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Chapter 13 Gene Regulation in Eukaryotes Chapter 13 Gene Regulation in Eukaryotes.

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Presentation on theme: "Chapter 13 Gene Regulation in Eukaryotes Chapter 13 Gene Regulation in Eukaryotes."— Presentation transcript:

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2 Chapter 13 Gene Regulation in Eukaryotes Chapter 13 Gene Regulation in Eukaryotes

3 Eukaryotic gene regulation occurs at several levels

4 Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation. Methylation occurs most often in symmetrical CG sequences. Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes. Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation. 1- Control at DNA level by DNA methylation

5 Acetylation ( 乙酰化 ) by histone acetyl transferases (HATs) and coactivators leads to euchromatin formation; p53 acetylation Methylation by HDACs (去 乙酰化酶) and corepressors leads to heterochromatin formation. Rb-E2F 2- Control at DNA level by Histone modifications(Chromatin Remodeling)

6 3-Control at DNA level by gene amplification Repeated rounds of DNA replication yield multiple copies of a particular chromosomal region.

7 4- Control at transcription initiation gene control region for gene X By using different sequences (promoter, enhancer or silencer sequences) and factors, the rate of transcription of a gene is controlled

8 Calcitonin gene-related peptide Control at mRNA splicing (alternate splicing) (four exons) 32 amino acids Reduces bone resorption 37 amino acids Vasodilator

9  Messenger RNA longevity can be influenced by several factors.  Poly(A) tails seem to stabilize mRNAs.  The sequence of the 3’untranslated region (3’UTR) preceding a poly(A) tail also seems to affect mRNA stability.  Several short-lived mRNAs have the sequence AUUUA repeated several times in their 3’untranslated regions. 6- Control at mRNA stability

10  When this sequence is artificially transferred to the 3’untranslated region of more stable mRNAs, they, too, become unstable.  Chemical factors, such as hormones, may also affect mRNA stablility.  In the toad Xenopus laevis( 非洲爪蟾 ), the vitellogenin gene( 卵黄生成素 ) is transcriptionally activated by the steroid hormone estrogen( 类固醇激 素 ). However, in addition to inducing transcription of this gene, estrogen also increases the longevity of its mRNA. 6- Control at mRNA stability

11 Recent research has revealed that the stability of mRNAs and the translation of mRNAs into polypeptides are also regulated by small, noncoding RNA molecules called microRNA (miRNAs). 6- Control at mRNA stability

12 7- Control at initiation of translation 5’ UTR 3’ UTR AUG UAA Specific sequences make specific secondary structures Specific protein factors bind to these secondary structures

13 COOH + NH 2 ATP CONH CONH ubiquitin protein ligase Doomed protein molecule 26S proteasome Ubiquitin-dependent proteolysis. Protein molecule is tagged for degradation by attachment of a 20 kDa protein, ubiquitin 蛋白酶体系统 (ubiquitin-proteasome system(UPS)) 主要由泛 素激活酶 (E1) 、泛素交联酶 (E2) 、泛素连接酶 (E3) 和 26S 蛋白酶体 组成, 是降解细胞内蛋白质的主要途径 对于许多细胞进程,包括细胞周期、基因表达的调控、氧化应激反应等, 都是必不可少的。 2004 年诺贝尔化学奖. 8-Regulation by protein stability

14 Similarity of regulation between eukaryotes and prokaryote 1.Principles are the same: signals ( 信号 ), activators and repressors ( 激活蛋白和阻遏蛋白 ) recruitment and allostery, cooperative 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.

15 Difference in regulation between eukaryotes and prokaryote***** 1. Pre-mRNA splicing adds an important step for regulation. (mRNA 前体的剪接 ) 2. The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. ( 真核转录机 器更复杂 ) 3. Nucleosomes and their modifiers influence access to genes. ( 核小体及其修饰体 ) 4. Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes. ( 真核基因有更多结合位点 )

16 15 A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration Bacteria Yeast Human

17 16 Promoter  Core promoter  in eukaryote: TATA-box, Initiator (Inr)  in prokaryote: -10 region, Inr  Proximal elements of promoter  in prokaryote: -35 region  in eukaryote: CAAT-box, GC-box UPE: upstream promoter element UAS: upstream activating sequence Terminator ( 终止子 ) : A DNA sequence just downstream of the coding segment of a gene, which is recognized by RNA polymerase as a signal to stop transcription. Cis-acting element

18 Enhancer ( 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 the same gene at different times and places in responsible to different signals. Activation at a distance is much more common in eukaryotes. Silencer (沉默子) A DNA sequence that helps to reduce or shut off the expression of a nearby gene. 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 bound at the enhancer, and therefore ensure activators work discriminately.

