現(xiàn)代分子生物學(xué)之真核細(xì)胞轉(zhuǎn)錄調(diào)控(二)培訓(xùn)資料

現(xiàn)代分子生物學(xué)之真核細(xì)胞轉(zhuǎn)錄調(diào)控(二)(a)TheDNA-bindingdomainofGal4,withoutthatprotein'sactivationdomain,canstillbindDNAbutcannotactivatetranscription.(b)AttachingtheactivationdomainofGal4totheDNA-bindingdomainofthebacterialproteinLexAcreatesahybridproteinthatactivatestranscriptionofageneinyeastifthatgenebearsabindingsiteforLexA.ExpressionismeasuredusingareporterplasmidinwhichtheGAL1promoterisfusedtotheE.colilacZgenewhoseproduct(b-galactosidase)isreadilyassayedinyeastcells.Transcriptionactivators

haveseparateDNAbindingandactivatingdomains:the"Domainswapexperiment"GAL1promoterBacterialregulatoryproteinsmostlyusethehelix-turn-helixmotif

tobindDNAtargetRegulatoryproteins:DNAbindingregionsHomeodomainOnehelix(forrecognition,#3)fitsinmajorgrooveandrecognizesspecificbasepairs.Theotherhelix(#2)makescontactswiththeDNAbackbone,positioningtherecognitionhelixproperlyandincreasingthestrengthofbindingEukaryoticregulatorsusearangeofDNAbindingdomains

Homeodomainproteins(HTH):helix-turn-helix

ZinccontainingDNA-bindingdomain:zincfinger/zinccluster

Leucinezippermotif

Helix-Loop-Helixproteins

HMG:interactwithminorgroove/alterDNAconformationzinc?ngerproteinsThezincatominteractswithcysteineandhistidineresiduesandservesastructuralroleessentialforintegrityoftheDNA-bindingdomainTheαhelixforrecognitionispresentedtoDNAbyβsheetontheright.ThezinciscoordinatedbythetwohistidineintheαhelixandtwocysteineintheβsheetThezinciscoordinatedbyfourcysteineresiduesLeucinezipperproteinmajorgrooveLeucinezippermotifThedimerizationsurfaceisformedfromtwohelicalregions:thefirstispartofthesamehelixinvolvedinDNArecognition;theotherisashorterαhelix.ThesetwohelicesareseparatedbyaflexibleloopthatallowsthemtopacktogetherHelix-Loop-HelixProteins*6ActivatingRegionsAreNotWell-DefinedStructuresTheyareadhesivesurfacescapableofinteractingwithotherproteinsurfaces;Notina“l(fā)ockandkey”manner;aregroupedonthebasisofaminoacidscontent.Acidicregion:containbothcriticalacidicAAsandhydrophobicAAs.Glutamine-rich

regionProline-richregionTranscriptionFactorSp1andit'sclosestrelatedfamilymembers.

Thechargeddomain(green),althoughnotessentialforDNAbinding,promotesthebindingofthezincfingerstotheirDNAtarget.OnlySp1containsamultimerizationdomain(orange)whichcanfacilitateinteractionbetweenmultipleSp1moleculesofSp1andpromotesuper-activationofgenes.Buttonheaddomain(Btd)isconservedindrosophilaSp1DNAbindingdomaincomprisesthreeCys2His2-likezincfingersTheactivationdomainsarecomposedofserine/threoninerichregions,flankedbyregionsrichinglutamine**Eukaryoticactivatorsrecruitpolymeraseindirectly1.Interactingwithpartsofthetranscriptionmachinery

(GTFs,otherproteinsforRNAPIIinnitiationandelogation,etc.)2.Recruitingnucleosomemodifiers

/remodelersthatalterchromatinstructure,tohelpinitiationGal4interactswithmediatorcomplex,whichdirectlyrecruitRNAPIItogenepromoters.Gal4bindstoTBPandrecruitstheTFIIDcomplexand,RNAPIItopromoter.Themediatoractasaco-activator(facilitategeneactivationbyTF,butitselfisneitherpartofthetranscriptionalmachinerynoraDNA-bindingprotein)Activatorsinteractwithpartsofthetranscriptionmachinery

