现代分子生物学之真核细胞转录调控课件

现代分子生物学之真核细胞转录调控UASsUASs(upstream activation sequencesupstream activation sequences)=)=enhancersenhancersEach UAS is 17bp long,bound by a Gal4 dimerThe GAL System The GAL System in in YeastYeast:1.When lacking galactose,GAL genes are silent.2.In presence of galactosepresence of galactose(and absence of glucoseand absence of glucose),GAL genes are induced.(a)The DNA-binding domain of Gal4,without that proteins activation domain,can still bind DNA but cannot activate transcription.(b)Attaching the activation domain of Gal4the activation domain of Gal4 to the DNA-binding domain of the bacterial DNA-binding domain of the bacterial protein LexAprotein LexA creates a hybrid proteinhybrid protein that activates transcription of a gene in yeastif that gene bears a binding site for LexA.Expression is measured using a reporter plasmid in which the GAL1 promoter is fused to GAL1 promoter is fused to the E.coli lacZ genethe E.coli lacZ gene whose product(b-galactosidase)is readily assayed in yeast cells.Transcription Transcription activatorsactivators have have separateseparate DNA binding DNA binding andand activating activating domainsdomains:the Domain sDomain sGAL1 GAL1 promoterpromoterBacterial regulatory proteins mostly use the helix-turn-helix motifhelix-turn-helix motif to bind DNA targetRegulatory proteins:DNA binding regionsHomeodomainHomeodomainOne helix(for recognition,#3)One helix(for recognition,#3)fits in major groove and recognizes specific base pairs.The other helix(#2)The other helix(#2)makes contacts with the DNA backbone,positioning the recognition helix properly and increasing the strength of binding Eukaryotic regulators use a range of DNA binding Eukaryotic regulators use a range of DNA binding domainsdomains1.1.Homeodomain proteins(HTH):Homeodomain proteins(HTH):helix-turn-helix helix-turn-helix 2.2.Zinc containing DNA-binding domain:zinc finger Zinc containing DNA-binding domain:zinc finger/zinc cluster/zinc cluster3.3.Leucine zipper motifLeucine zipper motif4.4.Helix-Loop-Helix proteinsHelix-Loop-Helix proteins 5.5.HMG:interact with minor groove/alter DNA HMG:interact with minor groove/alter DNA conformation conformation zinc finger proteinsThe zinc atomThe zinc atom interacts with cysteinecysteine and histidinehistidine residues and serves a serves a structural role structural role essential for integrity of the DNA-binding domainintegrity of the DNA-binding domainThehelix for recognition is presented to DNA by sheet on the right.The zinc is coordinated by the two histidine in thehelix and two cysteine in thesheetThe zinc is coordinated by four cysteine residuesLeucine zipper proteinmajor grooveLeucine zipper motifThe dimerization surface is formed from two helical regions:the first is part of the same helix involved in DNA recognition;the other is a shorter helix.These two helices are separated by a flexible loop that allows them to pack togetherHelix-Loop-Helix Proteins*7Activating Regions Are Not Well-Defined StructuresActivating Regions Are Not Well-Defined StructuresThey are adhesive surfaces capable of interacting with other protein surfaces;Not in a“lock and key”mannerNot in a“lock and key”manner;are grouped on the basis of amino acids content.Acidic region:contain both critical acidic AAs and hydrophobic AAs.Glutamine-rich regionProline-rich regionDNA binding domain comprises DNA binding domain comprises three Cys2His2-like zinc fingersthree Cys2His2-like zinc fingersThe activation domains are composed The activation domains are composed of of serine/threonineserine/threonine rich regions,rich regions,flanked by regions rich in flanked by regions rich in glutamineglutamine*Eukaryotic activators recruit polymerase indirectlyindirectly1.Interacting with partsparts of the transcription machinery(GTFs,other proteins for RNAPII innitiation and elogation,etc.)2.Recruiting nucleosome modifiersnucleosome modifiers /remodelersremodelers that alter chromatin structure,to help initiationGal4 interacts with mediator complex,which directly recruit RNAP II to gene promoters.Gal4 binds to TBP and recruits the TFIID complex and,RNAP II to promoter.The mediator mediator act as a co-activator(facilitate gene activation by TF,but itself is itself is neitherneither part of the part of the transcriptional machinery transcriptional machinery nornor a DNA-binding a DNA-binding protein)protein)Activators interact with Activators interact with parts of the transcription machineryparts of the transcription machinery Promoters bearing acetylated nucleosomes have higher affinity higher affinity for transcriptional machineryfor transcriptional machineryActivators recruit nucleosome modifiersmodifiers1.Acetylation can alter the DNA-histone interaction the octamer slide octamer slide along the DNA to a new position.2.can alter the interaction between adjacent nucleosomes a more open chromatin.a more open chromatin.3.creates specicreates specificfic binding sites binding sites on nucleosomes for proteins