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21 What is trans-acting factor? Usually they are proteins, that bind to the cis-acting elements to control gene expression.

22 These trans-acting factors can control gene expression in several ways:  may be expressed in a specific tissue  may be expressed at specific time in development  may be required for protein modification  may be activated by ligand binding

23 (1) RNA polymerase  prokaryotic RNA Pol  eukaryotic RNA Pol (2) Transcription factors  Basal/general TFs  Specific TFs

24 (3) Domains of trans-acting factors  DNA binding domain DBD DNA 结合结构域  transcription activating domain 转录活化结构域

25 Topic 1: Conserved Mechanisms of Transcriptional Regulation from Yeast ( 酵母 ) to Mammals ( 哺乳动物 ) Topic 1: Conserved Mechanisms of Transcriptional Regulation from Yeast ( 酵母 ) to Mammals ( 哺乳动物 ) 一、真核的转录激活蛋白的结构特征 The structure features of the eukaryotic transcription activators

26 25 The basic features of 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 eukaryotic activators works in a manner similar to the simplest bacterial case. Repressors work in a variety of ways.

27 1. 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.***** Gal4 bound to its site on DNA

28 The regulatory sequences of the Yeast GAL1 gene. Eukaryotic activators---Example 1: Gal4***** Gal4 is the most studied eukaryotic activator Gal4 activates transcription of the galactose genes in the yeast S. cerevisae. Gal4 binds to four sites (UAS G ) upstream of GAL1( 5'- CGGRNNRCYNYNYNCNCCG-3' ), and activates transcription 1,000-fold in the presence of galactose

29 Experimental evidences showing that Gal4 contains separate DNA binding and activating domains. 1. Expression of the N-terminal region (DNA-binding domain) of the activator produces a protein bound to the DNA normally but did not activate transcription. Domain swap experiment 2. Fusion of the C-terminal region (activation domain) of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. Domain swap experiment

30 29 Domain swap experiment***** Moving domains among proteins, proving that domains can be dissected into separate parts of the proteins. Many similar experiments shows that DNA binding domains and activating regions are separable.

31 30 Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4, respectively. Produce fusion proteins two hybrid Assay ( 双杂交 ) Box1 The two hybrid Assay ( 双杂交 ) to study protein- protein interaction and identify proteins interacting with a known protein in cells*****

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37 ***** 2. Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles as found in bacteria. ***** Helix-turn-helix ( HTH) ( 螺旋 - 转角 - 螺旋 ) Zinc finger (锌指) and zinc cluster Leucine zipper motif (亮氨酸拉链) Helix-Loop-Helix proteins (螺旋 - 突环 - 螺旋) : basic zipper and HLH proteins Transcription factor motifs

38 HTH (helix-turn-helix) α-helix (N-terminus)----specific α-helix (C-terminus)----non-specific

39 Bacterial regulatory proteins Most use the helix-turn-helix ( HTH :旋转 - 转角 - 旋转) motif to bind DNA target Most bind as dimers to DNA sequence: each monomer inserts an a helix into the major groove. Eukaryotic regulatory proteins 1. Recognize the DNA using the similar principles, with some variations in detail. 2. In addition to form homodimers ( 同源二聚体 ), some form heterodimers ( 异源二聚体 ) to recognize DNA, extending the range of DNA- binding specificity.

40 Zinc containing DNA-binding domains ( 锌指 结构域 ): Zinc finger proteins (TFIIIA) and Zinc cluster domain (Gal4)

41 Leucine Zipper Motif ( 亮氨酸拉链基序 ) : The Motif combines dimerization and DNA- binding surfaces within a single structural unit.