PromotersbearingacetylatednucleosomeshavehigheraffinityfortranscriptionalmachineryActivatorsrecruitnucleosomemodifiers1.AcetylationcanaltertheDNA-histoneinteraction→theoctamerslidealongtheDNAtoanewposition.2.......canaltertheinteractionbetweenadjacentnucleosomes→

amoreopenchromatin.3.......createsspecificbindingsitesonnucleosomesforproteinswithbromodomains(eg.,TFIID)Activatorsrecruitnucleosomeremodelers

to“Loosen”thechromatinstructureSlidingRequirementofcomponentsofthetranscriptionalmachineryvariesunderdifferentcircumstancesManyproteinscaninteractspecificallywiththeGal4-activationdomain:TBP,TFIIB,Gal11(acomponentofMediator),Cdk8,SWI/SNF(nucleosomeremodeler),SAGA(Spt-Ada-Gcn5-acetyltransferase),Srb4(anothercomponentofMediator)andproteasomecomponentsSug1andSug2----by2006EMBOreportsVOL7|NO5|2006Gal4functionsinmosteukaryotes,inmanyways*HSP70genefromDrosophila,activatedbyheatshock,iscontrolledbytwoactivatorsworkingtogether.TheGAGA-bindingfactorcanrecruitenoughofthetranscriptionmachinerytothepromoterfortranscriptioninitiation.But,intheabsenceofasecondactivator,HSF,mostoftheinitiatedpolymerasesstallsome25–50bpdown-streamfromthepromoter.Inresponsetoheatshock,HSFbindstospeci?csitesatthepromoterandrecruitsa

kinase,P-TEFb(positivetranscriptionelongationfactor,partofalargercomplex,theSEC,superelongationcomplex),tothestalledpolymerases.AstrongacidicactivatorlikeGal4isabletorecruitPTEFb/SECalongwiththerestofthemachinery.ThekinasephosphorylatestheSer2oftheCTDheptadrepeatofRNAPII,freeingtheenzymefromthestallandallowingtranscriptiontoproceedthroughthegene.*TranscriptionalRepressorIneukaryotes,mostrepressorsusually

donot

actbybindingtositesthatoverlapwiththepromoterandblockbindingofpolymerase.(Bacteriaoftendoso)1.

Mostcommon:

Compactingthenucleosomeby

directlyremoving/addingsomegroups

orby

recruitingnucleosomemodifiers

2.

Competingwiththeactivator

foranoverlappedbindingsite3.

Bindingtoasitedifferentfromthatoftheactivator,butphysicallyinteractswithanactivator

moleculeandblocksitsactivatingregion4.

Bindingtoasiteupstreamofthepromoter,physicallyinteractswiththetranscriptionmachineryatthepromotertoinhibittranscriptioninitiation.InthepresenceofglucoseRepressionoftheGAL1geneinyeastMig1bindstoasitebetweentheGal4-bindingsiteandtheGAL1promoterRecruitstheTup1proteincomplexTup1recruitshistonedeacetylasesDirectlyinteractswithtranscriptionmachineryRepresstranscription.

*Tomodulatetranscription,regulatoryproteinspossess

oneormoreofthefollowingfunctionaldomains:1.AdomainthatrecognizesaDNA

sequence(DNA

bindingsite)2.Adomainthatinteractswiththetranscriptionalapparatus(RNAPorprotein(s)associatedwithRNAP)3.Adomainthatinteractswithotherregulatoryproteins4.Adomainthatinfluenceschromatincondensation,directlyorindirectly5.AdomainthatactsasasensorforphysiologicalconditionswithinthecellSp1ishighlyregulatedbypost-translationalmodificationsthatbothpositivelyandnegativelyaffectSp1'sactivityonawidearrayofgenes.**Sp1bothactivatesandsuppressestheexpressionofanumberofessentialoncogenesandtumorsuppressors,aswellasgenesinvolvedinessentialcellularfunctions,includingproliferation,

differentiation,DNAdamageresponse,apoptosis,senescence,andangiogenesis,

inflammationandgenomicinstability,aswellasepigeneticsilencing.*21Studyprotein/nucleicacidinteraction/method/gel-shift-assays-emsaGelretardationassay/electrophoreticmobility-shiftassay

