第七章 真核基因表达调控

EukaryoticGeneExpression

andRegulation第七章真核基因表达调控本章主要内容基因表达与调控的基本概念与原理转录水平的调控(transcriptionalregulation):GeneticlevelEpigeneticlevel转录后水平的调控(post-transcriptionalregulation)：RNAinterference（RNAi）Proteindegradation（Ubiquitin/proteasome）第一节

基本概念与原理BasicConceptsandPrinciplesGenome(cell’srepertoireofDNA)Transcriptome(cell’srepertoireofRNAtranscripts)Proteome(cell’srepertoireofproteins)单个基因单个细胞中心法则一、基因表达的概念基因组(genome)一个细胞或病毒所携带的全部遗传信息或整套基因。基因表达(geneexpression)基因经过转录、翻译，产生具有特异生物学功能的蛋白质分子或RNA分子的过程。基因表达调控(generegulation,orregulationofgeneexpression)

基因表达是受严格调控的。RegulationofGeneExpressionChromatinepigeneticcontrolRNAsilencingProteindegradation一般而言的基因表达调控范畴二、基因表达的时间性及空间性（一）时间特异性按功能需要，某一特定基因的表达严格按特定的时间顺序发生，称之为基因表达的时间特异性(temporalspecificity)。多细胞生物基因表达的时间特异性又称阶段特异性(stagespecificity)。人体发育过程中不同类型β-珠蛋白的含量变化

（二）空间特异性基因表达伴随时间顺序所表现出的这种分布差异，实际上是由细胞在器官的分布决定的，所以空间特异性又称细胞或组织特异性(cellortissuespecificity)。在个体生长全过程，某种基因产物在个体按不同组织空间顺序出现，称之为基因表达的空间特异性(spatialspecificity)。ExpressionpatternofBARD1InSituhybridization（朱玉贤第六章课件）四种母源影响基因的mRNA和蛋白沿果蝇胚胎前-后轴分布的浓度变化图

APBICOIDCAUDALHUNCHBACKNANOS第十章（基因和发育）FactsIdenticalgenome:VirtuallyeverycellinanorganismcontainsacompletesetofgenesSpatialspecificity:ButtheyarenotallturnedonineverycellortissueTemporalspecificity:EachcellofanorganismexpressesadistinctivesubsetofgenesatdifferenttimeordevelopmentalstageTight

regulation:Duringdevelopmentdifferentcellsexpressdifferentsetsofgenesinapreciselyregulatedfashion三、基因表达的方式按对刺激的反应性，基因表达的方式分为：（一）组成性表达(constitutiveexpression)某些基因在一个个体的几乎所有细胞中持续表达，通常被称为管家基因(housekeepinggene)。这类基因表达又称为组成性基因表达(constitutivegeneexpression)。genesforessentialcellularstructuresandmetabolicpathways(e.g.rRNA,actin,tubulin)usuallyexpressedathighleveltheleveloftheirgeneexpressionmayvaryHousekeepinggenes（二）诱导和阻遏表达在特定环境信号刺激下，相应的基因被激活，基因表达产物增加，这种基因称为可诱导基因(inducible

genes)。

如果基因对环境信号应答是被抑制，这种基因是可阻遏基因(repressiblegenes)。基因表达调控大多数是对这些基因的转录和翻译速率的调节,从而导致其编码产物的水平发生改变，影响其功能。四、基因表达调控的生物学意义（一）维持细胞增殖、分化（二）维持个体生长、发育（三）适应环境变化第九、十章（基因与疾病、基因与发育）将要讲到1.Transcripts（转录本）beginandendbeyondthecodingregion(5’UTRand3’UTR)2.Theprimarytranscriptisprocessedby: 5’capping 3’formation/polyA

splicing3.Maturetranscriptsaretransportedtothecytoplasmfortranslation一般而言，基因表达调控主要发生在基因转录水平上的调节，既mRNA合成的多少transcription五、基因转录调节基本要素（一）RNA聚合酶(RNAPolymerase)（二）特异DNA序列(cis-actingelements)（三）调节蛋白(trans-actingfactors)GeneexpressionregulationatthelevelofDNA

(transcriptionalregulation) --highlysequence-dependent --variedregulationfordifferentgenescis-actingelements:promoters/regulatorysequencesofgenestrans-actingfactors:proteinsandRNAsthatbindcis-elementsandpromoteorrepressgeneexpression

(一)RNA聚合酶启动子、调节序列和调节蛋白通过DNA-蛋白质相互作用、蛋白质-蛋白质相互作用影响RNA聚合酶活性。RNA

Pol

I:

rRNA,相对活性50-70%RNA

Pol

II:

mRNA,相对活性20-40%RNAPolIII:tRNA,相对活性10%RNAPolIV:smallncRNA,相对活性??

