真核生物基因表达调控课件

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Regulation,第八章,真核基因表达调控,.Eukaryotic Gene Expression an,.,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,(transcriptional regulation),:,DNA level (Genetic),Chromatin level (Epigenetic),转录后水平的调控,(post-transcriptional regulation),：,RNA interference,（,RNAi,）,Protein degradation,（,Ubiquitin/proteasome,）,.本章主要内容,.,第 一 节基本概念与原理,Basic Concepts and Principles,.第 一 节基本概念与原理Basic Concepts,.,Genome,(cells repertoire of DNA),Transcriptome,(cells repertoire of RNA transcripts),Proteome,(cells repertoire of proteins),单个基因,单个细胞,中心法则,.GenomeTranscriptomeProteome单个,.,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,(gene expression),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,(gene regulation, or regulation of gene expression),基因表达是受内源及外源信号调控的。,.一、基因表达的概念基因组(genome)基因表达(gene,.,Regulation of Gene Expression,Chromatin,epigenetic,control,RNA silencing,Protein,degradation,一般而言的基因表达调控范畴,.Regulation of Gene Expression,.,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,(temporal specificity),。,多细胞生物基因表达的时间特异性又称,阶段特异性,(stage specificity),。,.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,.,人体发育过程中不同类型,-,珠蛋白的含量变化,.人体发育过程中不同类型-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,(cell or tissue specificity),。,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,(spatial specificity),。,.（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,.,BARD1 is expressed specifically in the apical domains of,Arabidopsis inflorescence (A), ovules (B), anthers,(C), and embryos (D).,In suit hybridization,A, B, C, D: antisense BARD1 probe;,E:,sense BARD1 probe as a negative control.,（朱玉贤第五章课件）,.BARD1 is expressed specifical,.,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,protein,BICOID,NANOS,第十章（基因和发育）,mRNA,.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,.,Facts,Identical genome,: Virtually every cell in an organism contains a complete set of genes,Spatial specificity:,But they are not all turned on in every cell or tissue,Temporal specificity,: Each cell of an organism expresses a distinctive subset of genes at different time or developmental stage,Tight,regulation,: During development different cells express different sets of genes in a precisely regulated fashion,.Facts Identical genome: Virtu,.,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,(constitutive expression),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,(housekeeping gene),。,.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：（,.,这类基因表达又称为,组成性基因表达,(constitutive gene expression),。,genes for,essential,cellular structures and metabolic pathways (e.g. rRNA, actin, tubulin),usually expressed at,high,level,the level of their gene expression may,vary,Housekeeping genes,rRNA, actin, tubulin,are commonly used as loading control in RT-PCR or Northern blot,.这类基因表达又称为组成性基因表达(constitutive,.,（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,(repressible genes),。,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。,.（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,.,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,.四、基因表达调控的生物学意义（一）维持细胞增殖、分化第九、,.,1. Transcripts,（转录本）,begin and end beyond the coding region (5,UTR and 3,UTR),2. The primary transcript is processed by:,5,capping,3,formation / polyA,splicing,3. Mature transcripts are transported to the cytoplasm for translation,一般而言，基因表达调控主要是发生在基因转录水平上的调节，即：,mRNA,合成的多少。,transcription,.1. Transcripts （转录本）begin an,.,五、基因转录调节基本要素,（一）,RNA,聚合酶,(RNA Polymerase),（二）特异,DNA,序列,(,cis,-acting elements),（三）调节蛋白,(,trans,-acting factors),Gene expression regulation at the level of DNA,(transcriptional regulation),-highly sequence-dependent,-varied regulation for different genes,.