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1、.,Eukaryotic Gene Expression and Regulation,第八章,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(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）,.,第 一 节基本概念与原理,Basic Concept
2、s and Principles,.,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,.,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
3、of gene expression) 基因表达是受内源及外源信号调控的。,.,Regulation of Gene Expression,RNA silencing,Proteindegradation,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,.,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
4、的空间特异性(spatial specificity)。,.,BARD1 is expressed specifically in the apical domains of Arabidopsis inflorescence (A), ovules (B), anthers (C), and embryos (D).,In suit hybridizationA, B, C, D: antisense BARD1 probe;E: sense BARD1 probe as a negative control.,（朱玉贤第五章课件）,.,四种母源影响基因的mRNA和蛋白沿果蝇胚胎前-后轴分布
5、的浓度变化图,protein,BICOID,NANOS,第十章（基因和发育）,mRNA,.,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 tissueTemporal specificity: Each cell of an organism expresses a distinctive subset of genes
6、 at different time or developmental stageTight regulation: During development different cells express different sets of genes in a precisely regulated fashion,.,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)。,.,这类基因表达又称为组成性基因表达(con
7、stitutive gene expression)。,genes for essential cellular structures and metabolic pathways (e.g. rRNA, actin, tubulin)usually expressed at high levelthe level of their gene expression may vary,Housekeeping genes,rRNA, actin, tubulin are commonly used as loading control in RT-PCR or Northern blot,.,（
8、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。,.,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）begin and end beyond the coding re
9、gion (5UTR and 3UTR),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,.,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
10、)（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,.,cis-acting elements: promoters/regulatory sequences of genestrans-acting factors: proteins and RNAs that bind cis-elements and
11、 promote or repress gene expression,.,(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?,.,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
12、达活性。 启动子 (promoter) 顺式作用元件又分 增强子 (enhancer) 沉默子 (silencer),转录起始点,（二）特异DNA序列,.,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,.,RNA聚合酶在转录因子帮助下，形成的转录起始复合物,holoenzyme,1. 基本转录因子
13、 (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,.,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
14、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。,.,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,.,Zinc Finger,bHLH,bZIP,Homeo
15、domain,1） TF最常见的DNA binding domain,.,(1) 锌指(zinc finger),常结合GC box,Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His,C-terminal: -helix binding DNA,.,(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,.,2） TF常见的trans-activation domain,.,(Activation domain is interchangeable),.,Interaction As
16、saysDesign of Two-hybrid / Three-hybrid /etc,separable functional domains,Two-hybrid assay(protein-protein),Tri-hybrid assay(protein-RNA),.,1. RNA polymerase II2. promoter and enhancers3. transcription factors,Eukaryotic gene expression is usually controlled at the level of initiation of transcripti
17、on.,真核基因转录起始的调控,.,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,.,TFIIB binds to DNA and contacts RNA polymerase near
18、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 proteinTAFs: TBP associated factors,Binding of TFIID (TBP + 11 TAFs, 800KD) to the TATA box is the first step in initiation.,.,CTD：RNA Pol II C-terminal domain,C
19、TD 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 Y1S2P3T4S5P6S7.S2 and S5 are major phosphorylation sites.CTD phosphorylation cause the conversion of proline isomerization states
20、.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
21、the C-terminal domain of RNA polymerase II (CTD).,.,PIC: PhosphoS5 is required for assembly of the PIC and facilitates mRNA capping via recruitment of capping enzymes. Elongation: S5 gradually becomes dephosphorylated, whereas S2 is phosphorylated. Terminating: PhosphoS2 ensures efficient 3-RNA proc
22、essing 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.,S2 & S5, the trigger for transcriptional process modulation,.,.,Fa
23、ctors 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.
24、CAATGC-box,.,Near the initiation site,A little far away,.,SP1 stimulates transcription in presence of TAFII110,GC boxes bound by DNA binding protein SP1 SP1 recruits TFIID by binding TAFII110 Partially reconstituted complex (TBP and 3 TAFs) in addition to other GTFs, Pol II leads to high levels of t
25、ranscription,SV40 early promoter,Near,.,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,.,What
26、 is the mechanism of activation?,Two models: Tethering holoenzyme (recruitment)Activating holoenzyme (allosteric),.,In favor of recruitment model,（勾引模型）,.,tat protein of HIV can stimulate transcription initiation without binding DNA at all The activating domain of the tat protein can stimulate trans
27、cription 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,.,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 ca
28、n 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
29、 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.,.,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
30、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。,.,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 machi
31、nery Multiple copies of the same activator also induce synergistic activation,.,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 as
32、sist assembly Remarkably increase binding affinity for both DNA and machinery,.,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,.,One possible strategy:,-Looping-,Cohesins help to stabilize enhancer-promoter interactions,How do enhancers act independent of distance and orientation?,.,An enhancer may functi
33、on 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 inte
34、ract 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-,.,Receptors for many steroid and thyroid hormones have a similar organization, with an individual N-
35、terminal region, conserved DNA-binding region, and a C-terminal hormone-binding region,Steroid receptors are transcription factors,Zinc finger TF,.,Glucocorticoids regulate gene transcription bycausing their receptor to transport into the nucleus and bind to an enhancer whose action is needed for pr
36、omoter function.,Activation of Glucocorticoid Receptor (GR),Nuclear shuttling,.,利用GR的特性构建可诱导表达融合蛋白系统,Dexamethasone（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
37、研究核蛋白的功能（包括转录因子）。,.,Activation Tagging approach in plants,Plant transformation,mutants screeninglocate 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，435S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-D
38、NA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；,.,XVELexA 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OLexA-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，
39、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（OLexA），VP16的转录激活域（V）激活LB旁边的基因高水平表达。,.,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。,
40、.,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,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
41、别任意靶DNA的目的，其应用受到一定的限制。,.,TALEN=Transcription Activator-Like Effector+FokI Nuclease 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
42、genes during pathogenesis.,codon,.,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,.,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。,.,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。,.,思考题：,1、如何通过实验的方法分析CTD上S2和S5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？,