42 Helix-Loop-Helix motif

43 myogenic factor: 生肌调节蛋白是 一种转录因子。

44 3. Activating regions ( 激活区域 ) are not well-defined structures*** The activating regions are grouped on the basis of amino acids content. yeast Gal4  Acidic activation region ( 酸性激活区域 ): contain both critical acidic amino acids and hydrophobic aa. yeast Gal4  Glutamine-rich region ( 谷氨酰胺富集区 ): mammalian activator SP1  Proline-rich region ( 脯氨酸富集区 ): mammalian activator CTF1

45 Topic 2: Recruitment of Protein Complexes to Genes by Eukaryotic Activators Topic 2: Recruitment of Protein Complexes to Genes by Eukaryotic Activators 二、真核转录激活蛋白的招募调控方式和远距调控特征 Activation of the eukaryotic transcription by recruitment & Activation at a distance

46 Eukaryotic 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 with parts of the transcription machinery. 2. Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.

47 eukaryotic transcriptional machinery The eukaryotic transcriptional machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TF Ⅱ D complex. Activators interact with one or more of these complexes and recruit them to the gene. 1. Activators recruit the transcription machinery to the gene.

48 Chromatin Immuno-precipitation (ChIP) ( 染色质免疫 共沉淀 ) ***** Chromatin Immuno-precipitation (ChIP) ( 染色质免疫 共沉淀 ) to visualize where a given protein (activator) is bound in the genome of a living cell*****.)

49 2. Activators also recruit modifiers that help the transcription machinery bind at the promoter Two types of Nucleosome modifiers : Those add chemical groups 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.

50 ***** How the nucleosome modification help activate a gene?***** 1. “Loosen” the chromatin structure by chromosome remodeling and histone modification such as acetylation, which uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome.

51 Local alterations in chromatin structure directed by activators Activators, capable of binding to their sites on DNA within a nucleosome are shown bound upstream of a promoter that is inaccessible within chromain. (a)The activator is shown recruiting a histone acetylase. That enzyme adds acetyl groups to residues within the histone tails. This alters the packing of the nucleosomes somewhat, and also creates binding sites for proteins carrying the appropriate recognition domains. (b)The activator recruits a nucleosome remodeller, which alters the structure of nucleosomes around the promoter, rendering it accessible and capable of binding the transcription machinery.

52 insulators Specific cis-acting elements called insulators ( 绝缘子 ) control the actions of activators, preventing the activating the non-specific genes 3. Action at a distance: loops and insulators

53 Insulators block activation by enhancers. a) A promoter activated by activators bound to an enhancer. b) An insulator is placed between the enhancer and the promoter. When bound by appropriate insulator- binding proteins, activation of the promoter by the enhancer is blocked, despite activators binding to the enhancer. c) The activator can activate another promoter nearby. d) The original promoter can be activated by another enhancer placed downstream.

54 Transcriptional Silencing ( 转录沉默 ) Transcriptional Silencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressor. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression 。

55 4 Appropriate regulation of some groups of genes requires locus control region (LCR). 1. Human and mouse globin genes are clustered in genome and differently expressed at different stages of development locus control region (LCR) 2. A group of regulatory elements collectively called the locus control region (LCR), is found 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 defined order.

56 Regulation by LCRs (a)The human globin genes and the LCR that ensures their ordered expression. (b) The globin genes from mice, which are also regulated by an LCR. (C) The HoxD gene cluster from the mouse controlled by an element called the GCR which like the LCRs appears to impose ordered expression on the gene cluster.

57 Topic 3: Transcriptional Repressor & its regulation Topic 3: Transcriptional Repressor & its regulation do not In 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) 三、真核转录阻遏蛋白(或抑制蛋白)及其调控

58 recruit nucleosome modifiers Commonly, eukaryotic repressors recruit nucleosome modifiers that compact the nucleosome or remove the groups recognized by the transcriptional machinery [contrast to the activator recruited nucleosome modifiers, histone deacetylases ( 组蛋白去乙酰化酶 ) removing the acetyl groups]. Some modifier adds methyl groups to the histone tails, which frequently repress the transcription. This modification causes transcriptional silencing.

59 Three other ways in which an eukaryotic repressor works include: (1) Competes with the activator for an overlapped binding site (1) Competes with the activator for an overlapped binding site. physically interacts with an activator (2) Binds to a site different from that of the activator, but physically interacts with an activator and thus block its activating region. physically interacts with the transcription machinery (3) Binds to a site upstream of the promoter, physically interacts with the transcription machinery at the promoter to inhibit transcription initiation.