with bromodomainsbromodomains(eg.,TFIID)Activators recruit Activators recruit nucleosome nucleosome remodelremodelersers to“Loosen”the chromatin structureSliding Sliding Requirement Requirement of components of the transcriptional machinery varies under different circumstancesvaries under different circumstancesMany proteinsMany proteins can interact specifically with the Gal4-activation domainGal4-activation domain:TBP,TFIIB,Gal11(a component of Mediator),Cdk8,SWI/SNF(nucleosome remodeler),SAGA(Spt-Ada-Gcn5-acetyltransferase),Srb4(another component of Mediator)and proteasome components Sug1 and Sug2-by 2006EMBO reports VOL 7|NO 5|2006Gal4 functions in most eukaryotes,in many waysGal4 functions in most eukaryotes,in many ways*HSP70 gene from Drosophila,activated by heat shock,is controlled by two activators working together.The GAGA-binding factor GAGA-binding factor can recruit enough of the transcription machinery to the promoter for transcription initiation.But,in the absence of a second activator,HSFa second activator,HSF,most of the initiated polymerases stall some 2550 bp stall some 2550 bp down-stream from the promoter.down-stream from the promoter.In response to heat shock,HSF binds to specific sites at the promoter and recruits a kinase,P-TEFb P-TEFb(positive transcription elongation factor,part of a larger complex,the SEC,super elongation complex),to the stalled polymerases.A strong acidic activator like Gal4 is able to recruit PTEFb/SEC along with the rest of the machinery.The kinase phosphorylates the Ser2 of The kinase phosphorylates the Ser2 of the CTD heptad repeat of RNAPII,the CTD heptad repeat of RNAPII,freeing the enzyme from the stall and allowing transcription to proceed through the gene.*Transcriptional RepressorTranscriptional RepressorIn eukaryotes,most repressors usually do do notnot act by binding to sites that overlap binding to sites that overlap with the promoterwith the promoter and block binding of polymerase.(Bacteria often do so)1.1.Most common:Most common:Compacting the nucleosome Compacting the nucleosome byby directly directly removing/removing/adding some groups or by recruiting nucleosome modifiersrecruiting nucleosome modifiers 2.2.Competing with the activatorCompeting with the activator for an for an overlapped binding siteoverlapped binding site3.Binding to a site different from that of the activator,but physically interacts with an physically interacts with an activatoractivator molecule and blocks its activating blocks its activating regionregion4.Binding to a site upstream of the promoter,physically interacts with the transcription physically interacts with the transcription machinerymachinery at the promoter to inhibit transcription initiation.In the presence of glucoseRepression of the GAL1 gene in yeastMig1 binds to a site between the Gal4-binding site and the GAL1 promoterRecruits the Tup1 protein complexTup1 recruits histone histone deacetylasesdeacetylasesDirectly interacts withDirectly interacts with transcription machinerytranscription machineryRepress transcription.*22Study protein/nucleic acid interactionGel retardation assayGel retardation assay/electrophoretic mobility-shift assayelectrophoretic mobility-shift assay can be used qualitatively to identify sequence-specific DNA-binding proteinssequence-specific DNA-binding proteins(such as transcription factors)Protein:DNA complexes migrate more Protein:DNA complexes migrate more slowly than free linear DNA fragments slowly than free linear DNA fragments in non-denaturing polyacrylamide or agarose gel electrophoresis However,if circular DNA is used,the protein:DNA complex may migrate faster than free DNAThe order of component addition for the binding reaction is often critical.Completed binding reactions are best electrophoresed immediately to preserve potentially labile complexes for detection.(A)Labelled oligonucleotide incubated with increasing amounts of V47R POU mutant protein.0,control DNA probe without protein.Gel retardation assay of DNA-protein complexes.(B)Quantification of gel retardation data.Amount of free DNA as a Amount of free DNA as a function of log protein function of log protein concentrationconcentration.Relative affinity of protein to octamer probe(ATGCAAATGA)can be determined FEBS Letters 412(1997)5-8protein-DNA complexfree DNA241）The protein protects DNA from attack by DNase.2）Treat the DNA-protein complex with DNase under mild conditions mild conditions,so that only one cut occur per DNA molecule on average.Electrophoresis,autoradiographDNase I footprinting:Identify the actual region of sequence with which the protein interacts.The three lanes represent DNA that was bound to 0,1,and 5 units of protein.The lane with no protein shows a regular ladder of fragments.The lane with one unit shows some protection,and the lane with 5 units shows complete protection in the middle.With sequence ladders,we can tell exactly where the protein bound.only one cutonly one cut occur per DNA moleculeoccur