Chapter 9-II - Transcriptional Control of Gene Expression
Chapter9-II- TranscriptionalControlofGeneExpression
TranscriptionalControlofGeneExpression
9.3RNAPolymeraseIIPromotersandGeneralTranscriptionFactors• ThreetypesofpromotersequencesineukaryoticDNA:
• TATAbox– prevalentinhighlytranscribedgenes• Initiatorpromotersinsomegenes• CpGislands,thepromotersforabout70percentofprotein-codinggenesinvertebrates,arecharacteristicofgenestranscribedatalowrate.
• Sequentialbindingofgeneraltranscriptionfactorsinitiatestranscriptionofprotein-codinggenesbyPolII.
Corepromoterelementsofnon-CpG islandpromotersinmetazoans
• RNApolymeraseIIpromotersequences:• SeveraldifferenttypesofgeneDNAsequences– TATAboxes,initiators,andCpG islands(notshown)
• SpecifywherepolymeraseinitiatestranscriptionofanRNAcomplementarytothetemplatestrandofthegeneDNA.
• TATAbox – singlebasechangedecreasesgenetranscription• Initiatorsequences– degeneratesequence• CpG islands(“p”representsthephosphatebetweentheCandGnucleotides):
• controlhousekeepinggenesexpressedatlowconstantlevels• hardertobendaroundnucleosomes– formnucleosome-freeregions
• (Largerfont– mostfrequentlyobservedbasesinthatposition;A+1 isthebaseatwhichtranscriptionstarts;Yisapyrimidine(CorT),Nisanyofthefourbases.)
AnalysisofelongatingRNApolymeraseIImoleculesinhumanfibroblasts.
• Nucleiisolatedfromculturedfibroblasts– incubatedinabufferwithanon-ionicdetergentthatallowsRNApolymeraseIIcontinuationoftranscriptionbutpreventsinitiationofnewtranscription
• Treatednuclei– incubatedwithATP,CTP,GTP,andBr-UTPfor5minutesat30°C– PolIIincorporates~100nucleotides
• Sensetranscripts:• peakat~+50– PolIIpausesinthe+50to+200regionbeforeelongatingfurther
• manytranscribelongerthan1kb• Antisensetranscripts:
• peakat~-250– PolIIpausesattheotherendofthenucleosome-freeregionsinCpG islandpromoters
• fewertranscriptslongerthan1kb
Whatdoyouknowwiththisdata?1. Transcriptionstartsite?2. Elongation?
Thechromatinimmunoprecipitationtechniquelocalizeswhereaproteinofinterestassociateswiththegenome.
• (a)Experiment:• Step1:Liveculturedcellsortissuesincubatedin1percentformaldehyde –covalentlycross-linksproteinstoDNAandproteinstoproteins
• Step2:Preparationsonicated – solubilizeschromatinandshearsitinto200–500bpfragmentsofDNA
• Step3:Anti-RNApolymeraseIIantibody–precipitatesRNApolymeraseIIanditscovalentlylinkedDNA
• Step4:Reversecross-linkingandisolateDNA• SequenceDNAbymassivelyparallelDNAsequencing.
• (b)Results(inmouseembryonicstemcells):• DataareplottedasthenumberoftimesaDNAsequenceina50-bpintervalwasfoundpermillionbasepairssequenced.
• (left)RNApolymeraseIItranscribinggeneinbothdirections
• (right)RNApolymeraseIItranscribinggeneonlyinthesensedirection
Think…
Seethefigure(b)anddiscuss1. Whatcanyouexpectthepromoter
typesofleftandrightpanels?Pleaseexplainthereasons
2. Youcanseethepeaksinbothpanels.Whatcanyouexplainthisinsenseoftranscriptionalelongation?
ModelforthesequentialassemblyofanRNApolymeraseIIpreinitiationcomplex.
