分子生物学教学课件：Part 4 Chapter 17 Gene Regulation in Eukaryotes

1、1Part IV RegulationCh 16: Transcriptional regulation in prokaryotesCh 17: Transcriptional regulation in eukaryotesCh18: Regulatory RNAsCh 19: Gene regulation in development and evolutionCh 20: Genome Analysis and Systems Biology2Chapter 17Gene Regulation in EukaryotesMolecular Biology Course3TOPIC 1

2、 Conserved Mechanisms of Transcriptional Regulation from Yeast to Human (2 techniques).TOPIC 2 Recruitment of Protein Complexes to Genes by Eukaryotic Activators. (2 techniques)TOPIC 3 Transcriptional RepressorsTOPIC 4 Signal Integration and Combinatorial Control.TOPIC 5 Signal Transduction and the

3、Control of Transcriptional Regulators.TOPIC 6 Gene Silencing by Modification of Histones and DNA.TOPIC 7 Epigenetic Gene Regulation. 1. Gene Expression is Controlled by Regulatory Proteins (调控蛋白)Gene expression is very often controlled by Extracellular Signals, which are communicated to genes by reg

4、ulatory proteins:Positive regulators or activators INCREASE the transcriptionNegative regulators or repressors DECREASE or ELIMINATE the transcriptionPrinciples of Transcription Regulation51. Principles are the same: signals (信号), activators and repressors (激活蛋白和阻遏蛋白)recruitment and allostery, coope

5、rative binding (招募，异构和协同结合)2. The gene expression steps subjected to regulation are similar, and the initiation of transcription is the most pervasively regulated step. Similarity of regulation between eukaryotes and prokaryote6Pre-mRNA splicing adds an important step for regulation. (mRNA前体的剪接)The

6、eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. (真核转录机器更复杂)Nucleosomes and their modifiers influence access to genes. (核小体及其修饰体) Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes. (真

7、核基因有更多结合位点) Difference in regulation between eukaryotes and prokaryote7The Regulatory Element of Bacterial, Yeast and Human Genes8Enhancer (激活元件) : a given site binds regulator responsible for activating the gene. Alternative enhancer binds different groups of regulators and control expression of th

8、e same gene at different times and places in responsible to different signals. Activation at a distance is much more common in eukaryotes. Insulators (绝缘子) or boundary elements (临界元件) are regulatory sequences between enhancers and promoters. They block activation of a linked promoter by activator bo

9、und at the enhancer, and therefore ensure activators work discriminately. 9CHAPTER 17 Gene Regulation in eukaryotesTopic 1:Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals一、真核的转录激活蛋白的结构特征The structure features of the eukaryotic transcription activators10The basic features of

10、gene regulation are the same in all eukaryotes, because of the similarity in their transcription and nucleosome structure.Yeast is the most amenable to both genetic and biochemical dissection, and produces much of knowledge of the action of the eukaryotic repressor and activator. The typical eukaryo

11、tic activators works in a manner similar to the simplest bacterial case.Repressors work in a variety of ways.111. Eukaryotic activators (真核激活蛋白) have separate DNA binding and activating functions, which are very often on separate domains of the protein.*1.1 Categories of ActivatorsActivators can sti

12、mulate or inhibit transcription by RNA polymerase IIStructure is composed of at least 2 functional domainsDNA-binding domainTranscription-activation domainMany also have a dimerization domainDNA-Binding DomainsProtein domain is an independently folded region of a proteinDNA-binding domains have DNA-

13、binding motifPart of the domain having characteristic shape specialized for specific DNA bindingMost DNA-binding motifs fall into 3 classesZinc-Containing Modules含锌组件There are at least 3 kinds of zinc-containing modules that act as DNA-binding motifsAll use one or more zinc ions to create a shape to

14、 fit an a-helix of the motif into the DNA major grooveZinc fingers（锌指）Zinc modules（锌组件）Modules containing 2 zinc and 6 cysteinesHomeodomains（同源结构域）These domains contain about 60 amino acidsResemble in structure and function the helix-turn-helix domains of prokaryotic proteinsFound in a variety of ac

15、tivatorsOriginally identified in homeobox proteins regulating fruit fly development12-16bZIP and bHLH MotifsA number of transcription factors have a highly basic DNA-binding motif linked to protein dimerization motifsLeucine zippers （亮氨酸拉链）Helix-loop-helix （螺旋-环-螺旋模体）Examples include:CCAAT/enhancer-