原核生物的特异DNA序列

原核生物的基因表达调控是通过操纵子机制实现的。

操纵子（operon）是由功能上相关联的多个编码序列（2个以上)及其上游的调控序列（包括操纵序列、启动序列和调节序列）等成簇串联在一起，构成的一个转录协调单位。

（二）特异DNA序列AB编码序列

启动序列

操纵序列

其他调节序列(promoter)(operator)操纵序列

——阻遏蛋白(repressor)的结合位点当操纵序列结合有阻遏蛋白时，会阻碍RNA聚合酶与启动序列的结合，或是RNA聚合酶不能沿DNA向前移动，阻碍转录。启动序列编码序列操纵序列pol阻遏蛋白真核生物的特异DNA序列真核生物基因组中含有可以调控自身基因表达活性的特异DNA序列，称为顺式作用元件(cis-actingelement)。

顺式作用元件能够被转录调节蛋白特异识别和结合，从而影响基因表达活性。启动子(promoter)

顺式作用元件又分增强子(enhancer)沉默子(silencer)En/SiProDNA编码序列转录起始点（三）调节蛋白原核生物的调节蛋白（3类）特异因子---promoter

决定RNA聚合酶对启动序列的特异识别和结合能力；

(RNA聚合酶的－因子)阻遏蛋白---operator

通过与操纵序列结合，阻遏基因转录；（由调节基因表达的阻遏蛋白）激活蛋白---regulatoryelement与启动子上游DNA序列结合，促进RNA聚合酶与启动序列结合，促进基因转录。（CAP:分解代谢物基因活化蛋白）

2.真核基因的调节蛋白

反式作用因子

(trans-actingfactor)

能直接或间接与顺式作用元件相互作用，进而调控基因转录的一类调节蛋白，统称为反式作用因子。按其功能不同，常有以下三类：

基本转录因子:识别promoter元件转录调节因子:识别enhancer或silencer共调节因子：不能进行DNA-蛋白质相互作用RNA聚合酶Ⅱ在转录因子帮助下，形成的转录起始复合物polⅡTFⅡHTAFTFⅡFTAFTAFTFⅡATFⅡBTBPTATADNATAF:TBP

associatedfactorsholoenzyme（1）基本转录因子(generaltranscriptionfactor)

是指能够直接或间接与启动子核心序列TATA盒特异结合、并启动转录的一类调节蛋白。TBP:TATA-boxbindingproteinTFII:polIIassociatedTF(2)转录调节因子(transcriptionfactor,TF)

这类调节蛋白能识别并结合转录起始点的上游序列和远端的增强子元件，通过DNA－蛋白质相互作用而调节转录活性。决定不同基因的时间、空间特异性表达.

转录激活因子(transcriptionalactivator)

转录阻遏因子(transcriptionalrepressor)（3）共调节因子(transcriptionalregulator/co-factor)首先与转录因子发生蛋白－蛋白相互作用，进而影响它们的分子构象，以调节转录活性,本身无DNA结合活性。如果与转录激活因子有协同作用——共激活因子；与转录阻遏因子有协同作用——共阻遏因子。常见转录因子的结构域(domain)DNA结合域（DNA

binding

domain)BasicAA(K/R)rich,positivelycharged转录激活域(trans-activationdomain)TF蛋白质-蛋白质结合域（dimerization,co-factors）

谷氨酰胺(Q)富含域酸性激活域(D/E-rich)脯氨酸(P)富含域最常见的DNA

bindingdomain锌指(zincfinger)C——CysH——His常结合GC

box典型的类固醇激素受体(steroidhormonereceptor)结构示意图(2ZincfingersintheDBdomain)

图8-20碱性亮氨酸拉链（bZIP:

basic

Leu

Zip)结构域转录激活因子序列比较

图8-21碱性亮氨酸拉链转录激活蛋白与调控区DNA相结合的示意图

图8-22bHLH蛋白(basicHelix-Loop-Helix)同源域(Homeodomain)蛋白通过其第三个螺旋与双链DNA的大沟相结合，其N端的延伸部分则与DNA的小沟相结合，提高了稳定性