canbeusedqualitativelytoidentifysequence-specificDNA-bindingproteins(suchastranscriptionfactors)Protein:DNAcomplexesmigratemoreslowlythanfreelinearDNAfragmentsinnon-denaturingpolyacrylamideoragarosegelelectrophoresisHowever,ifcircularDNAisused,theprotein:DNAcomplexmaymigratefasterthanfreeDNATheorderofcomponentadditionforthebindingreactionisoftencritical.Completedbindingreactionsarebestelectrophoresedimmediatelytopreservepotentiallylabilecomplexesfordetection.(A)LabelledoligonucleotideincubatedwithincreasingamountsofV47RPOUmutantprotein.0,controlDNAprobewithoutprotein.GelretardationassayofDNA-proteincomplexes.(B)Quantificationofgelretardationdata.AmountoffreeDNAasafunctionoflogproteinconcentration.Relativeaffinityofproteintooctamerprobe(ATGCAAATGA)canbedetermined

FEBSLetters412(1997)5-8protein-DNAcomplexfreeDNA231）TheproteinprotectsDNAfromattackbyDNase.

2）TreattheDNA-proteincomplexwithDNaseundermildconditions,sothatonlyonecutoccurperDNAmoleculeonaverage.Electrophoresis,autoradiographDNaseIfootprinting:Identifytheactualregionofsequencewithwhichtheproteininteracts.ThethreelanesrepresentDNAthatwasboundto0,1,and5unitsofprotein.

Thelanewithnoproteinshowsaregularladderoffragments.

Thelanewithoneunitshowssomeprotection,

andthelanewith5unitsshowscompleteprotectioninthemiddle.

Withsequenceladders,wecantellexactlywheretheproteinbound.onlyonecut

occurperDNAmoleculeYeasttwohybridsystem:

todiscoverprotein–proteininteractionsandprotein–DNAinteractionsbyusingyeastfusionproteins,transcriptionactivationsystem

and

reportergenes

TranscriptionfactorWhenthesetwodomanisareexpressedasseperateproteins,theBDwillstillbindtoDNA,buttheADisNOT

intherightplacetoactivatetranscriptionAsplittranscriptionfactorBDXYADpromotergeneHybridproteinsasmolecularglueTotestiftwoproteins(XandY)interact,theyareexpressedinfusionwiththeBDandtheADBDpromotergeneXYADIfproteinsXandYbindtoeachother,thetranscriptionfactorisreconstituted,andgeneexpressionisactivatedADBDGAL4-ADGAL4andReportergenesInmanycases,thereconstitutedtranscriptionfactoristheyeastGAL4transcriptionalactivator.reporter

geneGAL4-BDGAL4-UASTomonitortranscriptionalactivation,reporterproteinssuchasb-galactosidaseareexpressedunderthecontroloftheGAL4-upstreamactivatingsequence.Applicationoftheyeasttwo-hybridsystemIdentifiesnovelprotein-proteininteractions:Whichproteinscaninteractwithanknownprotein?NeedtoconstructbaitandpreyplasmidstoexpressfusionproteinsIdentifiesmutationsthataffectprotein-proteinbindingCanidentifyinterferingproteinsinknowninteractions/method/pull-down-assaysExploringprotein-proteininteractions:Pull-DownAssaysCHIP:chromatinimmunoprecipitationCHIP-on-chipGeneralMechanismsofTranscriptionalRegulationinEukaryotesRecruitmentofProteinComplexestoGenesbyEukaryoticActivators.TranscriptionalRepressorsSignalIntegrationandCombinatorialControlSignalTransductionandtheControlofTranscriptionalRegulatorsGeneSilencingbyModificationofHistonesandDNAEpigeneticGeneRegulation

Activatorsworktogethersynergistically(Whenaneffectisgreaterthanadditive)tointegratesignalsABCFEDSignalIntegrationandCombinatorialControlNumeroussignalsmayberequiredtoswitchageneon;Eachsignalistransmittedtothegenebyaseparateregulator.S1:

Multipleactivatorsrecruitasinglecomponentofthetranscriptionalmachinery,bytouchingthedifferentpartofthemediatorcomplex.