16、binding proteinMyoD proteinTranscription-Activating DomainsMost activators have one of these domainsSome have more than oneAcidic domains (酸性域）： such as yeast GAL4 with 11 acidic amino acids out of 49 amino acids in the domainGlutamine-rich domains（富含谷氨酸域）： include Sp1 having 2 that are 25% glutamin

17、eProline-rich domains (富含脯氨酸域）： such as CTF which has a domain of 84 amino acids, 19 proline1.2 Structures of the DNA-Binding Motifs of ActivatorsDNA-binding domains have well-defined structuresX-ray crystallographic studies have shown how these structures interact with their DNA targetsInteraction

18、domains forming dimers, or tetramers, have also been describedMost classes of DNA-binding proteins cant bind DNA in monomer formZinc FingersDescribed by Aaron Klug in TFIIIANine repeats of a 30-residue element:2 closely spaced cysteines followed 12 amino acids later by 2 closely spaced histidinesCoo

19、rdination of amino acids to the metal helps form the finger-shaped structureRich in zinc, enough for 1 zinc ion per repeatSpecific recognition between the zinc finger and its DNA target occurs in the major grooveStructure of Zinc FingersZnSSZnThe zinc finger is composed of: An antiparallel b-strand

20、contains the 2 cysteines2 histidines in an a-helixHelix and strand are coordinated to a zinc ionInteraction with DNAModules containing 2 zinc and 6 cysteines: The GAL4 ProteinThe GAL4 protein is a member of the zinc-containing family of DNA-binding proteinsIt does not have a zinc fingerEach GAL4 mon

21、omer contains a DNA-binding motif with:6 cysteines that coordinate 2 zinc ions in a bimetal thiolate cluster （双金属巯基簇）The recognition module contains a short a-helix that protrudes into the DNA major grooveThe Gal4 monomer also contains an a-helix that forms a parallel coiled coil（螺旋圈） as it interact

22、s with the a-helix on another GAL4 monomerDNA recognition module of Gal 4Zinc modules: The Nuclear ReceptorsA third class of zinc module is the nuclear receptorThis type of protein interacts with a variety of endocrine（内分泌）-signaling moleculesProtein plus endocrine molecule forms a complex that func

23、tions as an activator by binding to hormone response elements and stimulating transcription of associated genesType I Nuclear ReceptorsThese receptors reside in the cytoplasm bound to another proteinWhen receptors bind to their hormone ligands:Release their cytoplasmic protein partnersMove to nucleu

24、sBind to enhancersAct as activatorsGlucocorticoid Receptors糖皮激素受体DNA-binding domain with 2 zinc-containing modulesOne module has most DNA-binding residuesOther module has the surface for protein-protein interaction to form dimersHsp90Types II and III Nuclear ReceptorsType II nuclear receptors stay w

25、ithin the nucleusBound to target DNA sitesWithout ligands the receptors repress gene activityWhen receptors bind ligands, they activate transcriptionType III receptors are “orphan” whose ligands are not yet identifiedHomeodomainsHomeodomains contain DNA-binding motif functioning as helix-turn-helix

26、motifsA recognition helix fits into the DNA major groove and makes specific contacts thereN-terminal arm nestles in the adjacent minor grooveThe bZIP and bHLH DomainsbZIP proteins dimerize through a leucine zipperThis puts the adjacent basic regions of each monomer in position to embrace DNA target

27、like a pair of tongsbHLH proteins dimerize through a helix-loop-helix motifAllows basic parts of each long helix to grasp the DNA target sitebHLH and bHLH-ZIP domains bind to DNA in the same way, later have extra dimerization potential due to their leucine zippersStructure of leucine zipperbZIP prot

28、eins dimerize through a leucine zipperThis puts the adjacent basic regions of each monomer in position to embrace DNA target like a pair of tongsInteraction with DNAbZIP proteins dimerize through a leucine zipperThis puts the adjacent basic regions of each monomer in position to embrace DNA target l

29、ike a pair of tongsThe bZIP and bHLH DomainsbHLH proteins dimerize through a helix-loop-helix motifAllows basic parts of each long helix to grasp the DNA target site1.3 Independence of the domains of activatorsActivators can stimulate or inhibit transcription by RNA polymerase IIStructure is compose

30、d of at least 2 functional domainsDNA-binding domainTranscription-activation domainMany also have a dimerization domain Domain swap experiment域交换实验35 Domain swap experiment域交换实验The Yeast Two-Hybrid SystemWhat is the yeast two-hybrid system used for?Identifies novel protein-protein interactionsCan id

