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?