per DNA moleculeYeast two hybrid system:to discover proteinprotein interactions to discover proteinprotein interactions andand proteinDNA interactions proteinDNA interactions by using by using yeast fusion proteins,yeast fusion proteins,transcription transcription activation systemactivation system andand reporter genesreporter genes Transcription factorWhen these two domanis are expressed as seperate proteins,the BD will still bind to DNA,but the AD is NOT in the right place to activate transcriptionA split transcription factorBD XYADpromotergeneHybrid proteins as molecular glueTo test if two proteins(X and Y)interact,they are expressed in fusion with the BD and the ADBDpromotergene XYADIf proteins X and Y bind to each other,the transcription factor is reconstituted,and gene expression is activatedADBDGAL4-ADGAL4 and Reporter genesIn many cases,the reconstituted transcription factor is the yeast GAL4 transcriptional activator.reporter geneGAL4-BDGAL4-UASTo monitor transcriptional activation,reporter proteins such as b b-galactosidase-galactosidase are expressed under the control of the GAL4-upstream activating sequence.Application of the yeast two-hybrid systemIdentifies novel protein-protein interactions:Which proteins can interact with an known protein?Need to construct bait and prey plasmids to express fusion proteinsIdentifies mutations that affect protein-protein bindingCan identify interfering proteins in known interactions Exploring protein-protein interactions:Pull-Down AssaysCHIP:chromatin immuno precipitationCHIP-on-chip General Mechanisms of Transcriptional Regulation General Mechanisms of Transcriptional Regulation in Eukaryotes Recruitment of Protein Complexes to Genes by Eukaryotic Activators.Recruitment of Protein Complexes to Genes by Eukaryotic Activators.Transcriptional RepressorsTranscriptional Repressors Signal Integration and Combinatorial ControlSignal Integration and Combinatorial Control Signal Transduction and the Control of Transcriptional RegulatorsSignal Transduction and the Control of Transcriptional Regulators Gene Silencing by Modification of Histones and DNAGene Silencing by Modification of Histones and DNA Epigenetic Gene RegulationEpigenetic Gene Regulation 1.Activators work together synergistically(When an effect is greater than additive)to integrate signalsABCFEDSignal IntegrationSignal Integration and and Combinatorial ControlCombinatorial ControlNumerous signals may be required to switch a gene on;Each signal is transmittedtransmitted to the gene by a separate regulatorby a separate regulator.S1:Multiple activators recruit a single component of the transcriptional machinery,by touching the different part of the mediator complex.S2:Multiple activators each recruit a different component of the transcriptional machinery.These components binds to the These components binds to the promoter DNA promoter DNA inefficientlyinefficiently without help.without help.S3:Multiple activators help each other bind to their sites upstream of the gene they control.Strategies of the synergya.“Classical”cooperative binding.b.Both proteins interacting with a third protein.c.The first protein recruit a nucleosome remodeller whose action reveal a binding site for the second protein.d.Binding a protein unwinds the DNA from nucleosome a little,revealing the binding site for another protein.Example:the HO gene of yeast S.cerevisiae only expressed in mother cells and at certain point in cell cycle,and is controlled by two regulators:recruiting nucleosome modifiers and mediators.SWI5:acts only in the mother cellmother cell and binds unaidedunaided to multiple sites distant from the gene,which recruit enzymes to open the SBF binding sitesSBF:only active at the correct stagescorrect stages of the cell cycle,and cannot bind the sites unaided*InfectionExample:Cooperative binding of activators at human b b-interferon gene.Triggers three activators three activators(communicator)(communicator):NFkB,IRF,and Jun/ATFActivators bind cooperatively to an enhancer located about 1 kb upstream of the promoter,forming enhanceosomeThe human b-interferon genehuman b-interferon gene is activated in cells upon viral infectionviral infectionTranscription is activated to high level only when all the proteins are present and touching one another in just the right wayRole of HMGB as architectural factors*Binding sites of activators at the human b b-interferon gene The conservation of the interferon-b enhancer DNA The conservation of the interferon-b enhancer DNA sequences sequences across species separated by 100 million yearsacross species separated by 100 million years.The crystal structure of the enhanceosome*When all of the regulatory proteins are bound and interacting correctly,they form a“landing they form a“landing pad,”a high-affinity binding site for the protein pad,”a high-affinity binding site for the protein CBP,CBP,a co-activator protein that also recruits the transcriptional machinery.The large CBP protein