S2:

Multipleactivatorseachrecruitadifferentcomponentofthetranscriptionalmachinery.ThesecomponentsbindstothepromoterDNAinefficientlywithouthelp.S3:

Multipleactivatorshelpeachotherbindtotheirsitesupstreamofthegenetheycontrol.Strategiesofthesynergya.“Classical”cooperativebinding.b.Bothproteinsinteractingwithathirdprotein.c.Thefirstproteinrecruitanucleosomeremodellerwhoseactionrevealabindingsiteforthesecondprotein.d.BindingaproteinunwindstheDNAfromnucleosomealittle,revealingthebindingsiteforanotherprotein.Example:theHOgeneofyeastS.cerevisiae

onlyexpressedinmothercellsandatcertainpointincellcycle,andiscontrolledbytworegulators:recruitingnucleosomemodifiersandmediators.SWI5:actsonlyinthemothercellandbindsunaided

tomultiplesitesdistantfromthegene,whichrecruitenzymestoopentheSBFbindingsitesSBF:onlyactiveatthecorrectstagesofthecellcycle,andcannotbindthesitesunaided*InfectionExample:Cooperativebindingofactivatorsathumanb-interferongene.Triggersthreeactivators(communicator):NFkB,IRF,andJun/ATFActivatorsbindcooperativelytoanenhancerlocatedabout1kbupstreamofthepromoter,formingenhanceosomeThehumanb-interferongeneisactivatedincellsuponviralinfectionTranscriptionisactivatedtohighlevelonlywhenalltheproteinsarepresentandtouchingoneanotherinjusttherightwayRoleofHMGBasarchitecturalfactors*Bindingsitesofactivatorsatthehumanb-interferongene

Theconservationoftheinterferon-benhancerDNAsequencesacrossspeciesseparatedby100millionyears.Thecrystalstructureoftheenhanceosome*Whenalloftheregulatoryproteinsareboundandinteractingcorrectly,theyforma“l(fā)andingpad,”ahigh-affinitybindingsitefortheproteinCBP,aco-activatorproteinthatalsorecruitsthetranscriptionalmachinery.ThelargeCBPproteinalsocontainsanintrinsichistoneacetylaseactivitythatmodifiesnucleosomesandfacilitateshighlevelsoftranscription用肘輕推nuc2isattheTATAboxandtranscriptionstartsite37bpofftheTATAboxTheB-interferonenhanceosomeactstomovenucleosomesbyrecruitingtheSWI–SNFcomplex*Combinatorycontrol-------activatorsandrepressorsworktogether----------complexityanddiversityofeukaryotesExample:combinatorycontrolofthemating-typegenesfromS.cerevisiaeThecelltypes(theaandαhaploid,andthea/αdiploid)aredefinedbythesetsofgenestheyexpress-----determinedbymating-typeregulators:Oneubiquitousregulator(Mcm1)andthreecell-type-specificregulators(a1,α1,andα2),encodedbytheMATlocus.*2.SignalTransductionandtheControlofTranscriptionalRegulatorsSignalsTranscriptional

regulatorssignaltransductionpathwayMechanismsI:UnmaskingActivatingRegionby:

aconformationalchangeintheDNA-boundactivator,revealing

apreviouslyburiedactivatingregion,

orbyreleaseofamaskingprotein

thatpreviouslyinteractedwith,andeclipsed,anactivatingregion.

throughbinding

liganddirectlyorthroughaligand-dependentphosphorylation.Gal3bindsgalactoseandATPLessonsfromYeast-TheGALSystem:Whenlackinggalactose,GALgenesaresilent.Inpresenceofgalactose(andabsenceofglucose),GALgenesareinduced.GAL3,GAL4,and

GAL80encodeproteinsregulatingtheenzymegenes

expression*EMBOreportsVOL7|NO5|2006Gal80doesnotexhibitnucleocytoplasmicexchangeGalactosedoesnotcauseGal3entryintonucleustoaccountfortherapidGal4-mediatedgeneactivationNuclear-localizedGal3isrequiredforrapidinductionStableGal3–Gal80–Gal4complexisnotdetectableingalactose-inducedcellsGal80complexedwithGal4onDNAdoesnotexchangerapidlywithfreeGal80TransientassociationofGal3withGal4-associatedGal80:aplausiblemechanismoftheGALgeneswitch**Phosphorylationof

