Welcome Each of You to My Molecular

Biology Class

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Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods

2005-5-10

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Part IV RegulationCh 16: Transcriptional regulation in Ch 16: Transcriptional regulation in prokaryotesprokaryotesCh 17: Transcriptional regulation in Ch 17: Transcriptional regulation in eukaryoteseukaryotesCh18: Regulatory RNAsCh18: Regulatory RNAsCh 19: Gene regulation in Ch 19: Gene regulation in development and evolutiondevelopment and evolutionCh 20: Genome Analysis and Ch 20: Genome Analysis and Systems BiologySystems Biology

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Surfing the contents of Part IV

--The heart of the frontier biological

disciplines

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Chapter 17Gene Regulation

in Eukaryotes

•Molecular Biology Course

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TOPIC 1 TOPIC 1 Conserved Mechanisms of Conserved Mechanisms of Transcriptional Regulation from Yeast to Transcriptional Regulation from Yeast to Human.Human.

TOPIC 2TOPIC 2 Recruitment of Protein Complexes to Recruitment of Protein Complexes to Genes by Eukaryotic Activators.Genes by Eukaryotic Activators.

TOPIC 3TOPIC 3 Transcriptional Repressors Transcriptional RepressorsTOPIC 4TOPIC 4 Signal Integration and Combinatorial Signal Integration and Combinatorial

Control.Control.TOPIC 5TOPIC 5 Signal Transduction and the Control of Signal Transduction and the Control of

Transcriptional Regulators.Transcriptional Regulators.TOPIC 6 TOPIC 6 Gene Silencing by Modification of Gene Silencing by Modification of

Histones and DNA.Histones and DNA.TOPIC 7 TOPIC 7 Epigenetic Gene Regulation.Epigenetic Gene Regulation.

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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 regulatory proteins:

Positive regulators or activators INCREASE the transcription

Negative regulators or repressors

DECREASE or ELIMINATE the transcription

Principles of Principles of Transcription RegulationTranscription Regulation

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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.

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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 )

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A lot more regulator bindings sites in multicellular organisms reflects the more extensive signal integration

Fig. 17-1

Bacteria

Yeast

Human

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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.

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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Topic 1: Conserved Topic 1: Conserved Mechanisms of Mechanisms of

Transcriptional Regulation Transcriptional Regulation from from YeastYeast ( ( 酵母酵母 ) ) to to MammalsMammals ( ( 哺乳动物哺乳动物 ))

CHAPTER 17 Gene Regulation in eukaryotes一、真核的转录激活蛋白的结构特征

The structure features of the eukaryotic transcription activators

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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.

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1. Eukaryotic activators ( 真核激活蛋白 ) have separate DNA binding and activating functions 【与原核相似】 , which are very often on separate domains of the protein.

Fig. 17-2 Gal4 bound to its site on DNA

15Fig. 17-3 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 (UASG) upstream of

GAL1, and activates transcription 1,000-fold in the presence of galactose

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Eukaryotic activators---Example 1: Gal4Experimental 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.

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 experimentDomain swap experiment

实验介绍系列 1 － Experiment introduction series

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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.

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Box 1 The two hybrid Assay (two hybrid Assay ( 酵母双杂交酵母双杂交 )) is used to identify proteins interacting with each other. （实验介绍系列 2 ）

Fuse protein A and protein B genes to the DNA binding domain and activating region of Gal4, respectively.

Produce fusion proteins

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2. Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles as found in bacteria.

Homeodomain proteins Zinc containing DNA-binding

domain: zinc finger and zinc cluster Leucine zipper motif Helix-Loop-Helix proteins : basic

zipper and HLH proteins

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Bacterial regulatory proteins• Most use the helix-turn-helix motif

to bind DNA target• Most bind as dimers to DNA

sequence: each monomer inserts an helix into the major groove.

Eukaryotic regulatory proteins1. 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.

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Homeodomain proteins: The homeodomain ( 同源结构域 ) is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins.

Figure 17-5

What is the same?

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

Figure 17-6

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

Figure 17-7

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Dimerization ( 二聚化 ) is mediated by hydrophobic interactions between the appropriately-spaced leucine ( 亮氨酸 ) to form a coiled coil structure

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Helix-Loop-Helix motif: similar as in leucine zipper motif.