also contains an intrinsic intrinsic histone acetylase activityhistone acetylase activity that modifies nucleosomes and facilitates high levels of transcription用肘轻推用肘轻推nuc 2 is at the TATA box and transcription start site37 bp off the TATA box37 bp off the TATA boxThe B-interferon enhanceosome acts to move nucleosomes by recruiting the SWISNF complex*Combinatory control-activators and repressors work activators and repressors work togethertogether-complexity and diversity of eukaryotesExample:combinatory control of the mating-type genes from S.cerevisiaeThe cell types(the a and haploid,and the a/diploid)are defined by the sets of genes they express-determined by mating-type regulatorsdetermined by mating-type regulators:One ubiquitous regulator(Mcm1)and three cell-type-specific regulators(a1,1,and 2),encoded by the MAT locus.*2.2.Signal TransductionSignal Transduction and and the Control the Control of Transcriptional Regulatorsof Transcriptional Regulators Signals Transcriptional regulatorssignal transduction signal transduction pathwaypathwayMechanismsMechanisms I I:UnUnmasking masking Activating Region byActivating Region by:a conformational change in the a conformational change in the DNA-bound activator,DNA-bound activator,revealingrevealing a previously buried a previously buried activating region,activating region,or by release of a masking proteinor by release of a masking protein that previously interacted that previously interacted with,and eclipsed,an with,and eclipsed,an activating region.activating region.throughthrough binding binding ligand directly ligand directly oror through a ligand-dependent through a ligand-dependent phosphorylation.phosphorylation.Gal3 binds galactoseand ATPLessons from Yeast-The GAL System:Lessons from Yeast-The GAL System:1.When lacking galactose,GAL genes are silent.2.In presence of galactose(and absence of glucose),GAL genes are induced.*EMBO reports VOL 7|NO 5|20061.Gal80 does not exhibit nucleocytoplasmic exchange2.Galactose does not cause Gal3 entry into nucleus to account for the rapid Gal4-mediated gene activation3.3.Nuclear-localized Gal3 is required for rapid inductionNuclear-localized Gal3 is required for rapid induction4.Stable Gal3Gal80Gal4 complex is not detectable in galactose-induced cells5.Gal80 complexed with Gal4 on DNA does not exchange rapidly with free Gal80TransientTransient association of Gal3 with Gal4-associatedGal80:a plausible mechanism of the GAL gene switch*Phosphorylation ofPhosphorylation of Rb causes release of Rb causes release of it it from from E2F and activation of the genes.E2F and activation of the genes.Mechanism II:Mechanism II:T The masking protein he masking protein may may not only blocksnot only blocks activating regionactivating region but also is itself(or recruits)a but also is itself(or recruits)a deacetylasedeacetylaseactively represses theactively represses the gene.gene.Mutant Rb:Mutant Rb:-inactivating mutation-inactivating mutation-Rb gene deletion-Rb gene deletionT The repressor Rb(he repressor Rb(retinoblastoma protein)retinoblastoma protein)controls activity of E2F by controls activity of E2F by binding to it binding to it-both both blocking activation blocking activation andand recruiting recruiting a deacetylasea deacetylase T The mammalian activator E2Fhe mammalian activator E2F binds sitesbinds sites upstream of its target genesupstream of its target genes.uncontrolled cell cycleuncontrolled cell cycle2.Signal Transduction and the Control of Transcriptional Regulators2.Signal Transduction and the Control of Transcriptional RegulatorsMechanisms:Transport into and out of the nucleusMechanisms:Transport into and out of the nucleus:1.When not active,many activators and repressors are held in the cytoplasmare held in the cytoplasm through interaction with an inhibitory protein,or with the cell membrane.The signaling ligand causes them to move into the nucleusmove into the nucleus to activate transcription;2.2.A cascade of kinases causeA cascade of kinases cause the phosphorylation of regulatorthe phosphorylation of regulator in nucleus;in nucleus;3.3.The activated receptor is cleaved by cellular proteases,and The activated receptor is cleaved by cellular proteases,and the c-terminal the c-terminal portion of the receptorportion of the receptor enters the nucleus and activates the regulator enters the nucleus and activates the regulatorThe STAT pathwayThe MAP kinase pathwayThe signal is then relayed(分程传递分程传递)to the relevant transcriptional transcriptional regulatorregulatorThe transcriptional regulator control the target gene expressiontarget gene expressionThe signal is communicated to the intracellular domainintracellular domain of receptor(via an allosteric change or allosteric change or dimerizationdimerization)The ligand(“signal”)binds to an extracellular extracellular domaindomain of a specific cell surface receptor*Signaling mechanisms linking neuronal activity to gene expression and plasticity of the nervous sys