RbcausesreleaseofitfromE2Fandactivationofthegenes.MechanismII:Themaskingproteinmaynotonlyblocks

activatingregion

butalsoisitself(orrecruits)adeacetylase→activelyrepressesthe

gene.MutantRb:-inactivatingmutation-RbgenedeletionTherepressorRb(

retinoblastomaprotein)

—

controlsactivityofE2Fbybindingtoit---bothblockingactivationandrecruiting

adeacetylase

ThemammalianactivatorE2F

bindssites

upstreamofitstargetgenes.uncontrolledcellcycle2.SignalTransductionandtheControlofTranscriptionalRegulatorsMechanisms:Transportintoandoutofthenucleus:Whennotactive,manyactivatorsandrepressorsareheldinthecytoplasm

throughinteractionwithaninhibitoryprotein,orwiththecellmembrane.Thesignalingligandcausesthemtomoveintothenucleustoactivatetranscription;

Acascadeofkinasescause

thephosphorylationofregulator

innucleus;Theactivatedreceptoriscleavedbycellularproteases,andthec-terminalportionofthereceptorentersthenucleusandactivatestheregulatorTheSTATpathwayTheMAPkinasepathwayThesignalisthenrelayed(分程傳遞)totherelevanttranscriptionalregulatorThetranscriptionalregulatorcontrolthetargetgeneexpressionThesignaliscommunicatedtotheintracellulardomainofreceptor(viaanallostericchangeordimerization)Theligand(“signal”)bindstoanextracellulardomainofaspecificcellsurfacereceptor*Signalingmechanismslinkingneuronalactivitytogeneexpressionandplasticityofthenervoussystem-------AnnuRevNeurosci.;31:563-5902.Thesepathwaysconvergeonpreexistingtranscriptionfactorsinthenucleusandleadtotheiractivationthroughdirectposttranslationalproteinmodifications.1.Calciuminfluxthroughneurotransmitterreceptorsorvoltage-gatedcalciumchannelsleadstoactivationofcalcium-regulatedsignalingenzymes,settinginmotionseveralsignaltransductioncascades.3.Severaloftheactivity-regulatedgenesencodetranscriptionalregulators,whichpromotetranscriptionofadditionalactivity-regulatedgenes.4.Geneticmutationsingenesthatencodethesignalingmoleculesgiverisetoneurologicaldisordersinhumans(yellowboxes).3.Manyotheractivity-regulatedgenesencodeproteinsworkingindendritesoratsynapsesandtherebycoordinateactivity-dependentdendriticandsynapticremodelingwithintheneuron.*GeneralMechanismsofTranscriptionalRegulationinEukaryotesRecruitmentofProteinComplexestoGenesbyEukaryoticActivators.TranscriptionalRepressorsSignalIntegrationandCombinatorialControlSignalTransductionandtheControlofTranscriptionalRegulatorsGeneSilencingbyModificationofHistonesandDNAEpigeneticGeneRegulation