• Generaltranscriptionfactors:• BindtopromotersofgenestranscribedbyRNApolymeraseII
• PositionRNApolymeraseIIatstartsitesandassistintranscriptioninitiation
• Mechanism:• Preinitiationcomplex(PIC)– generaltranscriptionfactorsandpurifiedRNApolymeraseII(PolII)bindsequentiallytoTATAboxDNA
• TFIIHhelicaseATPhydrolysis– providesenergyforunwindingDNAatthetranscriptionstartsiteandpushingdownstreamDNAintothepolymerase
• ThePICTFIIDTBPsubunit– holdsDNAinpositionbybindingtotheTATAbox
• StrainonDNAstructure– assiststheTFIIBN-terminalregionandPolIItomelttheDNAatthetranscriptionstartsite,formingthetranscriptionbubble
• PolII:• initiatestranscriptionintheresultingopencomplex
• continuestranscriptionawayfromthepromoter
• TheTFIIHkinasedomainphosphorylatesthePolIICTD.
• Generaltranscriptionfactorsdissociatefromthepromoter.
Eukaryotictranscription
https://www.youtube.com/watch?v=icZjgZozkB8
Cryoelectron microscopicstructureoftheyeastRNApolymeraseIIpreinitiationcomplex.
• YeastPolIIpreinitiationcomplex (PIC):• Composedof33polypeptides(~sizeofaribosomalsubunit).
• RNApolymeraseII• Generaltranscriptionfactors
• TFIIH:• Ssl2subunit:
• helicasethatbindstheDNAandhelpsopentheDNAstrandsatthetranscriptionstartsite
• pushesDNAthatisboundupstreamtoTBP,TFIIB,andTFIIA– createstorsionalstressthatcontributestotranscriptionbubblemelting
• Kinasemodule– phosphorylatesserine5ofthePolIICTD
Elongation
Elongationregulation
NELF:negativeelongationfactor
The inhibition of Pol II elongation that results from NELF binding is relieved when DSIF, NELF, and serine 2 of the Pol II CTD repeat (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) are phosphorylated by a protein kinase with two subunits, CDK9-cyclin T, also called P-TEFb, which associates with the Pol II, NELF, DSIF complex
DSIF(DRBsensitivity-inducingfactor)
Modelofantitermination complexcomposedofHIVTatproteinandseveralcellularproteins
NELF:negativeelongationfactorDSIF(DRBsensitivity-inducingfactor)
• HIVTat:• Asequence-specificRNA-bindingprotein– bindstotheRNAcopyofTARsequencetoformastem-loopstructurenearthe5ʹendoftheHIVtranscript
• Functionsasanantitermination factor– permitsRNApolymeraseIItoreadthroughatranscriptionalblock
• ActivatescyclinT-CDK9• HIVtranscriptTARelement– bindsTatandcellularcyclinT• CyclinT
• ActivateskinaseCDK9• PositionsCDK9nearitscellularsubstrates– CTDofRNApolymeraseII,NELF,andDSIF
• PolIICTDphosphorylationofheptadrepeatserine2– requiredfortranscriptionelongation• CellularproteinsDSIFandtheNELFcomplex– involvedinregulatingPolIIelongation
TranscriptionalControlofGeneExpression
9.4RegulatorySequencesinProtein-CodingGenesandtheProteinsThroughWhichTheyFunction• Expressionofeukaryoticprotein-codinggenesisregulatedthroughmultipleprotein-bindingtranscription-controlregionslocatedatvariousdistancesfromthetranscriptionstartsite.
• PromotersdirectbindingofRNApolymeraseIItoDNA,determinethesiteoftranscriptioninitiation,andinfluencethefrequencyoftranscriptioninitiation.
• Promoter-proximalelementsandenhancersarecell-type-specific.
• Transcriptionactivatorsandrepressors– modularproteinscontainingasingleDNA-bindingdomainandoneorafewactivationorrepressiondomains
Linkerscanningmutationsidentifytranscriptioncontrolelements
• Results:• LSmutations1,4,6,7,and9– littleornoeffectonexpressionofthereportergene–regionscontainnocontrolelements
• LSmutations2,3,5,and8– reporter-geneexpressionsignificantlyreduced– regionscontaincontrolelements
Generalorganizationofcontrolelementsthatregulategeneexpressioninmulticellulareukaryotesandyeast.
upstreamactivatingsequence(UAS)
• MammaliangeneswithaCpG islandpromoter:• Transcription– initiatesatseveralsitesinboththesenseandantisensedirectionsfromtheendsoftheCpG-richregion
Findthedifferences…
Howtogettheregionwherethetranscriptionfactorsbind?
