现代分子生物学之真核细胞转录调控(二)PPT课件

.,0,GeneralMechanismsofTranscriptionalRegulationinEukaryotesEukaryoticTranscriptionalActivators:激活蛋白的结构、作用方式EukaryoticTranscriptionalRepressorsSignalIntegrationandCombinatorialControlSignalTransductionandtheControlofTranscriptionalRegulatorsGeneSilencingbyModificationofHistonesandDNAEpigeneticGeneRegulation,.,1,UASs(upstreamactivationsequences)=enhancersEachUASis17bplong,boundbyaGal4dimer,TheGALSysteminYeast:Whenlackinggalactose,GALgenesaresilent.Inpresenceofgalactose(andabsenceofglucose),GALgenesareinduced.,.,2,(a)TheDNA-bindingdomainofGal4,withoutthatproteinsactivationdomain,canstillbindDNAbutcannotactivatetranscription.(b)AttachingtheactivationdomainofGal4totheDNA-bindingdomainofthebacterialproteinLexAcreatesahybridproteinthatactivatestranscriptionofageneinyeastifthatgenebearsabindingsiteforLexA.ExpressionismeasuredusingareporterplasmidinwhichtheGAL1promoterisfusedtotheE.colilacZgenewhoseproduct(b-galactosidase)isreadilyassayedinyeastcells.,TranscriptionactivatorshaveseparateDNAbindingandactivatingdomains:theDomainswapexperiment,GAL1promoter,Bacterialregulatoryproteinsmostlyusethehelix-turn-helixmotiftobindDNAtarget,Regulatoryproteins:DNAbindingregions,HomeodomainOnehelix(forrecognition,#3)fitsinmajorgrooveandrecognizesspecificbasepairs.Theotherhelix(#2)makescontactswiththeDNAbackbone,positioningtherecognitionhelixproperlyandincreasingthestrengthofbinding,EukaryoticregulatorsusearangeofDNAbindingdomainsHomeodomainproteins(HTH):helix-turn-helixZinccontainingDNA-bindingdomain:zincfinger/zincclusterLeucinezippermotifHelix-Loop-HelixproteinsHMG:interactwithminorgroove/alterDNAconformation,.,4,zincngerproteins,ThezincatominteractswithcysteineandhistidineresiduesandservesastructuralroleessentialforintegrityoftheDNA-bindingdomain,ThehelixforrecognitionispresentedtoDNAbysheetontheright.Thezinciscoordinatedbythetwohistidineinthehelixandtwocysteineinthesheet,Thezinciscoordinatedbyfourcysteineresidues,.,5,Leucinezipperprotein,majorgroove,Leucinezippermotif,.,6,Thedimerizationsurfaceisformedfromtwohelicalregions:thefirstispartofthesamehelixinvolvedinDNArecognition;theotherisashorterhelix.Thesetwohelicesareseparatedbyaflexibleloopthatallowsthemtopacktogether,Helix-Loop-HelixProteins,*,7,ActivatingRegionsAreNotWell-DefinedStructuresTheyareadhesivesurfacescapableofinteractingwithotherproteinsurfaces;Notina“lockandkey”manner;aregroupedonthebasisofaminoacidscontent.,Acidicregion:containbothcriticalacidicAAsandhydrophobicAAs.,Glutamine-richregionProline-richregion,.,8,DNAbindingdomaincomprisesthreeCys2His2-likezincfingers,Theactivationdomainsarecomposedofserine/threoninerichregions,flankedbyregionsrichinglutamine,*,.,9,*,Eukaryoticactivatorsrecruitpolymeraseindirectly,1.Interactingwithpartsofthetranscriptionmachinery(GTFs,otherproteinsforRNAPIIinnitiationandelogation,etc.)2.Recruitingnucleosomemodifiers/remodelersthatalterchromatinstructure,tohelpinitiation,.,11,Gal4interactswithmediatorcomplex,whichdirectlyrecruitRNAPIItogenepromoters.,Gal4bindstoTBPandrecruitstheTFIIDcomplexand,RNAPIItopromoter.,Themediatoractasaco-activator(facilitategeneactivationbyTF,butitselfisneitherpartofthetranscriptionalmachinerynoraDNA-bindingprotein),Activatorsinteractwithpartsofthetranscriptionmachinery,Promotersbearingacetylatednucleosomeshavehigheraffinityfortranscriptionalmachinery,Activatorsrecruitnucleosomemodifiers,1.AcetylationcanaltertheDNA-histoneinteractiontheoctamerslidealongtheDNAtoanewposition.2.canaltertheinteractionbetweenadjacentnucleosomesamoreopenchromatin.3.createsspecificbindingsitesonnucleosomesforproteinswithbromodomains(eg.,TFIID),.,13,Activatorsrecruitnucleosomeremodelers,to“Loosen”thechromatinstructure,Sliding,.,14,Requirementofcomponentsofthetranscriptionalmachineryvariesunderdifferentcircumstances,ManyproteinscaninteractspecificallywiththeGal4-activationdomain:TBP,TFIIB,Gal11(acomponentofMediator),Cdk8,SWI/SNF(nucleosomeremodeler),SAGA(Spt-Ada-Gcn5-acetyltransferase),Srb4(anothercomponentofMediator)andproteasomecomponentsSug1andSug2-by2006,EMBOreportsVOL7|NO5|2006,Gal4functionsinmosteukaryotes,inmanyways,*,.,15,HSP70genefromDrosophila,activatedbyheatshock,iscontrolledbytwoactivatorsworkingtogether.TheGAGA-bindingfactorcanrecruitenoughofthetranscriptionmachinerytothepromoterfortranscriptioninitiation.But,intheabsenceofasecondactivator,HSF,mostoftheinitiatedpolymerasesstallsome2550bpdown-streamfromthepromoter.,Inresponsetoheatshock,HSFbindstospecicsitesatthepromoterandrecruitsakinase,P-TEFb(positivetranscriptionelongationfactor,partofalargercomplex,theSEC,superelongationcomplex),tothestalledpolymerases.,AstrongacidicactivatorlikeGal4isabletorecruitPTEFb/SECalongwiththerestofthemachinery.,ThekinasephosphorylatestheSer2oftheCTDheptadrepeatofRNAPII,freeingtheenzymefromthestallandallowingtranscriptiontoproceedthroughthegene.,*,.,16,TranscriptionalRepressor,Ineukaryotes,mostrepressorsusuallydonotactbybindingtositesthatoverlapwiththepromoterandblockbindingofpolymerase.(Bacteriaoftendoso),1.Mostcommon:Compactingthenucleosomebydirectlyremoving/addingsomegroupsorbyrecruitingnucleosomemodifiers2.Competingwiththeactivatorforanoverlappedbindingsite3.Bindingtoasitedifferentfromthatoftheactivator,butphysicallyinteractswithanactivatormoleculeandblocksitsactivatingregion4.Bindingtoasiteupstreamofthepromoter,physicallyinteractswiththetranscriptionmachineryatthepromotertoinhibittranscriptioninitiation.,.,17,Inthepresenceofglucose,RepressionoftheGAL1geneinyeast,Mig1bindstoasitebetweentheGal4-bindingsiteandtheGAL1promoter,RecruitstheTup1proteincomplex,Tup1recruitshistonedeacetylases,Directlyinteractswithtranscriptionmachinery,Represstranscription.,*,.,18,.,19,*,.,20,*,.,21,*,.,22,22,Studyprotein/nucleicacidinteraction,Gelretardationassay/electrophoreticmobility-shiftassaycanbeusedqualitativelytoidentifysequence-specificDNA-bindingproteins(suchastranscriptionfactors),Protein:DNAcomplexesmigratemoreslowlythanfreelinearDNAfragmentsinnon-denaturingpolyacrylamideoragarosegelelectrophoresis,However,ifcircularDNAisused,theprotein:DNAcomplexmaymigratefasterthanfreeDNA,Theorderofcomponentadditionforthebindingreactionisoftencritical.Completedbindingreactionsarebestelectrophoresedimmediatelytopreservepotentiallylabilecomplexesfordetection.,.,23,(A)LabelledoligonucleotideincubatedwithincreasingamountsofV47RPOUmutantprotein.0,controlDNAprobewithoutprotein.,GelretardationassayofDNA-proteincomplexes.,(B)Quantificationofgelretardationdata.AmountoffreeDNAasafunctionoflogproteinconcentration.Relativeaffinityofproteintooctamerprobe(ATGCAAATGA)canbedetermined,FEBSLetters412(1997)5-8,protein-DNAcomplex,freeDNA,.,24,24,1）TheproteinprotectsDNAfromattackbyDNase.2）TreattheDNA-proteincomplexwithDNaseundermildconditions,sothatonlyonecutoccurperDNAmoleculeonaverage.,Electrophoresis,autoradiograph,DNaseIfootprinting:Identifytheactualregionofsequencewithwhichtheproteininteracts.,ThethreelanesrepresentDNAthatwasboundto0,1,and5unitsofprotein.Thelanewithnoproteinshowsaregularladderoffragments.Thelanewithoneunitshowssomeprotection,andthelanewith5unitsshowscompleteprotectioninthemiddle.Withsequenceladders,wecantellexactlywheretheproteinbound.,onlyonecutoccurperDNAmolecule,.,25,Yeasttwohybridsystem:todiscoverproteinproteininteractionsandproteinDNAinteractionsbyusingyeastfusionproteins,transcriptionactivationsystemandreportergenes,Transcriptionfactor,Whenthesetwodomanisareexpressedasseperateproteins,theBDwillstillbindtoDNA,buttheADisNOTintherightplacetoactivatetranscription,Asplittranscriptionfactor,.,26,BD,X,Y,AD,promoter,gene,Hybridproteinsasmolecularglue,Totestiftwoproteins(XandY)interact,theyareexpressedinfusionwiththeBDandtheAD,BD,promoter,gene,X,Y,AD,IfproteinsXandYbindtoeachother,thetranscriptionfactorisreconstituted,andgeneexpressionisactivated,.,27,AD,BD,GAL4-AD,GAL4andReportergenes,Inmanycases,thereconstitutedtranscriptionfactoristheyeastGAL4transcriptionalactivator.,reportergene,GAL4-BD,GAL4-UAS,Tomonitortranscriptionalactivation,reporterproteinssuchasb-galactosidaseareexpressedunderthecontroloftheGAL4-upstreamactivatingsequence.,.,28,Applicationoftheyeasttwo-hybridsystem,Identifiesnovelprotein-proteininteractions:Whichproteinscaninteractwithanknownprotein?Needtoconstructbaitandpreyplasmidstoexpressfusionproteins,Identifiesmutationsthataffectprotein-proteinbindingCanidentifyinterferingproteinsinknowninteractions,.,29,Exploringprotein-proteininteractions:Pull-DownAssays,.,30,.,31,CHIP:chromatinimmunoprecipitation,CHIP-on-chip,.,32,GeneralMechanismsofTranscriptionalRegulationinEukaryotesRecruitmentofProteinComplexestoGenesbyEukaryoticActivators.TranscriptionalRepressorsSignalIntegrationandCombinatorialControlSignalTransductionandtheControlofTranscriptionalRegulatorsGeneSilencingbyModificationofHistonesandDNAEpigeneticGeneRegulation,Activatorsworktogethersynergistically(Whenaneffectisgreaterthanadditive)tointegratesignals,A,B,C,F,E,D,SignalIntegrationandCombinatorialControl,Numeroussignalsmayberequiredtoswitchageneon;Eachsignalistransmittedtothegenebyaseparateregulator.,.,34,S1:Multipleactivatorsrecruitasinglecomponentofthetranscriptionalmachinery,bytouchingthedifferentpartofthemediatorcomplex.,S2:Multipleactivatorseachrecruitadifferentcomponentofthetranscriptionalmachinery.ThesecomponentsbindstothepromoterDNAinefficientlywithouthelp.,S3:Multipleactivatorshelpeachotherbindtotheirsitesupstreamofthegenetheycontrol.,Strategiesofthesynergy,a.“Classical”cooperativebinding.b.Bothproteinsinteractingwithathirdprotein.c.Thefirstproteinrecruitanucleosomeremodellerwhoseactionrevealabindingsiteforthesecondprotein.d.BindingaproteinunwindstheDNAfromnucleosomealittle,revealingthebindingsiteforanotherprotein.,Example:theHOgeneofyeastS.cerevisiaeonlyexpressedinmothercellsandatcertainpointincellcycle,andiscontrolledbytworegulators:recruitingnucleosomemodifiersandmediators.,SWI5:actsonlyinthemothercellandbindsunaidedtomultiplesitesdistantfromthegene,whichrecruitenzymestoopentheSBFbindingsites,SBF:onlyactiveatthecorrectstagesofthecellcycle,andcannotbindthesitesunaided,*,Infection,Example:Cooperativebindingofactivatorsathumanb-interferongene.,Triggersthreeactivators(communicator):NFkB,IRF,andJun/ATF,Activatorsbindcooperativelytoanenhancerlocatedabout1kbupstreamofthepromoter,formingenhanceosome,Thehumanb-interferongeneisactivatedincellsuponviralinfection,Transcriptionisactivatedtohighlevelonlywhenalltheproteinsarepresentandtouchingoneanotherinjusttherightway,RoleofHMGBasarchitecturalfactors,*,.,37,Bindingsitesofactivatorsatthehumanb-interferongene,Theconservationoftheinterferon-benhancerDNAsequencesacrossspeciesseparatedby100millionyears.,Thecrystalstructureoftheenhanceosome,*,.,38,Whenalloftheregulatoryproteinsareboundandinteractingcorrectly,theyforma“landingpad