图8-24常见的转录活化结构域

主要在转录水平进行调节1）σ因子的特异启动作用

2）操纵子调控机制具有普遍性3）结构基因进行多顺反子转录4）阻遏蛋白阻遏转录具有普遍性六、原核基因表达调控的基本原理乳糖操纵子调节机制（一）乳糖操纵子(lacoperon)的结构

调控区CAP结合位点启动序列操纵序列

结构基因Z：β-半乳糖苷酶Y：透酶A：乙酰基转移酶ZYAOPDNAmRNA阻遏蛋白IDNAZYAOPpol没有乳糖存在时（二）阻遏蛋白的负性调节阻遏基因mRNA阻遏蛋白有乳糖存在时IDNAZYAOPpol启动转录mRNA乳糖半乳糖β-半乳糖苷酶++++转录无葡萄糖，cAMP浓度高时有葡萄糖，cAMP浓度低时（三）CAP的正性调节ZYAOPDNACAPCAPCAPCAPCAPCAPmRNA低半乳糖时高半乳糖时

葡萄糖低cAMP浓度高

葡萄糖高cAMP浓度低RNA-polOOOO（四）协调调节※当阻遏蛋白封闭转录时，CAP对该系统不能发挥作用（repressor

is

sufficient)；※如无CAP存在，即使没有阻遏蛋白，操纵子仍无转录活性(CAP

is

necessary)。单纯乳糖存在时，细菌利用乳糖作碳源；若有葡萄糖或葡萄糖/乳糖共同存在时，细菌首先利用葡萄糖。（一）真核生物基因的特点（1）基因组结构庞大。人类的单倍体基因组由3X109的核苷酸组成，含有大约3万个基因。（2）形成染色体结构。真核生物的基因组是以DNA和蛋白质结合形成染色体结构形式而存在于细胞核。（3）单顺反子。真核基因的转录产物一般是单顺反子，即一个编码基因转录生成一个RNA转录本。（4）重复序列。真核生物基因普遍存在重复序列和异染色质。（5）断裂基因。有外显子和内含子。（6）大多数为非编码区。七、真核基因表达调控的基本原理45Typicalstructureofaneukaryoticgeneenhancerorsilencer5’

UTR3’

UTR（二）真核基因表达的多级调控组蛋白修饰DNA甲基化转录调控转录后加工mRNA降解蛋白质降解蛋白质翻译翻译后修饰图8-1真核基因表达调控的主要步骤

染色质去组装蛋白质修饰生化功能1.染色质水平的调节（三）真核基因表达调控的机理

基因表达时（常常在调节蛋白结合位点附近）对核酸酶极度敏感TF染色质水平调节主要依赖于辅助调节因子对染色质结构进行修饰。辅助调节子通过三种方式对染色质结构起调节作用：1）依赖于ATP的核小体重建复合体（ADRC）：它们依靠水解ATP所产生的能量来改变核小体的相对位置，将DNA序列暴露出来，使转录因子能够与之结合。这是一个物理过程，染色质本身的结构并没有变化，只改变核小体的相对位置。2）组蛋白修饰：主要通过共价修饰组蛋白的末端来改变染色质结构。当构成染色质的组蛋白发生修饰时，就会影响染色质的构型，而结构的变化引起基因转录活性的变化。3）DNA甲基化：真核DNA约有5%的胞嘧啶被甲基化。一般而言，DNA甲基化抑制基因表达。

组蛋白发生修饰，碱基暴露等原因而引起核小体结构改变，使核小体不稳定性增加。Sequence-independentlinkerhistones:controlDNAcompactionandaccessibilitytotrans-actingfactorspost-translationalmodificationsofhistonetails:controlcompactionofDNAandserveasdockingsitesfortrans-actingfactors图8-14DNA甲基化对基因转录的抑制作用

2.DNA水平的调节

真核基因一般都处于阻遏状态，RNA聚合酶对启动子的亲和力很低。通过利用各种转录因子正性激活RNA聚合酶是真核基因调控的主要机制。真核基因转录调节是复杂的、多样的*不同的DNA元件组合可产生多种类型的转录调节方式。*多种转录因子又可结合相同或不同的DNA元件。*转录因子与DNA元件结合后，对转录激活过程所产生的效果各异，有正性调节或负性调节之分。HypotheticalmodelforthecontrolofAmy32ba-amylasegeneexpressioninbarleyaleuronecells.A,Negativeregulators(HvWRKY38,BPBF,HRT,andHvMCB1)bindtotheircorrespondingcis-actingelementsintheabsenceofGA.B,Positiveregulators(RAMY,SAD,HvGAMYB,andHvMYBS3)bindtocorrespondingcis-actingelementsinthepresenceofGA.Doublelinesbetweenproteinsindicatethattheirphysicalinteractionshavebeendetected.Thearrowdenotesthetranscriptionstartsite.3.RNA水平的调节转录后加工：真核基因大多为断裂基因，内含子和外显子一起被转录。转录后产物经剪接（包括可变剪切,alternative