DNaseIfootprinting revealstheregionofaDNAsequencewhereatranscriptionfactorbinds.
• (a)Experiment:• ADNAfragmentknowntocontainatranscription-control
element– labeledatoneendwith32P(reddot)• LabeledDNA– digestedwithDNaseIinthepresenceandin
theabsenceofproteinsamplescontainingasequence-specificDNA-bindingprotein
• DNaseIhydrolyzesthephosphodiesterbondsofDNAbetweenthe3ʹoxygenonthedeoxyriboseofonenucleotideandthe5ʹphosphateofthenextnucleotide.
• alowconcentrationofDNaseIisusedsothat,onaverage,eachDNAmoleculeiscleavedjustonce.
• (left)Noproteinbinding:• DNAfragmentiscleavedatmultiplepositions
betweenthelabeledandunlabeledendsoftheoriginalfragment.
• generatesmultiplefragments• (right)Boundprotein:
• protectsdigestionofDNAboundtoproteins• blocksgenerationofsomefragments
• DNA– separatedfromprotein,denaturedtoseparatethestrands,andelectrophoresed
• Autoradiography:• detectsonlylabeledstrands• revealsfragmentsextendingfromthelabeledendto
thesiteofcleavagebyDNaseI• missinggelbandsconstitutefootprintofregions
whereproteinswerebound
DNaseIfootprinting revealstheregionofaDNAsequencewhereatranscriptionfactorbinds.
• (b)Results:FootprintsproducedbyincreasingamountsofTBP(triangle)andofTFIIDonthestrongadenovirusmajorlatepromoter
Bindingoftranscriptionfactors?
?
Vs.
Howtodetectit???
Bindingoftranscriptionfactors?
?
Howtodetectit???
+
Someproteinsoraprotein
DNAelementfromgene
Theelectrophoreticmobilityshiftassaycanbeusedtodetecttranscriptionfactorsduringpurification.
• Proteinfractionsseparatedbycolumnchromatography– assayedforbindingtoaradiolabeledDNA-fragmentprobecontainingaknownregulatoryelement
Aninvivotransfectionassaymeasurestranscriptionactivitytoevaluateproteinsbelievedtobetranscriptionfactors
• Experimentalsystem– twoplasmids:• TFplasmid– containsgeneencodingtheputativetranscriptionfactor(ProteinX)
• Reportergeneplasmid– containsoneormorebindingsitesforproteinXandareportergene(e.g.,luciferase)
• Bothplasmids– simultaneouslyintroducedintocellsthatlackthegeneencodingproteinX
• Measureproductionofreporter-geneRNAtranscriptsorencodedprotein(luciferase)activity
• Results:comparereporter-genetranscriptioninthepresenceandabsenceoftheX-encodingplasmid:
• GreaterwithProteinX– ProteinXisactivator• LesswithProteinX– ProteinXisrepressor
Whichproteindomainhasacriticalroleforthetranscription?
Schematicdiagramsillustratingthemodularstructureofeukaryotictranscriptionactivators.
• TranscriptionfactorsmaycontainmorethanoneactivationdomainbutrarelycontainmorethanoneDNA-bindingdomain:
• Gal4andGcn4– yeasttranscriptionactivators
• Glucocorticoidreceptor(GR)–promotestranscriptionoftargetgeneswhenglucocorticoidhormonesbindtotheC-terminalactivationdomain
• SP1– bindstoCpG promoterelementsinalargenumberofmammaliangenes
Interactionofbacteriophage434repressorwithDNA.
• ProteinsbindingtospecificDNAsequences:• CommonlyinvolvesnoncovalentinteractionsbetweenatomsinanDNA-bindingdomainαhelixandatomsontheedgesofthebaseswithinthemajorgrooveintheDNA
• Mayinvolveionicinteractionsbetweenpositivelychargedrepressorresiduesarginineandlysineandnegativelychargedphosphatesinthesugar-phosphatebackboneandwithatomsintheDNAminorgroove
• Bacteriophage434repressor:• dimericprotein• helix-turnhelixmotif– interactsintimatelywithonesideoftheDNAmoleculeoveralengthof1.5turns
• arecognitionorsequence-readingαhelixfromeachmonomer– insertsintothemajorgrooveintheDNAhelix
DNAbindingmotifs
EukaryoticDNA-bindingdomainsthatusean𝛂 helixtointeractwiththemajorgrooveofspecificDNAsequences.