Figure 17-8

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myogenic factor: 生肌调节蛋白是一种转录因子。

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Because the region of the -helix that binds DNA contains baisc amino acids residues, Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins.

Both of these proteins use hydrophobic amino acid residues for dimerization.

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3. Activating regions ( 激活区域 ) are not well-defined structures

The activating regions are grouped on the basis of amino acids content.

Acidic activation region ( 酸性激活区域 ): contain both critical acidic amino acids and hydrophobic acids.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

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Topic 2: Recruitment of Topic 2: Recruitment of Protein Complexes Protein Complexes

to Genes by to Genes by Eukaryotic ActivatorsEukaryotic Activators

CHAPTER 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Activation of the eukaryotic transcription by recruitment & Activation at a distance

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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.

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1. Activators recruit the transcription machinery to the gene.

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

Figure 17-9

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Box 2 Chromatin Immuno-precipitation (ChIP) Chromatin Immuno-precipitation (ChIP) (( 染色质免疫共沉淀染色质免疫共沉淀 )) to visualize where a given protein (activator) is bound in the genome of a living cell.) （实验介绍系列 3 ）

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Activator Bypass Experiment Activator Bypass Experiment (( 越过激活子实验越过激活子实验 ))-Activation of transcription through direct tethering of mediator to DNA. （实验介绍系列 4 ）

Directly fuse the bacterial DNA-binding protein LexA protein to Gal11, a component of the mediator complex to activate GAL1 expression.

Figure 17-10

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At most genes, the transcription machinery is not prebound, and appear at the promoter only upon activation. Thus, no allosteric no allosteric activationactivation of the prebound polymerase has been evident in eukaryotic regulation 。

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2. Activators also recruit modifiers that help the transcription machinery bind at the promoterTwo 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.

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How the nucleosome modification help activate a gene?

1. “Loosen” the chromatin structure by 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.

39Fig 17-11 Local alterations in chromatin directed by activators

（组蛋白乙酰化酶）

uncover DN

A-binding sites

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2. Adding acetyl groups to histones helps 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 nucleosomes.

41Figure 7-39 Effect of histone tail modification

溴区结构域蛋白One component of TFIID complex bears bromodomains.

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Many enkaryotic activators － particularly in higher eukaryotes － work from a

distance. How?How?1. Some proteins help, for example Chip

protein in Drosophila. 2. The compacted chromosome structure

help. 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 insulators

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Specific cis-acting elements called insulatorsinsulators ( 绝缘子 ) control the actions of activators, preventing the activating the non-specific genes

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Insulators block

activation by

enhancers

Figure 17-12

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Transcriptional Silencing Transcriptional Silencing (( 转录沉默转录沉默 ))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 ( 绝缘子元件 ) can block this spreading, so insulators protect genes from both indiscriminate activation and repression.[ 绝缘子的概念到此才介绍完毕 ]

ApplicationApplication ： A gene inserted at random into the mammalian genome is often “silenced”, and placing insulators upstream and downstream of that gene can protect the gene from silencing.

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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

2. A group of regulatory elements collectively called the locus control locus control region (LCR)region (LCR), 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 defined order.

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Figure 17-13

Please compare LCR with the Lac operon controlled gene expression in bacteria

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Another group of mouse genes whose expression is regulated in a temporarily and spatially ordered sequence are called HoxDHoxD genes genes. They are controlled by an element called the GCR (global control region)GCR (global control region) in a manner very like that of LCR.

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Topic 3: TranscriptionalTopic 3: TranscriptionalRepressor & its regulationRepressor & its regulation

CHAPTER 17 Gene Regulation in eukaryotes

In eukaryotes, most repressors do do notnot 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

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Commonly, eukaryotic repressors recruit nucleosome modifiersrecruit 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 modifier adds methyl groups to the histone tails, which frequently repress the transcription.

This modification causes transcriptional silencing.

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Three other ways in which an eukaryotic repressor works include:

(1)(1)Competes with the activator for an Competes with the activator for an overlapped binding siteoverlapped binding site.

(2)Binds to a site different from that of the activator, but physically physically interacts with an activatorinteracts with an activator and thus block its activating region.

(3)Binds to a site upstream of the promoter, physically interacts with physically interacts with the transcription machinerythe transcription machinery at the promoter to inhibit transcription initiation.

52Figure 17-19 ： Ways in which eukaryotic repressor work

Competes for the activator binding

Inhibits the function of the activator.