splicing）、加帽、加尾等加工修饰，才能转变为成熟的mRNA。RNA降解：包括非特异性降解（RNase,

exosome)和特异性降解(NMD，RNAi,下次课具体讲)。图8-9可变剪接导致α-淀粉酶基因在不同组织中的表达差异

siRNAmiRNARNAactsasaregulatorofgeneexpression----genesilencing4.蛋白水平的调节蛋白质合成:ribosome蛋白翻译，构像折叠，细胞内定位蛋白质修饰:包括磷酸化(phosphorylation),乙酰化(acetylation)，甲基化(methylation),糖基化(glycosylation),etc蛋白降解：包括非特异性降解（protease,

peroxisome，vacuole)和特异性降解(Ubiquitin/proteasomesystem,下下次课具体讲)。Historicalconceptofgeneexpressionregulation•Inclassicalgenetics:→gene=aunitofinheritancethattransmittedasingle

characteristicfromparenttooffspring•theDNAencodinggeneswereseenasdiscretebeadsona

longstring,withonegeneencodingoneprotein...but,isthisconceptofone-gene-one-proteinoutdated?Contemporaryconceptofgeneexpressionregulation

InformationencodedinDNAismorecomplexthanpreviouslyrealized:•Alternativepromoters•RNAsplicing•Non-codingrepetitiveDNA•SeparategeneproductswithoverlappingDNAsequences•Antisensegenetranscripts•Multiplegeneproductscombinetoformasingleprotein•Trans-actinggeneenhancerslocatedondifferentchromosomes•GenesalsoencodefunctionalRNAs(non-proteincoding)•RNAbasedinheritancemechanismsAlternativepromoters&RNAsplicing-essentiallydisprovedthe

oldernotionthatasinglegenesequenceencodedasingleproteinNon-codingsequencerepresentsthevastmajorityofnuclearDNASeparategenescanhaveoverlappingDNAsequencesTranscriptioncanoccurfrombothstrandsofDNA,inoppositedirections,generatingbothsenseandantisensetranscriptsofasinglegene

15-25%ofmouseandhumangenesexpressantisensetranscripts,~50%ofwhicharesplicedFunctionalproteinscanbemadeofseveralsubunitsthatare

derivedfromseparategenes

Embryonichemoglobin:zeta(2),epsilon(2)alpha(2),epsilon(2)zeta(2)gamma(2)

Fetalhemoglobin(HbF):alpha(2),gamma(2)

Adulthemoglobin:alpha(2),beta(2)alpha(2),delta(2)Combinationof2dimers(andassociatedhemeprostheticgroups)resultsinafunctionalhemoglobinproteinGenescanalsoencodefunctionalRNAtranscripts(e.g.tRNA,

rRNA,miRNA)

MicroRNAs(miRNAs)canregulategeneexpressionNatureReviewsMolecularCellBiology8:23–36(2007)TranscriptionalRegulationofEukaryoticGenes第二节真核基因的转录调控

1.

RNA

polymerase

II

2.

promoter

and

enhancers

3.

transcription

factorsEukaryoticgeneexpressionisusuallycontrolledatthelevelofinitiationof

transcription.·AlleukaryoticRNApolymeraseshave~12subunitsandare

aggregatesof>500

kD.·SomesubunitsarecommontoallthreeRNApolymerases.·ThelargestsubunitinRNApolymeraseIIhasaCTD(carboxy-terminaldomain)

consistingofmultiplerepeatsofaheptamer.TheCTDcanbehighly

phosphorylatedonserineorthreonineresidues;thisisinvolvedintheinitiationreactionandRNAprocessing.RNApolymeraseIIsynthesizesmRNAinthenucleoplasm（核胞质）KeyTermsAbasal(general)

factorisatranscriptionfactorrequiredbyRNApolymeraseIItoformthe

initiationcomplexatallpromoters.FactorsareidentifiedasTFIIX,whereXisa

number.Thebasaltranscriptionapparatusisthecomplexoftranscriptionfactorsthat

assemblesatthepromoterbeforeRNApolymeraseisbound.Anenhancer

isacis-actingsequencethatincreasestheutilizationof(some)

eukaryoticpromoters,andcanfunctionineitherorientationandinanylocation

(upstreamordownstream)relativetothepromoter.ActivatorsandtheirmanytargetsTBPTFIIDTFIIBTFIIATFIIHTFIIEPolIIMediatorSAGA/ChromatinmodifiersManymore…