• (a)C2H2 zincfinger– mostcommonDNA-bindingmotifencodedinthehumanandothermammaliangenomes
• Zn2+-bindingmotif:• 23–26consensussequenceresidues• containstwoconservedcysteine(C)andtwoconservedhistidine(H)residues,whosesidechainsbindoneZn2+ ion
• GL1DNA-bindingdomain:• monomeric• containsfiveC2H2 zincfingers– fingers2–5interactwithDNA
• (b)C4 zincfingerproteins:• Motiffoundin50humantranscriptionfactors,includingnuclearhormonereceptors
• FourconservedcysteinesbindZn2+• BindsDNAashomodimer– oneαhelixineachmonomerinteractswiththeDNA
• (c)Leucine-zipperproteins:• (d)bHLH proteins:
Combinatorialpossibilitiesduetoformationofheterodimerictranscriptionfactors
(a)HeterodimerictranscriptionfactorsinwhichtheactivationdomainofeachmonomerrecognizesthesameDNAsequence
• (b)HeterodimerictranscriptionfactorsinwhichtheactivationdomainofeachmonomerrecognizesdifferentDNAsequences:
Cooperativebindingoftwounrelatedtranscriptionfactorstoneighboringsitesinacompositecontrolelement
• (a)NFAT-AP1regulationofIL-2gene:• MonomericNFATandheterodimericAP1transcriptionfactors– eachalonehaslowaffinityfortheirrespectivebindingsitesintheIL-2promoter-proximalregion
• NFATandAP1interaction–• twoproteinsbindcooperativelytothecompositesite
• increasesoverallstabilityoftheNFAT-AP1-DNAcomplex
TranscriptionalControlofGeneExpression
9.5MolecularMechanismsofTranscriptionRepressionandActivation• Eukaryotictranscriptionactivators/repressorsaffectgeneexpressionbybindingtomultisubunitco-activators/co-repressorsthatmodulatechromatinstructureorinteractwithPolIIandgeneraltranscriptionfactors.
• Thehighlycooperativeassemblyofpreinitiationcomplexesinvivorequiresseveralactivators.Acellproducesthespecificsetofactivatorsrequiredfortranscriptionofaparticulargenetoexpressthatgene.
• Mediatorcomplex– formsamolecularbridgebetweenactivatorboundtocognateDNAsiteandPolII
Mediator(coactivator) isa multiproteincomplex thatfunctionsasa transcriptional coactivator inall eukaryotes.Itwasdiscovered inthelabof RogerD.Kornberg,winnerofthe2006 NobelPrizeinChemistry.Mediatorcomplexesinteractwith transcriptionfactors and RNApolymeraseII.Themain(butnotexclusive)functionofmediatorcomplexesistotransmitsignalsfromthetranscriptionfactorstothepolymerase.
Howtotransmitthesignal?
Signal
Receptor
Nucleus
DNA
ActivatorMediator
PolII
Promoter
Enhancer
ModelofseveralDNA-boundactivatorsinteractingwithasingleMediatorcomplex.• IndividualMediatorsubunitsbindtospecificactivationdomains
• MultipleactivatorsmayinfluencetranscriptionfromasinglepromoterbyinteractingwithaMediatorcomplexsimultaneouslyorinrapidsuccession.
• DifferentMediatorsubunitsinteractingwithspecificactivationdomains– maycontributetointegrationofsignalsfromseveralactivatorsatasinglepromoter
Points1. Mediator2. Twoactivators3. LoopofDNA
Cis-actingelement:enhancer
Signal
Receptor
Nucleus
DNA
ActivatorMediator
PolII
Promoter
Enhancer
Cis-actingelements
ConsensussequencesofDNAresponseelementsthatbindfivenuclearreceptors.
palindromic sequence
Example>CREB-mediatedtranscription
Activator
Signal
Receptor
Nucleus
DNA
ActivatorMediator
PolII
Promoter
Enhancer
CREB
CREB (cAMP responseelement-bindingprotein) isacellular transcriptionfactor.Itbindstocertain DNA sequencescalled cAMP responseelements (CRE),therebyincreasingordecreasingthe transcription ofthe downstream genes.
cAMP-responsivetranscriptionfactor
e.g.SNAT2gene
CREB
CREBrecruitsotherregulatoryproteins.