60 Ways in which eukaryotic repressor work Competes for the activator binding Inhibits the function of the activator.

61 Binds to the transcription machinery *** Recruits nucleosome modifiers (most common***)

62 recruits histone deacetylases directly interacts with the transcription machinery In the presence of glucose, Mig1 binds to a site between the USA G and the GAL1 promoter, and recruits the Tup1 repressing complex. Tup1 recruits histone deacetylases, and also directly interacts with the transcription machinery to repress transcription. A specific example: Repression of the GAL1 gene in yeast

63 Topic 4: Signal Transduction ( 信号传导 ) and the Control of Transcriptional Regulators 四、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.

64 communicated 1. Signals are often communicated to transcriptional regulators through signal transduction pathway 信号传导途径 信号经常通过信号传导途径被运输到转录调节蛋白

65 Environmental Signals/Information Environmental Signals/Information ( 信号 ) 1. Small molecules such as sugar, histamine ( 组胺 ). 2. Proteins released by one cell and received by another. most signals are communicated to genes through signal transduction pathway (indirect) In eukaryotic cells, most signals are communicated to genes through signal transduction pathway (indirect), in which the initiating ligand is detected by a specific cell surface receptor.

66 transcriptional regulator. 3*. The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulator. Signal transduction pathway*** extracellular domain 1. The initial ligand (“signal”) binds to an extracellular domain of a specific cell surface receptor intracellular domain 2. The signal is thus communicated to the intracellular domain of receptor (via an allosteric change or dimerization ) target gene expression 4. The transcriptional regulator control the target gene expression.

67 JAK activation occurs upon ligand-mediated receptor multimerization because two JAKs are brought into close proximity, allowing trans- phosphorylation. The activated JAKs subsequently phosphorylate additional targets, including both the receptors and the major substrates, STATs. STATs are latent transcription factors that reside in the cytoplasm until activated.

68 MAP kinases are activated within the protein kinase cascades called “MAPK cascade”. Each one consists of three enzymes, MAP kinase, MAP kinase kinase (MKK, MEK, or MAP2K) and MAP kinase kinase kinase (MKKK, MEKK or MAP3K) that are activated in series. A MAP3K that is activated by extracellular stimuli phosphorylates a MAP2K on its serine and threonine residues, and this MAP2K activates a MAP kinase through phosphorylation on its threonine and tyrosine residues (Tyr-185 and Thr-183 of ERK2).

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70 2. Signals control the activities of eukaryotic transcriptional regulators in a variety of ways. 信号通过不同方式控制转录调节蛋白的活性

71 Mechanism 1: unmasking an activating region Mechanism 1: unmasking an activating region: (1) A conformational change to reveal the previously buried activating region. (2) Releasing of the previously bound masking protein. Example: the activator Gal4 is controlled by the masking Gal80). block recruit (3) Some masking proteins not only block the activating region of an activator but also recruit a deacetylase enzyme to repress the target genes. Example: Rb represses the function of the mammalian transcription activator E2F in this way. Phosphorylation of Rb releases E2F to activate the target gene expression***.

72 masking protein Activator Gal4 is regulated by a masking protein Gal80*** Gal4

73 Mechanism 2: Transport into and out of the nucleus Mechanism 2: Transport into and out of the nucleus: W hen not active, many activators and repressors are held in the cytoplasm. The signaling ligand causes them to move into the nucleus where they activate transcription

74 Other Mechanisms #1: A cascade of kinases that ultimately cause the phosphorylation of regulator in nucleus (new Other Mechanisms #1: A cascade of kinases that ultimately cause the phosphorylation of regulator in nucleus (new) (Fig.19-4a).

75 Other Mechanisms #2: The activated receptor is cleaved by cellular proteases ( 蛋白酶 ), and the c-terminal portion of the receptor enters the nuclease and activates the regulator (new Other Mechanisms #2: The activated receptor is cleaved by cellular proteases ( 蛋白酶 ), and the c-terminal portion of the receptor enters the nuclease and activates the regulator (new): (Fig.19-4c).


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