五、基因转录调节基本要素Gene expression r,.,cis,-acting elements,:,promoters/regulatory,sequences of genes,trans,-acting factors,:,proteins,and,RNAs,that bind cis-elements and,promote,or,repress,gene expression,.cis-acting elements: promote,.,(,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%,RNA Pol III: tRNA,相对活性,10%,RNA Pol IV: small ncRNA,相对活性,?,. (一) RNA聚合酶,.,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-acting element),。,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/Si,Pro,DNA,编码序列,转录起始点,（二）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,.,反式作用因子,(trans-acting factor),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,（三）真核基因的调节调节蛋白,.反式作用因子 (trans-acting factor)（,.,RNA,聚合酶,在转录因子帮助下，形成的转录起始复合物,pol,TFH,TAF,TFF,TAF,TAF,TFA,TFB,TBP,TATA,DNA,TAF: TBP,associated factors,holoenzyme,1.,基本转录因子,(general transcription factor, GTF),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-box binding protein,TFII: pol II associated TF,.RNA聚合酶在转录因子帮助下，形成的转录起始复合物pol,.,2.,转录调节因子,(transcription factor, 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,(transcriptional activator),转录阻遏因子,(transcriptional repressor),3.,共调节因子,(transcriptional regulator/ 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。,.2. 转录调节因子 (transcription fact,.,常见转录因子的结构域,(domain),DNA,结合域,（,DNA,binding,domain),Basic AA (K/R) rich, positively charged,转录激活域,(trans-activation domain),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,.常见转录因子的结构域 (domain)DNA结合域 （DN,.,Zinc Finger,bHLH,bZIP,Homeodomain,1,）,TF,最常见的,DNA,binding domain,.Zinc FingerbHLHbZIPHomeodomai,.,(1),锌指,(zinc finger),常结合,GC,box,C,ys-X,2-4,-,C,ys-X,3,-Phe-X,5,-Leu-X,2,-,H,is-X,3,-,H,is,C-terminal:,-,helix,binding DNA,.(1) 锌指(zinc finger)常结合GC boxC,.,(2),碱性亮氨酸拉链,bZIP,.(2) 碱性亮氨酸拉链 bZIP,.,bHLH,蛋白,(basic Helix-Loop-Helix),(3),碱性螺旋,-,环,-,螺旋,bHLH,.bHLH蛋白(basic Helix-Loop-Helix,.,2,）,TF,常见的,trans-activation domain,.2） TF常见的trans-activation doma,.,(Activation domain is interchangeable),.(Activation domain is interch,.,Interaction Assays,Design of Two-hybrid / Three-hybrid /etc,separable functional domains,Two-,hybrid,assay,(protein-protein),Tri-hybrid assay,(protein-RNA),.Interaction Assaysseparable f,.,1.,RNA,polymerase,II,2.,promoter,and,enhancers3.,transcription,factors,Eukaryotic gene expression is usually controlled at the level of,initiation of,transcription,.,真核基因转录起始的调控,.1. RNA polymerase II2. pro,.,Holoenzyme,- a supramolecular complex comprising Pol II,most GTFs, and Mediator/Srb complex,In yeast, a 2MDa holoenzyme + TBP suffices for transcription,Ordered Assembly and Pol II Holoenzyme,TFIID,TFIID,one-step,multiple-step,. Holoenzyme - a supramolecu,.,TFIIB binds to DNA and contacts RNA,polymerase near the RNA exit site and at the active center, and,orients it on DNA.,+25bp,Q: prok -10bp vs euk -25bp?,Sequential Assembly,TBP,:,TATA binding protein,TAFs,:,TBP associated factors,Binding of TFIID (,TBP,+ 11,TAFs, 800KD) to the TATA box is the first step in initiation.,.TFIIB binds to DNA and contac,.,CTD,：,RNA Pol II C-terminal domain,CTD is an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II.,It comprises from 25 to 52 tandem copies of the consensus repeat heptad Y,1,S,2,P,3,T,4,S,5,P,6,S,7,.,S,2,and S,5,are major phosphorylation sites.,CTD phosphorylation cause the conversion of proline isomerization states.,Phosphorylation patterns on the CTD repeats determine different sets of associated factors, so that provide a dynamic platform to recruit different regulators of the transcription apparatus.,In eukaryotic cells, the transcription of genes is accurately orchestrated both spatially and temporally by the C-terminal domain of RNA polymerase II (CTD).,.CTD：RNA Pol II C-terminal dom,.,PIC:,PhosphoS,5,is required for assembly of the PIC and facilitates mRNA capping via recruitment of capping enzymes.,Elongation:,S,5,gradually becomes dephosphorylated, whereas S,2,is phosphorylated.,Terminating:,PhosphoS,2,ensures