Holoenzyme---asupramolecularcomplexcomprisingPolII, mostGTFs,andMediator/SrbcomplexInyeast,a2MDaholoenzyme+TBPsufficesfortranscriptionOrderedAssemblyvsPolIIHoloenzymeTFIIDTFIIDone-stepmultiple-stepBindingofTFIID(TBP+11TAFs,800KD)totheTATAboxisthefirststepininitiation.Othertranscriptionfactorsbindtothecomplexinadefinedorder,extendingthelengthoftheprotectedregiononDNA.WhenRNApolymeraseIIbindstothecomplex,itisreadytoinitiatetranscription.SequentialAssemblyTBP:TATAbindingproteinTAFs:TBPassociatedfactorsTFIIBbindstoDNAandcontactsRNA

polymeraseneartheRNAexitsiteandattheactivecenter,and

orientsitonDNA.+25bpQ:prok-10bpvseuk-25bp?PhosphorylationoftheCTDbythekinase

activityofTFIIHmaybeneededtoreleaseRNApolymeraseto

starttranscription.·TFIIEandTFIIHarerequiredtomeltDNAtoallowpolymerasemovement.·PhosphorylationoftheCTD(byTFIIHandotherkinases)is

requiredforelongationtobegin—firethePolII.·TheCTDmaycoordinateprocessingofRNAwithtranscription.CTD：RNAPolIIC-terminaldomainCTDPiRNAPolIIBasaltranscriptionalapparatus(TBP,TFIIs)TheCTDmayalsobeinvolvedinprocessingRNAafterithasbeensynthesized.ThecappingenzymebindstothephosphorylatedCTD:thismaybeimportantinenablingittomodifythe5’endassoonasitissynthesized.AsetofproteinscalledSCAFsbindtotheCTD,andtheymayinturnbindtosplicingfactors.Thismaybeameansofcoordinatingtranscriptionandsplicing.Somecomponentsofthecleavage/

polyAapparatusalsobindtotheCTD.soRNApolymeraseisallreadyforthe3’endprocessingreactionsassoonasitsetsout!CTDmaybeageneralfocusforcouplingotherprocesses(mRNA

maturation)

with

transcriptionFactorsinvolvedingeneexpressionincludeRNA

polymeraseandthebasalapparatus,activatorsthatbinddirectly

to,co-activatorsthatbind

tobothactivatorsandthebasalapparatus,andregulatorsthat

actonchromatinstructure(chromatinremodelingcomplex).ManyTranscriptionalActivatorsi.e.CAATGC-boxModularorganizationofTranscriptionFactorsDNAbindingDomain:

Znfingers,Homeodomains,bHLH,bZip,MADS,…OligomerizationDomain:

homo/heterooligomers---usuallydimers,

though trimersarenotuncommon:

HSF,NF-Y…RegulatoryDomain:

Activation-acidic,Q-rich,P-rich,RNA-binding,… Repression-basic,HDACbindingpeptides,…OtherDomains(notinallfactors):

includingnuclearlocalization,protein-interaction domains,ligandbinding,signal-responsivesites…ZincFingerbHLHbZIPHomeodomainSP1stimulatestranscriptioninpresenceofTAFII110GCboxesboundbyDNAbindingproteinSP1SP1recruitsTFIIDbybindingTAFII110Partiallyreconstitutedcomplex(TBPand3TAFs)inadditionto otherGTFs,PolIIleadstohighlevelsoftranscription

SV40earlypromoterMediatorcomplexistargetedbyanactivatorMediatorisastablecomplexcontainingseveralproteins(20-50)MediatorbindstotheRNApolIIandtranscriptionfactors(activators orrepressors)and‘mediates’theregulatorysignalstopolII

(中介复合体）TetheringtheMediatorcomplex

Inyeast:SRBcomplex(suppressorsofRNP

B).Itcontainsfactorsthatarenecessaryfortranscriptionfrommanyormost

promoters.

