethe limitedamountofMandarinthatIcanspeakineverydayconversation. More importantly,theyshowedmethesimilaritiesofChinaandAmericansocietybutalsothe differencesbetweenthetwocultures. Thisexposuretoadifferentculturehasbeenoneof themostrewardingexperiencesintheMuzyczkalab. RodneyandOpiehavealwaysbeenmylabmatesandhavemadetheexperience onetolongremember. Theyhavebeenagreatdealofentertainmentandhavemadethe processofgettingaPh.D.agreatdealoffun. Iwouldchoosenooneelsetospendthese yearswithatUFandtheywillalwaysmakemelaugh. KenandKevinhavebeengreatmentorseventhroughwearethesameage. I respecttheiropinionsandhavehadnobettertimethandiscussinganyaspectofAAV iv biologywiththesetwogreatpostdocs. TheyhavetaughtmemorethantheyknowandI knowtheywilldogreatintheirfuturecareers. Withoutquestion,theenduringsupportofmyparents,JohnandSally,has enabledmetocompletethisPh.D.dissertation. Duringmanyphonecalls,theirown interestinmyworkhasinspiredmetoexplainmanydifferentaspectsofbasicAAVand Adenovirusbiology. Icannotthankthemenoughforeverythingtheyhavedoneforme. Iwouldliketogiveaveryimportantacknowledgementandthankstomywife, Dr.CariAspacherLackner. Fromthebeginningofmygraduatecareer,shehasbeena greatsourceofhappinessandagreatcompanioninscientificresearch. Ihavealwayshad herasaresearchconsultantandaspiredtobeherequalasabenchscientist. v TABLEOFCONTENTS page ACKNOWLEDGMENTS iv ABSTRACT viii INTRODUCTION 1 EukaryoticGeneExpression 1 TheChromatinTemplate 1 ActivationandRepression 5 ChromatinModification 9 BasalTranscriptionFactors 11 RNAPolymeraseIIHoloenzyme 14 Adeno-AssociatedVirus 15 AdenovirusHelperFunctions 18 AAVRepProteins 19 AAVTranscriptionwithoutAdenovirus 20 AAVTranscriptionwithAdenovirus 20 ModelforActivationofanAAVProductiveInfectionfromLatency 28 MATERIALSANDMETHODS 35 CellLinesandVirus 35 Plasmids 35 Transfections 39 CATReporterAssays 40 RESULTS 41 AnalysisofAdeno-AssociatedVirusTranscription 41 DefineRepBindingElementasEnhancerorProximalPromoterElement 41 Thep5RBEisnotanupstreamactivationsignal 50 Activationofpi9isnotduetoreadthroughfromp5 54 Transactivationofpl9canbemodeledintheabsenceofRep 60 Whichp5elementisprimarilyresponsibleforRepmediatedinductionofpl9?....64 TranscriptionalAnalysisofMutantRepProteins 67 GenerationofRepMutantPlasmids 68 TranscriptionalActivationofthepi9PromoterbyRepMutants 71 vi 9 Repressionofthep5promoter 75 DISCUSSION 79 TheRBEisnotatypicalupstreamactivationsignal 79 Thep5RBEisprobablyanarchitecturalelementdesignedtobringthep5andthepi promoterstogether 81 RepProteinDomainsinAAVTranscriptionalRegulation 87 FutureDirections 96 Conclusions 99 TABLEOFABBREVIATIONS 104 LISTOFREFERENCES 105 BIOGRAPHICALSKETCH 125 vii AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy ADENOVIRUSINDUCEDADENO-ASSOCIATEDVIRUSGENEEXPRESSIONIS NOTDEPENDENTONAAVNON-STRUCTURALREPPROTEIN By DanielFrancisLackner August2002 Chairman: NicholasMuzyczka,Ph.D. MajorDepartment: MolecularGeneticsandMicrobiology ThenormallifecycleofAdeno-AssociatedVirus(AAV)alternatesbetweena latent,repressedprovirusandanactivelytranscribingviralgenome. Thisswitchingene expressionisthecombinationofcellularandviralfactorstopromoteAAVreplication. PreviousstudiesofAAVhaveindicatedregulatorypathways,whichmaintainthecorrect