31、entify protein cascades Identifies mutations that affect protein-protein bindingCan identify interfering proteins in known interactions (Reverse Two-Hybrid System)How does it work?391.4. Activating regions (激活区域) are not well-defined structures*The activating regions are grouped on the basis of amin

32、o acids content.Acidic activation region (酸性激活区域): contain both critical acidic amino acids and hydrophobic aa. yeast Gal4Glutamine-rich region (谷氨酰胺富集区): mammalian activator SP1 Proline-rich region (脯氨酸富集区): mammalian activator CTF140CHAPTER 17 Gene Regulation in eukaryotes二、真核转录激活蛋白的招募调控方式和远距调控特征A

33、ctivation of the eukaryotic transcription by recruitment & Activation at a distanceTopic 2: Recruitment of Protein Complexes to Genes by Eukaryotic Activators41Eukaryotic activators (真核激活蛋白) also work by recruiting (招募) as in bacteria, but recruit polymerase indirectly in two ways:1. Interacting wit

34、h parts of the transcription machinery.2. Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.1. Activators recruit the transcription machinery to the gene.The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complex

35、es, such as the Mediator and the TFD complex. Activators interact with one or more of these complexes and recruit them to the gene.432. Activators also recruit nucleosome modifiers that help the transcription machinery bind at the promoterTwo types of Nucleosome modifiers :Those add chemical groups

36、to the tails of histones (在组蛋白尾上加化学基团), such as histone acetyl transferases (HATs)Those remodel the nucleosomes (重塑核小体), such as the ATP-dependent activity of SWI/SNF.表观遗传学( epigenetics) 是研究没有DNA序列变化的可遗传的基因表达的改变。真核生物染色质是一切遗传学过程的物质基础，染色质构型局部和整体的动态改变，是基因功能调控的关键因素。染色体重塑是指染色质位置和结构的变化，主要涉及在能量驱动下核小体的置换或重新

37、排列，它改变了核小体在基因启动子区的排列，增加了基因转录装置和启动子的可接近性。染色质重塑的发生和组蛋白N 端尾巴修饰密切相关，尤其是对组蛋白H3 和H4 的修饰。修饰直接影响核小体的结构，并为其他蛋白提供了和DNA 作用的结合位点。染色质重塑主要包括2 种类型: 一类是含有组蛋白乙酰转移酶和脱乙酰酶的化学修饰； 另一类是依赖ATP 的物理修饰，利用ATP 水解释放的能量解开组蛋白和DNA 的结合，使转录得以进行。 45How the nucleosome modification help activate a gene?*“Loosen” the chromatin structure b

38、y chromosome remodeling (Fig. 17-11b) and histone modification such as acetylation (Fig. 17-11a), which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome.46Local alterations in chromatin directed by activators（组蛋白乙酰化酶） 472. Adding acetyl groups to histones help

39、s the binding of the transcriptional machinery. One component of TFIID complex bears bromodomains that specifically bind to the acetyl groups. Therefore, a gene bearing acetylated nucleosomes at its promoter have a higher affinity for the transcriptional machinery than the one with unacetylated nucl

40、eosomes. 48Figure 7-39 Effect of histone tail modification溴区结构域蛋白One component of TFIID complex bears bromodomains.49 Many enkaryotic activatorsparticularly in higher eukaryoteswork from a distance. How?Some proteins help, for example Chip protein in Drosophila. The compacted chromosome structure he

41、lp. DNA is wrapped in nucleosomes in eukaryotes. So sites separated by many base pairs may not be as far apart in the cell as thought.3. Action at a distance: loops and insulators50Specific cis-acting elements called insulators (绝缘子) control the actions of activators, preventing the activating the n

42、on-specific genes51Insulators block activation by enhancersFigure 17-12524. Appropriate regulation of some groups of genes requires locus control region (LCR).Human and mouse globin genes are clustered in genome and differently expressed at different stages of developmentA group of regulatory elemen

43、ts collectively called the locus control region (LCR,位点控制区域), is found 30-50 kb upstream of the cluster of globin genes. It binds regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators that control expression of the individual genes in a defin

44、ed order.Figure 17-13Please compare LCR with the Lac operon controlled gene expression in bacteria54 Another group of mouse genes whose expression is regulated in a temporarily and spatially ordered sequence are called HoxD genes. They are controlled by an element called the GCR (global control regi