efficient 3-RNA processing by triggering recruitment of 3-RNA processing machinery.,Ending:,CTDs are free of phosphate groups; non-phosphorylated CTDs are required for RNA polymerase II to recycle and bind a promoter for the next cycle of transcription.,S,2,& S,5, the trigger for transcriptional process modulation,.PIC: PhosphoS5 is required fo,.,.,.,Factors involved in gene expression include,RNA,polymerase,and the,basal apparatus,activators,that bind directly,to,co-activators,that bind,to both activators and the basal apparatus, and,regulators,that,act on chromatin structure (,chromatin remodeling complex,).,Many Transcriptional Activators,i.e. CAAT,GC-box,.Factors involved in gene expr,.,Near the initiation site,A little far away,.Near the initiation siteA lit,.,SP1 stimulates transcription in presence of TAF,II,110,GC boxes bound by DNA binding protein SP1,SP1,recruits TFIID,by binding TAF,II,110,Partially reconstituted complex (TBP and 3 TAFs) in addition to other,GTFs, Pol II leads to high levels of transcription,SV40 early promoter,Near,.SP1 stimulates transcription,.,Mediator complex is targeted by an activator,Mediator is a stable complex containing several proteins (20-50),Mediator binds to the RNA pol II and transcription factors (activators or repressors) and mediates the regulatory signals to pol II,(,中介复合体）,Far,.Mediator complex is targeted,.,What is the mechanism of activation?,Two models:,Tethering holoenzyme,(,recruitment),Activating holoenzyme (,allosteric,),(interaction,activation),?,.What is the mechanism of acti,.,In favor of,recruitment,model,（,勾引模型,）,.In favor of recruitment model,.,tat protein of HIV can stimulate,transcription initiation without binding DNA at all,The activating domain of the,tat,protein can stimulate transcription if it is tethered in the vicinity of promoter by binding to the RNA product (,tar,sequence) of a previous round of transcription.,tar,tat,.tat protein of HIV can stimul,.,DNA-binding domain is to bring the activation domain into the vicinity of the startpoint.,And activation is independent of the means of,tethering,.,we can think of DNA-binding (or RNA-binding in the case of tat) domain as providing a ,tethering, function,whose main purpose is to ensure that the activation domain is in the vicinity of the initiation complex.,The notion of,tethering,is a more general idea that initiation requires a high concentration of transcription factors in the vicinity of the promoter.,This may be achieved when activators bind to enhancers, upstream promoter elements, or in an extreme case by tethering to a newly-made RNA product.,.DNA-binding domain is to brin,.,总结,所有激活因子的共性：识别靶位点（启动子、增强子）的特异性由,DNA,结合域决定。,DNA,结合域将转录激活域带到基础转录区域附近。,直接作用的激活因子具有,DNA,结合域和转录激活域。,没有转录激活域的激活因子可能与具有转录激活域的共激活因子一起行使功能。,基础转录区域中许多元件是（共）激活因子的靶位点,RNA,聚合酶可以和多种不同的转录因子相互作用，形成全酶复合物行使功能。,.总结,.,Synergy,High levels of transcription induced by multiple factors,Transcription factors can enhance transcription in a,non-linear,manner,Synergisitic activation occurs due to multiple contacts with the machinery,Multiple copies of the same activator also induce synergistic activation,.Synergy High levels of tra,.,Interferon enhancer,Enhancers often have binding sites for several transcription,factors,Transcription factors can bind,cooperatively,at adjacent sites,Architectural factors (with no regulatory domains, i.e. HMG1) can,assist assembly,Remarkably increase binding affinity for both DNA and machinery,.Interferon enhancer Enhance,.,HMG1,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,.HMG1肩并肩、手挽手，根基稳、魅力足香肩并立、玉指紧扣，,.,One possible strategy:,-Looping-,Cohesins,help to stabilize enhancer-promoter interactions,How do enhancers act independent of distance and orientation?,.One possible strategy: -Lo,.,An enhancer may function by bringing proteins