It

provides

interaction

surfaces

for

many

transcriptional

activators

or

repressors,

thus

mediates

both

activation

and

repression

of

transcription.

tatproteinofHIVcanstimulate

transcriptioninitiationwithoutbindingDNAatall

TheactivatingdomainofthetatproteincanstimulatetranscriptionifitistetheredinthevicinityofpromoterbybindingtotheRNAproduct(tarsequence)ofapreviousroundoftranscription.tartatDNA-bindingdomainistobringtheactivationdomainintothevicinityofthestartpoint.

Andactivationisindependentofthemeansoftethering.wecanthinkofDNA-binding(orRNA-bindinginthecaseoftat)domainasprovidinga"tethering"function,whosemainpurposeistoensurethattheactivationdomainisinthevicinityoftheinitiationcomplex.Thenotionoftetheringisamoregeneralideathatinitiationrequiresahighconcentrationoftranscriptionfactorsinthevicinityofthepromoter.Thismaybeachievedwhenactivatorsbindtoenhancers,upstreampromoterelements,orinanextremecasebytetheringtoanewly-madeRNAproduct.InterchangeableModules(Activationdomainisinterchangeable)InteractionAssaysDesignofTwo-hybrid/Three-hybrid/etc…separablefunctionaldomainsTwo-hybridassay(protein-protein)Tri-hybridassay(protein-RNA)Summary

TheprinciplethatgovernsthefunctionofallactivatorsisthataDNA-bindingdomaindeterminesspecificityforthetargetpromoterorenhancer.TheDNA-bindingdomainisresponsibleforlocalizingatranscription-activatingdomainintheproximityofthebasalapparatus.AnactivatorthatworksdirectlyhasaDNA-bindingdomainandanactivatingdomain.

Anactivatorthatdoesnothaveanactivatingdomainmayworkbybindingacoactivatorthathasanactivatingdomain.Severalfactorsinthebasalapparatusaretargetswithwhichactivatorsorcoactivatorsinteract.RNApolymerasemaybeassociatedwithvariousalternativesetsoftranscriptionfactorsintheformofaholoenzymecomplex.Whatisthemechanismofactivation?Twomodels:

Tetheringholoenzyme

(recruitment)Activatingholoenzyme(allosteric)(interactionactivation)??Infavorofrecruitment

modelactivatorsrecruittranscriptionmachineryGal4-bindingsitesareboundbyGal4

Pre-bindingbyGal4isnecessaryforrecruitmentofthemachineryRoleinre-initiationaswell‘Synergy’

HighlevelsoftranscriptioninducedbymultiplefactorsTranscriptionfactorscanenhancetranscriptioninanon-linearmannerSynergisiticactivationoccursduetomultiplecontactswiththemachineryMultiplecopiesofthesameactivatoralsoinducesynergisticactivationInterferonßenhancerEnhancersoftenhavebindingsitesforseveraltranscription

factorsTranscriptionfactorscanbind

cooperativelyatadjacentsitesArchitecturalfactors(withnoregulatorydomains,i.e.HMG1)canassistassemblyRemarkablyincreasebindingaffinityforbothDNAandmachineryHMG1肩并肩、手挽手，根基稳、魅力足香肩并立、玉指紧扣，脚如磐石、面若桃花RegulatorymechanismfromadistanceCompactionSlidingLoopingWhydoenhancersactindependentofdistanceandorientation?Anenhancermayfunctionbybringingproteinsintothevicinityof

thepromoter.Anenhancerdoesnotactonapromoterattheoppositeendofalong

linearDNA,butbecomeseffectivewhentheDNAisjoinedintoacirclebyaprotein

bridge.An

enhancerandpromoteronseparatecircularDNAsdonotinteract,butcan

interactwhenthetwomoleculesarecatenated.Twoexperimentssupporttheloopingmodel--Theessentialroleoftheenhanceris