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Binds to the transcription machinery

Recruits nucleosome modifiers (most common)

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In the presence of glucose, Mig1 binds to a site between the USAG and the GAL1 promoter, and recruits the Tup1 repressing complex. Tup1 recruits recruits histone deacetylaseshistone deacetylases, and also directly directly interacts with the transcription interacts with the transcription machinerymachinery to repress transcription.

A specific example: Repression of the GAL1 gene in yeast

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Topic 4: Signal Integration Topic 4: Signal Integration and Combinatorial and Combinatorial

ControlControl

CHAPTER 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 control

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1. 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

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Review the Lac operon control in bacteria. Two signals are integrated to control Lac expression

GlucoseLactose

Figure 16-6

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In multicellular organisms, signal In multicellular organisms, signal integration (integration ( 信号整合信号整合 ) ) is used is used extensively.extensively. In some cases, numerous signals are required to switch a gene on. However, each signal is transmitted to the gene by a separate regulator, and therefore, multiple activators often work multiple activators often work together, and together, and they do so synergistically (two activators working together is greater than the sum of each of them working alone.)

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Three strategies of the synergy ( 协作的三种方式 ):S1: Multiple activators recruit a single component of the transcriptional machinery. For example, by touching the different part of the mediator complex ( 中介体复合物 ). The combined binding energy has an exponential effect on recruitment.

S2: Multiple activators each recruit a different component of the transcriptional machinery. These components binds to the promoter DNA inefficiently without help.

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S3: Multiple activators help each other bind to their sites upstream of the gene they control. (Figure 17-14)

61Figure 17-14: Cooperative binding of activators

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 the second protein.d. Binding a protein unwinds the DNA from nucleosome a little, revealing the binding site for another protein.

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2. Signal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator. [S3 策略的 example]The HO gene is only expressed in

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 mother cell and cell cycle conditionsconditions (signals)(signals) are communicated to the the HOHO gene gene (target)(target) by two activators: SWI5 and SWI5 and SBF (communicators)SBF (communicators).

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SWI5: acts only in the mother cellmother 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 stagescorrect stages of the cell cycle, and cannot bind the sites unaided.

Alter the nucleosome

Figure 17-15

( 组蛋白乙酰化酶 )

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Figure 17-14: Cooperative binding of activators. (c) The first protein recruit a nucleosome remodeller whose action reveal a binding site for the second protein.

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3. Signal integration: Cooperative binding of activators at the human -interferon gene. [S3 策略的example] The human human -interferon gene-interferon gene (target (target

gene)gene) is activated in cells upon viral viral infection (signal)infection (signal). Infection triggers three activatorsthree activators (communicator)(communicator): NFB, IRF, and Jun/ATF. Activators bind cooperatively to sites adjacent to one another within an enhancer located about 1 kb upstream of the promoter, which forms a structure called enhanceosome.

66Figure 17-16

增强体

( 人的干干干干干干干干干干 )增强子

1. Activators interact with each other

2. HMG-I binds within the enhancer and aids the binding of the activators (bends the DNA to promote the activator interaction)

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HMG-IHMG-I is constitutively active in the cells, and play an architectural role in the INF- gene activation process.

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Figure 17-14: Cooperative binding of activators. (a)“Classical” cooperative binding through direct interaction between the two proteins.

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4. 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。

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A regulator (CAP) works together with different repressors at different genes, this is an example of Combinatorial Control.

In fact, CAP acts at more than 100 genes in E.coli, working with an array of partners.

7: Combinatorial Control ( 组合调控 ): CAP controls other genes as well

Page 499 & Slide 47 in Ch 16Review

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In complex multicellular organisms, combinatorial control involves many more regulators and genes than shown above. Both activatorsactivators and repressorsrepressors can be involved.

Four signal

s

Three signal

s

Figure 17-18

There is extensive combinatorial control in eukaryotes.---A generic picture

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5. An example: combinatory control of the mating-type genes from S. cerevisiae ( 啤酒酵母的交配型的组合调控 )

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The yeast S. cerevisiae exists in three forms: ---two haploid cells ( 单倍体 ) of different mating types － aaand .---the diploid cells ( 双倍体 ) (a/a/) formed when an a and an cell mate and fuse.

Cells of the two mating typesCells of the two mating types (aaand ) differ because they express different sets of genes: aa specific genesspecific genes and specific genes specific genes.