,”ahigh-affinitybindingsitefortheproteinCBP,aco-activatorproteinthatalsorecruitsthetranscriptionalmachinery.ThelargeCBPproteinalsocontainsanintrinsichistoneacetylaseactivitythatmodifiesnucleosomesandfacilitateshighlevelsoftranscription,用肘轻推,nuc2isattheTATAboxandtranscriptionstartsite,37bpofftheTATAbox,TheB-interferonenhanceosomeactstomovenucleosomesbyrecruitingtheSWISNFcomplex,*,Combinatorycontrol-activatorsandrepressorsworktogether-complexityanddiversityofeukaryotes,Example:combinatorycontrolofthemating-typegenesfromS.cerevisiae,Thecelltypes(theaandhaploid,andthea/diploid)aredefinedbythesetsofgenestheyexpress-determinedbymating-typeregulators:Oneubiquitousregulator(Mcm1)andthreecell-type-specificregulators(a1,1,and2),encodedbytheMATlocus.,*,2.SignalTransductionandtheControlofTranscriptionalRegulators,Signals,Transcriptionalregulators,signaltransductionpathway,MechanismsI:UnmaskingActivatingRegionby:aconformationalchangeintheDNA-boundactivator,revealingapreviouslyburiedactivatingregion,orbyreleaseofamaskingproteinthatpreviouslyinteractedwith,andeclipsed,anactivatingregion.throughbindingliganddirectlyorthroughaligand-dependentphosphorylation.,Gal3bindsgalactoseandATP,.,42,LessonsfromYeast-TheGALSystem:Whenlackinggalactose,GALgenesaresilent.Inpresenceofgalactose(andabsenceofglucose),GALgenesareinduced.,*,.,43,EMBOreportsVOL7|NO5|2006,Gal80doesnotexhibitnucleocytoplasmicexchangeGalactosedoesnotcauseGal3entryintonucleustoaccountfortherapidGal4-mediatedgeneactivationNuclear-localizedGal3isrequiredforrapidinductionStableGal3Gal80Gal4complexisnotdetectableingalactose-inducedcellsGal80complexedwithGal4onDNAdoesnotexchangerapidlywithfreeGal80,TransientassociationofGal3withGal4-associatedGal80:aplausiblemechanismoftheGALgeneswitch,*,.,44,*,.,45,PhosphorylationofRbcausesreleaseofitfromE2Fandactivationofthegenes.,MechanismII:Themaskingproteinmaynotonlyblocksactivatingregionbutalsoisitself(orrecruits)adeacetylaseactivelyrepressesthegene.,MutantRb:-inactivatingmutation-Rbgenedeletion,TherepressorRb(retinoblastomaprotein)controlsactivityofE2Fbybindingtoit-bothblockingactivationandrecruitingadeacetylase,ThemammalianactivatorE2Fbindssitesupstreamofitstargetgenes.,uncontrolledcellcycle,2.SignalTransductionandtheControlofTranscriptionalRegulators,Mechanisms:Transportintoandoutofthenucleus:Whennotactive,manyactivatorsandrepressorsareheldinthecytoplasmthroughinteractionwithaninhibitoryprotein,orwiththecellmembrane.Thesignalingligandcausesthemtomoveintothenucleustoactivatetranscription;Acascadeofkinasescausethephosphorylationofregulatorinnucleus;Theactivatedreceptoriscleavedbycellularproteases,andthec-terminalportionofthereceptorentersthenucleusandactivatestheregulator,.,47,TheSTATpathway,TheMAPkinasepathway,Thesignalisthenrelayed(分程传递)totherelevanttranscriptionalregulator,Thetranscriptionalregulatorcontrolthetargetgeneexpression,Thesignaliscommunicatedtotheintracellulardomainofreceptor(viaanallostericchangeordimerization),Theligand(“signal”)bindstoanextracellulardomainofaspecificcellsurfacereceptor,*,.,48,Signalingmechanismslinkingneuronalactivitytogeneexpressionandplasticityofthenervoussystem-AnnuRevNeurosci.;31:563-590,2.Thesepathwaysconvergeonpreexistingtranscriptionfactorsinthenucleusandleadtotheiractivationthroughdirectposttranslationalproteinmodifications.,1.Calciuminfluxthroughneurotransmitterreceptorsorvoltage-gatedcalciumchannelsleadstoactivationofcalcium-regulatedsignalingenzymes,settinginmotionseveralsignaltransductioncascades.,3.Severaloftheactivity-regulatedgenesencodetranscriptionalregulators,whichpromotetranscriptionofadditionalactivity-regulatedgenes.,4.Geneticmutationsingenesthatencodethesignalingmoleculesgiverisetoneurologicaldisordersinhumans(yellowboxes).,3.Manyotheractivity-regulatedgenesencodeproteinsworkingindendritesoratsynapsesandtherebycoordinateactivity-dependentdendriticandsynapticremodelingwithintheneuron.,*,.,49,GeneralMechanismsofTranscriptionalRegulationinEukaryotesRecruitmentofProteinComplexestoGenesbyEukaryoticActivators.TranscriptionalRepressorsSignalIntegrationandCombinatorialControlSignalTransductionandtheControlofTranscriptionalRegulatorsGeneSilencingbyModificationofHistonesandDNAEpigeneticGeneRegulation,Gene“Silencing”byModificationofHistonesandDNA,TranscriptionalSilencing:aspecializedformofrepressionthatcanspreadalongchromatin,switchingoffmultiplegenes,eventhosequitedistantfromtheinitiatingevent,withouttheneedforeachtobearbindingsitesforspecificrepressor.,Transcriptionalsilencingisapositioneffect:Ageneissilencedbecauseofwhereitislocated,notinresponsetoaspecificenvironmentalsignal.,Transcriptionalsilencingisassociatedwithmodificationofnucleosomesthatalterstheaccessibilityofagenetothetranscriptionalmachineryandotherregulatoryproteins.,SIR2,3,and4havebeenfoundasregulatorsofsilencing(SIRstandsforSilentInformationRegulator).Rap1recruitsSircomplextothetemomere.Sir2deacetylatesnearbynucleosome,Thespreadingofsilencingisrestricted/controlledbyinsulatorsandotherkindofhistonemodificationsthatblockbindingoftheSir2proteins.,Transcriptioncanalsobesilencedthroughchromatinmethylationbyhistonemethyltransferase,MethylationofH3K9isamodificationassociatedwithsilencedheterochromatininhighereukaryotesandintheyeastS.pombe.Incontrast,methylationofH3K4areassociatedwithincreasedtranscription.,InS.cerevisiae,methylationofspecificlysineresiduesinthetailsofhistonesH3andH4helprepressionofsomegenesandblockspreadingofSir2-mediatedsilencinginothers.,.,53,WhenmammaliancellDNAisstainedwithfluorescentdye,heterochromatin,whichisadenselycondensedassemblyofchromatin,becomesvisibleunderbrightlight(left).Thebasicstructureofheterochromatinisalsofoundinfissionyeast(right).,Themostcommonformofsilencing:adenseformofchromatin-“heterochromatin”.Usuallyatparticularregionsofthechromosome,eg.,thetelomeres,andthecentromeres:constitutiveheterochromatinInmammaliancells,about50%ofthegenomeisestimatedtobeinsomeformofheterochromatin.,organizedinto30-nmchromatinfibers,.,55,Variegation:Insteadofbeingsilencedinallcellsallthetime,thegeneswitchesbetweenthesilencedandexpressedstateatrandom,being“on”insomecellsand“off”inothers.,TheSu(Var)3-9orClr4proteinisaH3K9methyltransferase,Differenttypesofmodificationatthehistonescanbeinvolvedindistinctgeneregulation,Multipleformsofmodification:the“HistoneCode”:Thedirecteffectsofmodificationsonchromatindensityandform.Particularpatternofmodificationsatanygivenlocationwouldrecruitspecificproteins,DNAMethylationIsAssociatedwithSilencedGenesinMammliancells,ThemethylatedDNAsequencesalonecandisruptbindingofthetranscriptionmachineryandactivatorsinsomecases;theycanalsoberecognizedbyDNA-bindingproteinsthatrecruithistonedeacetylasesandhistonemethylases,whichthenmodifynearbychromatin.,Initsunmodifiedstate,themammaliangeneexpressionisneverfirmlyshutoffitisleaky,DNAmethyltransferasemethylatecytosineswithinthepromotersequence,thegeneitself,orupstreamactivatorbindingsites,DNAmethylationcancauseImprinting:Inadiploidcell,asamegenefromfatherormotherisdifferentiallyexpressed,ICR:imprintingcontrolregion,aninsulator,ThemethylationstateoftheinsulatorelementdetermineswhetherornottheICRbindingprotein(CTCF)canbindandblockactivationofthe