toincreasetheconcentrationofactivatorin

thevicinityofthepromoter---(Signaltransductionandgeneregulation)How

isageneregulatedbysignals?Theregulatoryregionofahumanmetallothioneingenecontainsregulatorelementsinbothitspromoterandenhancer.Thepromoterhaselementsformetalinduction;anenhancerhasanelementforresponsetoglucocorticoid.Promoterelementsareshownabovethemap,andproteinsthatbindthemareindicatedbelow.EachgenecontainsmultipleresponseelementsTheactivityofaregulatorytranscriptionfactormaybecontrolledbysynthesisofprotein,covalentmodificationofprotein,ligandbinding,orbindingofinhibitorsthatsequestertheproteinoraffectitsabilitytobindtoDNA.SteroidreceptorsaretranscriptionfactorsSteroidreceptorsareexamplesofligand-responsiveactivatorsthatareactivatedbybindingasteroid.ThereareseparateDNA-bindingandligand-bindingdomains.TheDNAbindingdomainisatypeofzincfingerthathasCysbutnotHisresidues.TheybindtoDNAasdimers.Glucocorticoidandestrogenreceptorseachhavetwozincfingers,thefirstofwhichdeterminestheDNAtargetsequence.BindingofligandtotheC-terminaldomainincreasestheaffinityoftheDNA-bindingdomainforitsspecifictargetsiteinDNA.Receptorsformanysteroidandthyroid

hormoneshaveasimilarorganization,withanindividualN-terminalregion,conservedDNA-bindingregion,anda

C-terminalhormone-bindingregionThefirstfingerofasteroidreceptorcontrolswhichDNAsequenceisbound(positionsshowninred);thesecondfingercontrolsspacingbetweenthesequences(positionsshowninblue).DiscriminationbetweenGREandEREtargetsequencesisdeterminedbytwoaminoacidsatthebaseofthefirstzincfingerinthereceptor.GREEREGlucocorticoidsregulategenetranscriptionbycausingtheir

receptortotransportintothenucleusandbindtoanenhancerwhoseactionis

neededforpromoterfunction.(ordexamethasone)ActivationofGlucocorticoidReceptor(GR)NuclearshuttlingGlucocorticoidreceptor(GR)fusionproteininductionCortisolDexamethasoneGRGRGRXXXDexbindstoGR,exposesnucleuslocalizationsignal(NLS),allowsfusionproteintotransportintothenucleustoactivatetranscription----Agoodsystemtoactivatethefunctionofnuclear-localizedproteins(includingtranscriptionfactors)ActivationTaggingapproach

inplantsPlanttransformationphenotypiccharacterizationofmutants

locateT-DNAinsertionsiteinArabidopsisgenome(how?)identifytherightgeneconferringmutantphenotype(how?)functionalstudyofthegeneGeneticscreenXVE，wasassembledbyfusionoftheDNA-bindingdomainofLexA(X),theacidicactivationdomainofVP16(V),andtheregulatoryregionofthehumanestrogenreceptor(E)

Achemical-inducibleactivationtaggingvectorpER16inplants

T-DNAfragment(canintegrateintoplantgenome)其工作原理为：在G10-90启动子控制下，XVE融合转录因子组成型表达；当加入雌激素，雌激素和其受体结合，导致XVE融合蛋白构象发生变化，并由细胞质转移进入核内；在细胞核内,LexA的DNA结合域特异性识别LexA操纵子区，以至使VP16的转录激活域激活-4635S启动子，高水平表达其下游可能的目标基因。TRandRARbindtheSMRTcorepressorinthe

absenceofligand.The

promoterisnotexpressed.WhenSMRT

isdisplacedbybindingofligand,thereceptorbindsa

coactivatorcomplex.Thisleadstoactivationoftranscriptionby

thebasalapparatus.Corepressor/coactivatorswitchSummary:

1)Cis-elementsmarkthesitesoftranscription

2)SomeboundbyGTFsandothersbyRegulators(TFs)

3)RegulatoryTFsaretriggedbycellularsignals

4)RegulatoryTFscanbepositiveornegative

5)TFsaremodular:

-DBDbindsEnhancersanddefinesgenesthataretargeted

-Regulatorydomaincontrolstheexpressionofthegene(s)

6)Simplearchitectureofregulatoryproteinshasledtothe

creationofpowerfultools(2-hybridandATFs)

诺贝尔化学奖得主Roger

Kornberg北大演讲被授予“北大名誉教授”罗杰•科恩伯格,斯坦福大学教授，因对“真核转录的分子基础所作的研究”而荣获2006年诺贝尔化学奖。科恩伯格发现了构成染色体的基本单位——核小体Ifnucleosomesformatapromoter,transcriptionfactors(andRNApolymerase)cannotbind.Iftranscriptionfactors(andRNApolymerase)bindtothepromotertoestablishastablecomplexforinitiation,histonesareexcluded.ThedynamicmodelfortranscriptionofchromatinreliesuponfactorsthatcanuseenergyprovidedbyhydrolysisofATPtodisplacenucleosomesfromspecificDNAsequences.ChromatinremodelingChromatinremodelingisundertakenbylargecomplexesthatuseATPhydrolysistoprovidetheenergyforremodeling.TheheartoftheremodelingcomplexisitsATPasesubunit.RemodelingcomplexesareusuallyclassifiedaccordingtothetypeofATPasesubunittwomajortypesofchromatinremodelingcomplex:SWI/SNFandISW(imitationSWI)Remodelingcomplexescancausenucleosomesto