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aa cells cells make the regulatory protein aa11,

cellscells make the protein 11 and 22. Both cell typesBoth cell types express the fourth

regulator protein Mcm1Mcm1 that is also involved in regulatory the mating-type specific genes.

How do these regulators work together to keep a cell in its own type? [Figure 17-19]

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Figure 17-19: Control of cell-type specific genes in yeast

Cooperative binding

Cooperative binding

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Topic 5: Signal Topic 5: Signal Transduction (Transduction ( 信号传导信号传导 ) )

and the Control of and the Control of Transcriptional RegulatorsTranscriptional Regulators

CHAPTER 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.

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1. Signals are often communicated to transcriptional regulators through signal transduction pathway

Topic 4: Signal Transduction and the Control of Transcriptional Regulators

信号经常通过信号传导途径信号传导途径被运输到转录调节蛋白

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Environmental Signals/InformationEnvironmental 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 signals are most signals are

communicated to genes through signal communicated to genes through signal transduction pathway (indirect)transduction pathway (indirect), in which the initiating ligand is detected by a specific cell surface receptor.

What about in bacteria?

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3*. The signal is then relayed ( 分程传递 ) to the relevant transcriptional regulator.transcriptional regulator.

Signal transduction pathway1. The initial ligand (“signal”) binds to an extracellular domainextracellular domain of a specific cell surface receptor2. The signal is thus communicated to the intracellular domainintracellular domain of receptor (via an allosteric change or dimerization )

4. The transcriptional regulator control the target gene target gene expression expression (topic 2-4).

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a. The STAT pathway

b. The MAP kinase pathway

Figure 17-22 ： Signal transduction pathway

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2. Signals control the activities of eukaryotic transcriptional regulators in a variety of ways---The mechanisms of signal transduction.

Topic 4: Signal Transduction and the Control of Transcriptional Regulators

信号传导的机制：信号经常通过不同方式控制转录调节蛋白的活性In eukaryotes, a signala signal can be communicated, directly or indirectly, to a transcriptional a transcriptional regulatorregulator.

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Mechanism 1: unmasking an activating Mechanism 1: unmasking an activating regionregion (Topic 2 & 3):

(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) (Fig.17-23).

(3) Some masking proteins not only blockblock the activating region of an activator but also recruitrecruit 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.

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Activator Gal4 is regulated by a masking proteinmasking protein Gal80

Gal4

Figure 17-23

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Mechanism 2: Transport into and out Mechanism 2: Transport into and out of the nucleus of the nucleus (Fig.17-21) :When 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 (Fig.19-4b).

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

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

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Topic 6: Gene “Silencing” Topic 6: Gene “Silencing” by Modification of by Modification of Histones and DNAHistones and DNA

CHAPTER 17 Gene Regulation in eukaryotes

Transcriptional silencing Transcriptional silencing is a position is a position effect. (1) A gene is silenced effect. (1) A gene is silenced because of where it is located, not in because of where it is located, not in response to a specific environmental response to a specific environmental signal. (2) Silencing can spread over signal. (2) Silencing can spread over large stretches of DNA, switching off large stretches of DNA, switching off multiple genes, even those quite multiple genes, even those quite distant from the initiating event. distant from the initiating event.

The most common form of silencing The most common form of silencing is associated with a dense form of is associated with a dense form of chromatin called chromatin called “heterochromatin”. “heterochromatin”.

Heterochromatin is frequently Heterochromatin is frequently associated with particular regions associated with particular regions of the chromosome, notably the of the chromosome, notably the telomeres, and the centromeres. telomeres, and the centromeres.

In mammalian cells, about 50% of In mammalian cells, about 50% of the genome is estimated to be in the genome is estimated to be in some form of heterochromatin.some form of heterochromatin.

Transcriptional silencing is Transcriptional silencing is associated with associated with Modification of Modification of nucleosomes that alters the nucleosomes that alters the accessibility of a gene accessibility of a gene to the to the transcriptional machinery and transcriptional machinery and other regulatory proteins. other regulatory proteins.

The modification enzymes for The modification enzymes for silencing include silencing include deacetylases, deacetylases, DNA methylases.DNA methylases.