AAVtranscriptionlevelsforproductiveinfection. AutoregulationoftheAAVgenomeis controlledbyRepproteininteractionswithcellularproteins. TheadditionofAdenovirus helperactivitiesinducechangesinbothcellularandAAVtranscriptionalregulation. Thiscombinationofregulatoryfactorsallowsthedissectionofacomplextranscriptional regulationmechanisminarelativelysimplegeneticsystem. ThedissectionoftheAAV transcriptionalregulationwasperformedbyaseriesofpromoterconstructsthatwould accessthefactorsrequiredfortranscriptionalactivationoftheAAVpi9promoter. Initially,thefirstpromoterconstructscontainingasingleAAVRepproteinbindingsite wereusedtodetermineifthenonstructuralRepproteinwouldfunctionasa viii 9 transcriptionalactivatorofAAVgeneexpression. TheRepproteinwasdeterminednot tobeatranscriptionalactivatorbutinsteadtofunctionasaposition-dependentinhibitor ofpi9promoteractivity. Thisposition-dependentinhibitionoftheAAVpi9promoter activitysuggestedthatthepreviouslycharacterizedRepandSplinteractionmightnotbe thedirectcauseofRep-mediatedtransactivation. Inordertoprovethattheprevious characterizedRepandSplinteractionwasnotresponsibleforinductionofthepl9 promoter,anovelsystemofhybridtranscriptionfactorswasusedtoreplacetheRepand Splproteins. Thenovelhybridtranscriptionfactorsmodifiedfromtheyeasttwo-hybrid system,consistedoftwodifferentproteinscontainingaGAL4-DNAbindingdomain fusedtoeitheraninteractiondomainfromp53orT-antigen. ThereplacementoftheRep andSplbindingsiteswithGAL4bindingsequencesinthepi9promotershowedthat RepandSplwerenotrequiredfortransactivation. WeconcludethattheRepandSpl transcriptionfactorsfunctionasarchitecturalproteinsfacilitatingaDNAlooptoform betweentheRepbindingsiteinthep5promoterandtheSplsitewithinthepi promoter. ix CHAPTER 1 INTRODUCTION EukaryoticGeneExpression Theregulationofeukaryoticgeneexpressionhasbeenshowntobefarmore complexthantheoriginalproteinfractionationsforRNApolymeraseactivities suggested. InitialfractionationprotocolsdiscoveredthreedistinctRNApolymerasesand theirrespectiveGeneralorBasalTranscriptionFactors(GTFs). However,purificationof thesefactorsdidnotrestorethecomplexregulationoftranscriptioninvitroindicating thatmorefactorswererequiredforpropertranscriptionalregulation. Morerecentstudies haveobservedthattheRNApolymeraseIIholoenzyme(RNAPII)isaboutthesamesize astheribosomeandmanyotherfactorsarecriticalforcorrectgeneexpression. DNA condensationintochromatinhasbeenshowntoregulategeneexpressionandthe exposureofDNAsequencesallowstheassemblyoftranscriptionalregulatorstorecruit thecompleteRNApolymeraseIIholoenzyme. TheChromatinTemplate TheDNAtemplateofmostpromotersforprotein-encodinggenescontainsthree importantfeatures. Thefirstcomponentisthetranscriptioninitiationsitethatdirectsthe initiationofpolymerizationofmRNA,whichiscatalyzedbytheRNApolymeraseII holoenzyme. ThesecondcomponentistheTATAboxwhichregulatesthebindingofthe TATAbindingprotein(TBP). Thelastcomponentistheregulatorysequences 1

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136 Pages·2002·4.4 MB·English
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