slidealongDNA,candisplacenucleosomesfromDNA,orcan

reorganizethespacingbetweennucleosomes.Remodelingcomplexesarerecruitedtopromotersbysequence-specificactivators.Agenomicsurveysuggestedthatmostsitesthatbindtranscriptionfactorsarefreeofnucleosome.Aremodelingcomplexbindstochromatinviaanactivator(orrepressor)HormonereceptorandNF1cannotbindsimultaneouslytotheMMTVpromoterintheformoflinearDNA,butcanbindwhentheDNAispresentedonanucleosomalsurface.TheMMTVpromoterrequiresachangeinrotationalpositioningofanucleosometoallowanactivatortobindtoDNAonthenucleosome.组蛋白的化学修饰组蛋白化学修饰发生在组蛋白N端尾部，尤其是组蛋白H3和H4的修饰起始了染色质结构的变化。组蛋白尾部由20个氨基酸组成，并且从DNA转弯处的核小体间延伸出来。组蛋白化学修饰的类型：组蛋白（去）乙酰化De/Acetylation组蛋白甲基化Methylation组蛋白磷酸化Phosphorylation组蛋白泛素化Ubiquitination组蛋白糖基化ADP-Rybosilation组蛋白与Swi/Snf复合体的结合Swi/Snfcomplex,which,invitro,usestheenergyofATPhydrolysistodisrupthistone-DNAinteractions多数化学修饰的化学基团可以减少组蛋白的正电性，从而使其与DNA结合变疏松，使染色质结构发生变化。AcetylationofH3andH4isassociatedwithactivechromatin,whilemethylationisassociatedwithinactive

chromatin.HistonemodificationisakeyeventSitesofpost-translationalmodificationsonthehistonetailsZhangY.,ReinbergD.GenesDev.2001;15:2343-2360Mostmodifiedsitesinhistoneshaveasingle,specifictypeofmodification,butsomesitescanhavemorethanonetypeofmodification.Individual

functionscanbeassociatedwithsomeofthemodifications.HistoneCodeKKAcetylation=TranscriptionalCompetenceMethylation

=TranscriptionallySilenced·Histoneacetylationoccurstransientlyatreplication.·Histoneacetylationisassociatedwithactivationofgeneexpression.Histoneacetylationoccursintwo

circumstancesHistoneacetyltransferase(HAT)enzymesmodifyhistonesbyadditionofacetyl

groups;sometranscriptionalcoactivatorshaveHATactivity.Adeacetylaseisanenzymethatremovesacetylgroupsfromproteins.Histonedeacetyltransferase(HDAC)enzymesremoveacetylgroupsfrom

histones;theymaybeassociatedwithrepressorsoftranscription.组蛋白乙酰化酶复合体的特点：HAT是一个大复合体的一部分(如SAGA复合体中的GCN5)，且多种HAT可同时存在于一个辅助激活复合体中，并具有不同的特异性(如P300,ACTR与PCAF的共同作用)PCAFACTRP300TFEF组蛋白乙酰化过程是动态的，被乙酰化的组蛋白位点可以通过组蛋白去乙酰化酶（histonedeacetylase,HDAC）将乙酰基去掉。组蛋白去乙酰化酶(HDAC)可分为4类。

(1)第一类：为酵母转录调控子RPD3蛋白的同类物，这是第一个被鉴定出来的HDAC。它包括HDAC1~3、6、7、9、18，19，可与转录因子结合。

(2)第二类：为酵母HDAl类蛋白质，包括HDAC4、5、10。它们的催化区域与酵母的HDAl蛋白同源，特异性地与细胞分化有关。

(3)第三类：最近发现酵母中的沉默信息调节因子2(silentinformationregulator2，SIR2)也是一种组蛋白去乙酰化酶，人细胞中的SIR2同源物也被鉴定出来。因此，SIR2及其相关蛋白构成了高等真核生物的第三类组蛋白去乙酰化酶。

(4)第四类：HD2类，仅在植物当中发现。Arepressorcomplexcontainsthreecomponents