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6-1. Silencing in yeast is mediated by deacetylation and methylation of the histones

Topic 6: Gene “Silencing” by Modification of Histones and DNA

在酵母中的沉默是通过对组蛋白的去乙酰化去乙酰化和和甲基化甲基化介导的

The telomeres, the silent mating-type locus, The telomeres, the silent mating-type locus, and the rDNA genes are all “silent” regions and the rDNA genes are all “silent” regions in in S. cerevisiaeS. cerevisiae..

Three genes encoding regulators of silencing, Three genes encoding regulators of silencing, SIR2SIR2, , 33, and , and 44 have been found ( have been found (SIRSIR stands stands for for SSilent ilent IInformation nformation RRegulator).egulator).

Fig. 17-24. Silencing at the yeast telomere

Rap1Rap1 recruits recruits Sir complex to Sir complex to the temomere.the temomere.Sir2Sir2 deacetylates deacetylates nearby nearby nucleosome.nucleosome.

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Silencing specificity Silencing specificity is determined is determined by Rap1, the telomere DNA-binding by Rap1, the telomere DNA-binding protein. It can also be determined protein. It can also be determined by RNA molecules using RNAi by RNA molecules using RNAi machinery (Chapter 18).machinery (Chapter 18).The spreading of silencing is The spreading of silencing is restricted/controlled restricted/controlled by insulators by insulators and other kind of histone and other kind of histone modifications that block binding of modifications that block binding of the Sir2 proteins.the Sir2 proteins.

Transcription can also be Transcription can also be silenced by silenced by methylation of DNAmethylation of DNA by histone by histone methyltransferase (H3 and H4, methyltransferase (H3 and H4, Chapter 7). Chapter 7).

This enzyme have been recently This enzyme have been recently found in yeast, but is common in found in yeast, but is common in mammalian cells. Its function is mammalian cells. Its function is better understood in higher better understood in higher eukaryotes.eukaryotes.

In higher eukaryotesIn higher eukaryotes, silencing is , silencing is typically associated with chromatin typically associated with chromatin containing histones that both containing histones that both deacetylated and methylated.deacetylated and methylated.

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6-2. In Drosophila, HP1 recognizes Methylated Histones and Condense Chromatin.

Topic 6: Gene “Silencing” by Modification of Histones and DNA

在果蝇中， HP1 蛋白识别甲基化甲基化的组蛋白和浓缩的染色质。

HP1 proteinHP1 protein is a component of is a component of heterochromatin in Drosophila heterochromatin in Drosophila that binds to methylated residue that binds to methylated residue in histone H3.in histone H3.

Such a histone modification is Such a histone modification is produced by Su(Var)3-9.produced by Su(Var)3-9.

Different types of modification at Different types of modification at the histones can be involved in the histones can be involved in distinct geene regulationdistinct geene regulation. What . What will happen when multiple forms will happen when multiple forms of modification occur? ---A of modification occur? ---A “Histone Code” hypothesis. “Histone Code” hypothesis.

Box 17-4 Box 17-4 Is there aIs there a histone code?histone code? According to this idea, different According to this idea, different

patterns of modification on histone patterns of modification on histone tails can be “read” to mean different tails can be “read” to mean different things. The “meaning” would be the things. The “meaning” would be the result of the direct effects of these result of the direct effects of these modifications on chromatin density modifications on chromatin density and form. and form.

But in addition, the particular pattern But in addition, the particular pattern of modifications at any given location of modifications at any given location would recruit specific proteins.would recruit specific proteins.

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6-3. DNA Methylation Is Associated with Silenced Genes in Mammlian cells.

Topic 6: Gene “Silencing” by Modification of Histones and DNA

DNA 甲基化甲基化与哺乳动物细胞的沉默基因相关联。【注 DNA 甲基化不是组蛋白甲基化】

Some mammalian genes are kept Some mammalian genes are kept silent by methylation of nearby DNA silent by methylation of nearby DNA sequences.sequences.

Large regions of mammalian genome Large regions of mammalian genome are marked by are marked by methylation of DNA methylation of DNA sequencessequences, which is often seen in , which is often seen in heterochromaticheterochromatic regions. [Why?] regions. [Why?]

The methylated DNA sequences are The methylated DNA sequences are often often

recognized by DNA-binding proteins recognized by DNA-binding proteins (such as MeCP2) that recruit histone (such as MeCP2) that recruit histone decetylases and histone methylases, decetylases and histone methylases, which then modify nearby chromatin.which then modify nearby chromatin.