into the vicinity of,the promoter. An enhancer does not act on a promoter at the opposite end of a long,linear DNA, but becomes effective when the DNA is joined into a circle by a protein,bridge. An,enhancer and promoter on separate circular DNAs do not interact, but can,interact when the two molecules are catenated.,Two experiments support the,looping,model,-The essential role of the enhancer is,to increase the concentration of activator in,the vicinity of the promoter-,.An enhancer may function by b,.,Receptors for many steroid and thyroid,hormones have a similar organization, with an individual N-terminal region, conserved DNA-binding region, and a,C-terminal hormone-binding region,Steroid receptors are transcription factors,Zinc,finger,TF,.Receptors for many steroid an,.,Glucocorticoids regulate gene transcription by,causing their,receptor to transport into the nucleus and bind to an enhancer whose action is,needed for promoter function.,Activation of Glucocorticoid Receptor (GR),Nuclear shuttling,.Glucocorticoids regulate gene,.,利用,GR,的特性构建可诱导表达融合蛋白系统,Dexamethasone,GR,GR,GR,X,X,X,D,examethasone,（,DEX,）,:,地塞米松,氟美松,(,抗炎药,),，合成的一种糖皮质激素；,通过分子克隆的方法将,GR,和要研究的核蛋白,X,构建成融合蛋白，转基因到酵母、动物细胞或者植物中；,不施加外源,DEX,时，融合蛋白与,HSP90,形成复合物，由于构象和空间位阻等原因，融合蛋白存在于胞质中，不能定位到细胞核；,添加,DEX,时，,DEX,扩散入胞与,GR,相结合，融合蛋白改变构象后核定位信号暴露，即可入核行使功能；,用以研究核蛋白的功能（包括转录因子）。,.利用GR的特性构建可诱导表达融合蛋白系统Dexametha,.,Activation Tagging approach,in plants,Plant transformation,mutants screening,locate T-DNA insertion site in Arabidopsis genome (,how?,),identify the right gene conferring mutant phenotype (,how?,),构建,T-DNA,序列，其中包括,4,倍重复的,CaMV,的,35S,增强字序列，,4,35S,元件可以大大增强相邻基因的转录；,通过转基因的方法将,T-DNA,片段整合到植物基因组中；,植物基因组上与,T-DNA,插入位点相近的基因表达量增高，即得到这个基因,gain-of-function,的转基因植物；,.Activation Tagging approach i,.,XVE,LexA,的,DNA,结合域,(,X,),VP16,转录激活域,(,V,),human estrogen receptor,调控域,(,E,),NPTII,；转基因筛选标记,O,LexA-46,：,LexA,操纵子,CaMV 35S,基本启动子（而非增强子）,A chemical-inducible activation tagging vector pER16 in plants,RB-LB: T-DNA fragment (,可以插入到基因组中,),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，雌激素和受体调控域（,E,）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，,XVE,中的,LexA,DNA,结合域（,X,）特异识别,LexA,操纵子区（,O,LexA,），,VP16,的转录激活域（,V,）激活,LB,旁边的基因高水平表达。,.XVELexA 的DNA结合域(X)VP16转录激活域,.,Enhancer Trap,Enhancer trap,的质粒包含一个报告基因（,lacZ,）和基本启动子，这段启动子不足以启动报告基因的表达，但是对增强子非常敏感。将这样的质粒整合到基因组中，如果插入位点附近有增强子报告基因就会表达。因此只要通过观测报告基因的表达情况（比如时空上的特异性）就可以知道这个增强子的作用，进而研究由这个增强子调控的内源基因的表达特性。,.Enhancer TrapEnhancer trap的质粒,.,ZFN,技术原理,锌指核酸酶（,Zinc-finger nucleases, ZFN,）是人工改造的限制性核酸内切酶，利用不同的锌指结构识别特异,DNA,序列，利用核酸酶切断靶,DNA,。,锌指结构中每一个螺旋可以特异识别,3-4,个碱基；,人工设计识别特异,DNA,序列的螺旋采用如上的通用序列，通过改变其中,7,个,X,来实现识别不同的三联体碱基，,TGEK,是多个螺旋间的连接序列；,构建成对人工锌指结构域和,FokI,融合蛋白（,ZFN,）可以在指定区域切断,DNA,双链。,.ZFN 技术原理锌指核酸酶（Zinc-finger nuc,.,ZFN,技术,锌指核酸酶介导的定向染色体删除,研究人员可以利用,ZFN,技术进行各种基因编辑，比如基因敲除。已建立有,ZFN,库，识别多种,DNA,序列，但还不能达到识别任意靶,DNA,的目的，其应用受到一定的限制。,.ZFN 技术锌指核酸酶介导的定向染色体删除研究人员可以利用,.,TALEN=,T,ranscription,A,ctivator-,L,ike,E,ffector+FokI,N,uclease fusion protein,TALEN,技术,长度为,34aa,的重复肽段中的第,12,、,13,个氨基酸可以特异识别,DNA,单个碱基，形成,2aa-1bp,的特殊,coden,。利用这个特性可以人工设计识别任意碱基序列的,TALE,蛋白。,TALE,: transcription activator-like effector from,Xanthomonas, TALE can specifically bind and regulate plant genes during pathogenesis.,codon,.TALEN=Transcription Activator,.,设计,TALE,结合位点接上报告基因（,mCherry,），同时构建一个特异识别这种,TALE,结合序列的的,TALE,蛋白，将两种质粒共同转入细胞，那么这个人工,TALE,蛋白可以启动报告基因,mCherry,的表达,.设计TALE结合位点接上报告基因（mCherry），同时构,.,特异,TALE,蛋白与核酸内切酶,FokI,的融合蛋白可以切割特异识别序列下游,9-13bp,。基于这个原理，可以设计转基因融合蛋白敲除指定内源基因。设计一对识别果蝇,tnikb,基因的,TALEN,蛋白（,left and right,），内源,tnikb,将被,FokI,切开，转基因果蝇子代将有,tnikb,基因突变的个体。,.特异TALE蛋白与核酸内切酶FokI的融合蛋白可以切割特异,.,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。,.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,.,思考题：,1,、如何通过实验的方法分析,CTD,上,S,2,和,S,5,不同磷酸化,pattern,的功能？,2,、如何鉴定,activation tagging,的转基因植物中是哪个基因的表达上调而导致所观测的表型？,.思考题：1、如何通过实验的方法分析CTD上S2和S5不同磷,