45、on) in a manner very like that of LCR.55Topic 3: TranscriptionalRepressor & its regulationCHAPTER 17 Gene Regulation in eukaryotesIn eukaryotes, most repressors do not repress transcription by binding to sites that overlap with the promoter and thus block binding of polymerase. (Bacteria often do so

46、)三、真核转录阻遏蛋白（或抑制蛋白）及其调控56Commonly, eukaryotic repressors recruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery contrast to the activator recruited nucleosome modifers, histone deacetylases (组蛋白去乙酰化酶) removing the acetyl groups. Some

47、modifier adds methyl groups to the histone tails, which frequently repress the transcription.This modification causes transcriptional silencing.Three other ways in which an eukaryotic repressor works include: Competes with the activator for an overlapped binding site. Binds to a site different from

48、that of the activator, but physically interacts with an activator and thus block its activating region. Binds to a site upstream of the promoter, physically interacts with the transcription machinery at the promoter to inhibit transcription initiation.58Competes for the activator bindingInhibits the

49、 function of the activator.59Binds to the transcription machineryRecruits nucleosome modifiers (most common*)60In the presence of glucose, Mig1 binds to a site between the USAG and the GAL1 promoter, and recruits the Tup1 repressing complex. Tup1 recruits histone deacetylases, and also directly inte

50、racts with the transcription machinery to repress transcription. A specific example: Repression of the GAL1 gene in yeast61CHAPTER 17 Gene Regulation in eukaryotes四、真核转录调控的特色：信号整合和组合调控Features of the eukaryotic transcriptional regulation: signal integration and combinatorial controlTopic 4: Signal I

51、ntegration and Combinatorial Control621. Activators work together synergistically (协作地) to integrate signals.激活蛋白一起协作来整合多种信号a.“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

52、 the second protein.d. Binding a protein unwinds the DNA from nucleosome a little, revealing the binding site for another protein.642. Signal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator. The HO gene is only expressed in

53、mother cells and only a certain point in the cell cycle, resulting in the budding division feature of yeast S. cerevisiae (啤酒酵母).The mother cell and cell cycle conditions (signals) are communicated to the HO gene (target) by two activators: SWI5 and SBF (communicators).65SWI5: acts only in the mothe

54、r cell and binds to multiple sites some distance from the gene unaided, which recruit enzymes to open the SBF binding sites. SBF: only active at the correct stages of the cell cycle, and cannot bind the sites unaided.Alter the nucleosome(组蛋白乙酰化酶)663. Signal integration: Cooperative binding of activa

55、tors at the human b-interferon gene. The human b-interferon gene (target gene) is activated in cells upon viral infection (signal). Infection triggers three activators (communicator): NFkB, IRF, and Jun/ATF. Activators bind cooperatively to sites adjacent to one another within an enhancer located ab

56、out 1 kb upstream of the promoter, which forms a structure called enhanceosome.684. Combinatory control (组合调控) lies at the heart of the complexity and diversity of eukaryotes, in which Both activators and repressors work together.组合调控是真核生物复杂性和多样性的核心。它属于协作调控的一种具体方式。在组合调控中，激活蛋白和阻遏蛋白共同起作用。69Topic 5: Si

57、gnal Transduction (信号传导) and the Control of Transcriptional RegulatorsCHAPTER 17 Gene Regulation in eukaryotes五、基于真核转录调控的前沿学科：信号传导Signal transduction-A life science frontier centered on the eukaryotic transcriptional regulation. 701. Signals are often communicated to transcriptional regulators throu

58、gh signal transduction pathwayTopic 4: Signal Transduction and the Control of Transcriptional Regulators信号经常通过信号传导途径被运输到转录调节蛋白71Environmental Signals/Information (信号) 1. Small molecules such as sugar, histamine (组胺). 2. Proteins released by one cell and received by another.In eukaryotic cells, most

59、signals are communicated to genes through signal transduction pathway (indirect), in which the initiating ligand is detected by a specific cell surface receptor. 723*. The signal is then relayed (分程传递) to the relevant transcriptional regulator.Signal transduction pathway*1. The initial ligand (“sign

60、al”) binds to an extracellular domain of a specific cell surface receptor2. The signal is thus communicated to the intracellular domain of receptor (via an allosteric change or dimerization )4. The transcriptional regulator control the target gene expression.73a. The STAT pathwayb. The MAP kinase pa