Gene“Silencing”byModificationofHistonesandDNATranscriptionalSilencing:aspecializedformofrepressionthatcanspreadalongchromatin,switchingoffmultiplegenes,eventhosequitedistantfromtheinitiatingevent,withouttheneedforeachtobearbindingsitesforspecificrepressor.Transcriptionalsilencingisapositioneffect:Ageneissilencedbecauseofwhereitislocated,notinresponsetoaspecificenvironmentalsignal.Transcriptionalsilencingisassociatedwithmodificationofnucleosomesthatalterstheaccessibilityofagenetothetranscriptionalmachineryandotherregulatoryproteins.SIR2,3,and4havebeenfoundasregulatorsofsilencing(SIRstandsforSilentInformationRegulator).Rap1recruitsSircomplextothetemomere.Sir2deacetylatesnearbynucleosomeThespreadingofsilencingisrestricted/controlledbyinsulatorsandotherkindofhistonemodificationsthatblockbindingoftheSir2proteins.TranscriptioncanalsobesilencedthroughchromatinmethylationbyhistonemethyltransferaseMethylationofH3K9isamodificationassociatedwithsilencedheterochromatininhighereukaryotesandintheyeastS.pombe.Incontrast,methylationofH3K4areassociatedwithincreasedtranscription.InS.cerevisiae,methylationofspecificlysineresiduesinthetailsofhistonesH3andH4helprepressionofsomegenesandblockspreadingofSir2-mediatedsilencinginothers.WhenmammaliancellDNAisstainedwithfluorescentdye,heterochromatin,whichisadenselycondensedassemblyofchromatin,becomesvisibleunderbrightlight(left).Thebasicstructureofheterochromatinisalsofoundinfissionyeast(right).Themostcommonformofsilencing:adenseformofchromatin-----“heterochromatin”.Usuallyatparticularregionsofthechromosome,eg.,thetelomeres,andthecentromeres:constitutiveheterochromatinInmammaliancells,about50%anizedinto30-nmchromatinfibersVariegation:Insteadofbeingsilencedinallcellsallthetime,thegeneswitchesbetweenthesilencedandexpressedstateatrandom,being“on”insomecellsand“off”inothers.TheSu(Var)3-9orClr4proteinisaH3K9methyltransferaseDifferenttypesofmodificationatthehistonescanbeinvolvedindistinctgeneregulationMultipleformsofmodification:the“HistoneCode”:Thedirecteffectsofmodificationsonchromatindensityandform.ParticularpatternofmodificationsatanygivenlocationwouldrecruitspecificproteinsDNAMethylationIsAssociatedwithSilencedGenesinMammliancellsThemethylatedDNAsequencesalonecandisruptbindingofthetranscriptionmachineryandactivatorsinsomecases;

theycanalsobe

recognizedbyDNA-bindingproteins

thatrecruithistonedeacetylasesandhistonemethylases,whichthenmodifynearbychromatin.Initsunmodifiedstate,themammaliangeneexpressionisneverfirmlyshutoff—itisleakyDNAmethyltransferasemethylatecytosineswithinthe

promotersequence,thegeneitself,or

upstreamactivatorbindingsitesDNAmethylation

cancause

Imprinting:

Inadiploidcell,asamegenefromfatherormotherisdifferentiallyexpressedICR:imprintingcontrolregion,aninsulatorThemethylationstateoftheinsulatorelementdetermineswhetherornottheICRbindingprotein(CTCF)canbindandblockactivationoftheH19genefromthedownstreamenhancerTheH19geneisfurtherrepressedonthepaternalchromosomebythebindingofMeCP2tothemethylatedICR.Manystepsarerequiredforimprinting

Soonafterfertilization,mammalssetasidecellsthatwillbecomegermcells.Imprintsareerasedbeforethegermcellsform→epigeneticallyequivalent.Astheprimordialgermcellsbecomefullyformedgametes,imprintedgenesreceivesex-specificmarkdeterminingwhethertheywillbeactiveorsilentafterfertilization.Cloningisextremelyinefficientinallspeciestested:KnowingthegenomesequenceisonlythefirststepinunderstandinghowgenesareregulatedEpigeneticGeneRegulationTheinheritanceofgeneexpressionpatterns,intheabsencetheinitiatingsignalAfter

specificgenes

expressioninasetofcellsareswitchedon,byasignal,

thesegenesmayremainswitchedonformanycellgenerationsinthesecellseveniftheoriginalsignal

isnolongerpresent.Whenthelysogeniccelldivides,eachdaughtercellinheritsa

copyofthephageDNAandsomerepressorprotein,sufficienttostimulatefurtherrepressorsynthesis.Muchofgeneregulationduringdevelopmentofmuticellularorganismsworksinjustthisway:Afeedback-balancesystemCellhasits"memory"CertainDNAmethylases

(recruitedbycertainsignal)canmethylate,atlowfrequency,previouslyunmodifiedDNA;butfarmoreefficiently,themaintenancemethylasesmodifyhemimethylatedDNA,afterreplicationoffullymethylatedDNAPatternsofDNA/Nucleosomemodifications

canbemaintainedthroughcelldivision70%~80%ofallCGdinucleotidesaremethylatedgenome-wide.MostoftheunmethylatedCGarefoundinclustersnearpromoters:the