CREBincAMP signaling
Interactingwithoutsideenvironment.
Interactingwithoutsideworld.
Signal
Receptor
Nucleus
DNA
ActivatorMediator
PolII
Promoter
Enhancer
Decondensation ofChromatin
• Histoneacetylationanddeacetylation• Epigenetics…
Chromatinandhistone
HowtobindtheDNA?Charge?
StructureofHistone
Whichchargeisnecessary?
Histone
AllhistoneshaveahighlypositivelychargedN-terminuswithmany lysine and arginine residues.
Q.HowtodetachtheDNAfromhistone?
Charge?
AcetylationandDeacetylation"histoneacetyltransferase"(HAT)or"histonedeacetylase"(HDAC)
Acetylationremovesthepositivechargeonthehistones,therebydecreasingtheinteractionoftheNterminiofhistoneswiththenegativelychargedphosphategroupsof DNA.
AcetylationandDeacetylation
https://www.youtube.com/watch?v=Tze3XR4Kcj4
Proposedmechanismofhistonedeacetylationandhyperacetylation inyeasttranscriptionalcontrol.
(a)Ume6repressor-directeddeacetylationofhistoneN-terminaltails
(b)Gcn4activator-directedhyperacetylation ofhistoneN-terminaltails
CREB-CBP=>Acetylation
Expressionoffusionproteinsdemonstrateschromatindecondensation inresponsetoanactivationdomain.
• Culturedhamstercellline– engineeredtocontainmultiplecopiesofatandemarrayofE.colilacoperatorsequencesintegratedintoachromosomeinaregionofheterochromatin
• (a)lacrepressor(LacI)expressionvectortransfectedintothesecells– cellsexpresslacrepressor:
• lacrepressorantibody(red)–showslacrepressorbindingtolacoperatorsitesinaregionofcondensedchromatin(DNAstainedwithDAPI[blue])
• diagram– condensedchromatin• (b)LacI fusedtoanactivationdomainand
transfectedintocells–• Activationdomaincauses
chromatindecondensation intoathinnerchromatinfiber– fillsamuchlargervolumeofthenucleus
• Diagram– decondensed chromatinwithboundLacI-VP16activationdomain(AD)fusionsandassociatedchromatinremodelingandhistoneacetylase complexes
9-3HistonePost-TranslationalModificationsAssociatedwithActiveandRepressedGenes
Associationofarepressedtransgenewithheterochromatin.
• DAPI(blue)– stainsallDNA– heterochromatinstainsbrighter(DNAconcentrationishigherthanineuchromatin)
• (left)(–)hormone–• Recombinantrepressorretainedinthecytoplasm• Transgene– transcribedandassociatedwitheuchromatin
• (right)(+)hormone–• Recombinantrepressorinthenucleus• Transgene– expressionrepressedandassociatedwithheterochromatin
• atransgenecontainingbindingsitesforanengineeredrepressorfusionprotein
MaintenanceofhistoneH3lysine9methylationduringchromosomereplication
H3lysine9methylationismaintainedfollowingchromosomereplication.
• Xinactivation:• Controlledbya~100-kbdomainontheXchromosome– X-inactivationcenter
• X-inactivationcenter– encodesseverallncRNAs requiredfortherandominactivationofoneentireXchromosomeearlyinthedevelopmentoffemalemammals.
Discussionwithfriends1. An electrophoretic mobility shift assay (EMSA) was performed using a radiolabeled DNA fragment from the sequence upstream of gene X. This DNA probe was incubated with (+) or without (-) nuclear extract isolated from tissues A (bone); B (lung); C (brain); and D (skin). The DNA;protein complexes were then fractionated on nondenaturing polyacrylamide gels. The gels were exposed to autoradiographic film; the results are presented in the figure.
a. Which tissues contain a binding activity that recognizes the sequence upstream of gene X? Is the transcription factor the same in each tissue?b. If the binding activity was purified, what test could be done to verify that thisfactor is in fact a transcription factor?
2.FindtheXchromosomeinactivationanditsmechanisms.
3.WhatarethedifferencesbetweenhistonemethylationandDNAmethylation?