Thus, methylation of DNA can mark Thus, methylation of DNA can mark sites where heterochromatin sites where heterochromatin subsequently forms (Fig. 17-25).subsequently forms (Fig. 17-25).

Fig. 17-25 Switching a gene off through DNA Fig. 17-25 Switching a gene off through DNA methylation and the subsequent histone methylation and the subsequent histone modification modification

DNA methylation lies at the DNA methylation lies at the heart of heart of ImprintingImprinting

Imprinting- in a diploid cell, one Imprinting- in a diploid cell, one copy of a gene from the father or copy of a gene from the father or mother is expressed while the mother is expressed while the other copy is silenced.other copy is silenced.

Two well-studied examples: human Two well-studied examples: human H19H19 and insulin-like growth factor and insulin-like growth factor 2 (2 (Igf2Igf2) genes.) genes.

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Figure 17-26 Figure 17-26 ImprintingImprinting

Enhancer: Enhancer: activate both gene transcription activate both gene transcription ICR: ICR: an insulator binds CTCF protein and blocks an insulator binds CTCF protein and blocks the activity of the enhancer on the activity of the enhancer on Igf2Igf2.Methylation .Methylation of ICR allows the enhancer to activateof ICR allows the enhancer to activate Igf2 Igf2. . H19H19 repression is mediated by DNA methylation repression is mediated by DNA methylation and the subsequent MeCP2 binding to the and the subsequent MeCP2 binding to the methylated ICR methylated ICR

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Topic 7: Epigenetic Topic 7: Epigenetic RegulationRegulation

CHAPTER 17 Gene Regulation in eukaryotes

Patterns of gene expression must Patterns of gene expression must sometimes be inherited. sometimes be inherited. After the expression of specific genes in After the expression of specific genes in a set of cells are switched on by a signal, a set of cells are switched on by a signal, these genes may have to remain these genes may have to remain switched on for many cell generations, switched on for many cell generations, even if the signal that induced them is even if the signal that induced them is present only fleetingly (present only fleetingly ( 飞快地飞快地 ). ). The inheritance of gene expression The inheritance of gene expression patterns, in the absence of both patterns, in the absence of both mutation and the initiating signal, mutation and the initiating signal, is called is called epigenetic regulationepigenetic regulation..

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7-1. Some States of Gene Expression Are Inherited through Cell Division Even When the Initiating Signal Is No Longer Present

Topic 7: Epigenetic Regulation

有些基因表达状态在起始信号不存在时仍然在细胞分裂中遗传。The maintenance of a bacteriophage lysogen is an example of epigenetic regulation. [Figure 17-27]

Establishment of lysogeny: Establishment of lysogeny: synthesis of the essential synthesis of the essential lysogenic lysogenic repressor repressor is is establishedestablished by transcription from by transcription from one promoter and then one promoter and then maintainedmaintained by transcription from by transcription from another one.another one.

DNA methylationDNA methylation provides a provides a mechanism of mechanism of epigenetic regulationepigenetic regulation. . DNA methylationDNA methylation is reliably inherited is reliably inherited throughout cell division [Fig. 17-28].throughout cell division [Fig. 17-28].

Certain DNA methylases Certain DNA methylases can can methylate, at low frequency, methylate, at low frequency, previously unmodified DNA; but far previously unmodified DNA; but far more efficiently, the so-called more efficiently, the so-called maintenance methylasesmaintenance methylases modify modify hemimethylated DNAhemimethylated DNA －－ the very the very substrate provided by replication of substrate provided by replication of fully methylated DNA.fully methylated DNA.

[A link with reprogramming of somatic cells [A link with reprogramming of somatic cells into Embryonic Stem-like cells. Box 17-6]into Embryonic Stem-like cells. Box 17-6]

Figure 17-28 Patterns of DNA methylation Figure 17-28 Patterns of DNA methylation can be maintained through cell divisioncan be maintained through cell division

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1. The structure features of the eukaryotic transcription activators.

2. Activation of the eukaryotic transcription by recruitment & Activation at a distance.

3. Transcriptional repressor & its regulation

4. Signal integration and combinatorial control

5. Signal transduction: communicating the signals to transcriptional regulators.

6. Gene silencing and Epigenetic regulation.

7. 4 experimental methods introduced (yeast two-hybrid and ChIP).

Key points of the chapter