Welcome Each of You to My Molecular Biology Class

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 Genome Part IV: Regulation Part V: Methods
2005-5-10
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Part IV Regulation
Ch 16: Transcriptional regulation in prokaryotes Ch 17: Transcriptional regulation in eukaryotes Ch18: Regulatory RNAs

Ch 19: Gene regulation in development and evolution Ch 20: Genome Analysis and Systems Biology
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Surfing the contents of Part IV

--The heart of the frontier biological disciplines

Molecular Biology Course

Chapter 17 Gene Regulation in Eukaryotes

TOPIC 1 Conserved Mechanisms of Transcriptional Regulation from Yeast to Human. TOPIC 2 Recruitment of Protein Complexes to Genes by Eukaryotic Activators. TOPIC 3 Transcriptional Repressors TOPIC 4 Signal Integration and Combinatorial Control. TOPIC 5 Signal Transduction and the Control of Transcriptional Regulators. TOPIC 6 Gene Silencing by Modification of Histones and DNA. TOPIC 7 Epigenetic Gene Regulation.
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1. Gene Expression is Controlled by Regulatory Proteins ( )

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
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Principles of Transcription Principles of Transcription

Similarity of regulation between eukaryotes and prokaryote 1.Principles are the same: signals ( ), activators and repressors ( ) recruitment and allostery, cooperative binding ( ) 2. The gene expression steps subjected to regulation are similar, and the initiation of transcription is the most pervasively regulated step. 8

Difference in regulation between eukaryotes and prokaryote

1. 2.

3. 4.

Pre-mRNA splicing adds an important step for regulation. (mRNA ) The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart. ( ) Nucleosomes and their modifiers influence access to genes. ( ) Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes. ( ) 9

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

Yeast

Human

10 Fig. 17-1

Enhancer ( ) : 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 ( ) or boundary elements ( ) are regulatory sequences between enhancers and promoters. They block activation of a linked promoter by activator bound at the enhancer, and therefore ensure activators 11

CHAPTER 17 Gene Regulation in eukaryotes

The structure features of the The structure features of the eukaryotic transcription activators eukaryotic transcription activators

Topic 1: Conserved Mechanisms of Transcriptional Regulation from Yeast ( ) to Mammals ( )

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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 ( ) have separate DNA binding and activating functions , which are very often on separate domains of the protein.

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

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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

Fig. 17-3 The regulatory sequences of the Yeast GAL1 gene.

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Eukaryotic activators---Example 1: Gal4 Experimental evidences showing that Gal4 contains separate DNA binding and activating domains. 1. Expression of the N-terminal region (DNAbinding 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 experiment
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 17 separable.

Box 1 The two hybrid Assay ( ) is used to identify proteins interacting with each other. 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 proteins 1. Recognize the DNA using the similar principles, with some variations in detail. 2. In addition to form homodimers ( ), some form heterodimers ( ) to recognize DNA, extending the range of DNA-binding specificity. 20

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

Figure 17-5

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

Figure 17-6

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Leucine Zipper Motif ( ) : The Motif combines dimerization and DNAbinding surfaces within a single structural unit.
Figure 17-7

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Dimerization ( ) is mediated by hydrophobic interactions between the appropriately-spaced leucine ( ) 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:

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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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The activating regions are grouped on the basis of amino acids content. Acidic activation region ( ): contain both critical acidic amino acids and hydrophobic acids.yeast Gal4 Glutamine-rich region ( ): mammalian activator SP1 Proline-rich region ( ): mammalian activator CTF1 29

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

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

CHAPTER 17 Gene Regulation in eukaryotes

Topic 2: Recruitment of Protein Complexes to Genes by Eukaryotic Activators

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Eukaryotic activators ( ) also work by recruiting ( ) 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 machinery contains polymerase and numerous proteins being organized to several complexes, such as the Mediator and the TF D 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) ( ) to visualize where a given protein (activator) is bound in the genome of a living cell.) 3

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Activator Bypass Experiment ( )-Activation of transcription through direct tethering of mediator to DNA.
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Directly fuse the bacterial DNAbinding 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 activation 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 promoter Two types of Nucleosome modifiers :
Those add chemical groups to the tails of histones ( ), such as histone acetyl transferases (HATs) Those remodel the nucleosomes ( ), 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.
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uncover DNA-binding sites

Fig 17-11 Local alterations in chromatin

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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.
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One component of TFIID complex bears bromodomains.

Figure 7-39 Effect of histone tail modification
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3. Action at a distance: loops and insulators

Many enkaryotic activators particularly in higher eukaryotes work from a distance.
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 42 cell as thought.

Specific cis-acting elements called insulators ( ) control the actions of activators, preventing the activating the non-specific genes
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Insulat ors block activat ion by enhanc Figure 17-12 ers

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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. [ ] Application 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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Transcriptional Silencing ( )

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

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 HoxD genes. They are controlled genes by an element called the GCR (global control region) in a manner very like that of LCR.

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CHAPTER 17 Gene Regulation in eukaryotes

Topic 3: Transcriptional Repressor & its regulation

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)
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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 modifers, histone deacetylases ( ) 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) Competes with the activator for an overlapped binding site. site (2) Binds to a site different from that of the activator, but physically interacts with an activator and thus block its activating region. (3) Binds to a site upstream of the promoter, physically interacts with the transcription machinery at the promoter to inhibit transcription initiation.
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Competes for the activator binding

Inhibits the function of the activator.

Figure 17-19 Ways in which eukaryotic repressor work

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

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A specific example: Repression of the GAL1 gene in yeast

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 histone deacetylases, and also directly deacetylases interacts with the transcription machinery to repress transcription.
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 Features of the eukaryotic transcriptional Features of the eukaryotic transcriptional regulation: signal integration and combinatorial regulation: signal integration and combinatorial control control

CHAPTER 17 Gene Regulation in eukaryotes

Topic 4: Signal Integration and Combinatorial Control

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1. Activators work together synergistically ( ) to integrate signals.

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

Glucose Lactose

57 Figure 16-6

In multicellular organisms, signal integration ( ) is used 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 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 (

): S1: Multiple activators recruit a single component of the transcriptional machinery.

For example, by touching the different part of the mediator complex ( ). 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)

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Figure 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 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 ( ). The mother cell and cell cycle conditions (signals) are communicated to the HO gene (target) by two activators: SWI5 and SBF (communicators). (communicators)

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SWI5: acts only in the mother cell and binds to

multiple sites some distance from the gene unaided, which recruit enzymes to open the SBF binding sites. cycle, and cannot bind the sites unaided.

SBF: only active at the correct stages of the cell

Alter the nucleosome
( )

Figure 17-15

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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 example]

The human -interferon gene (target gene) is activated in cells upon viral infection (signal). (signal) Infection triggers three activators (communicator): NFB, IRF, and Jun/ATF. (communicator) 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.
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( )

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)

Figure 17-16 66

HMG-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 ( ) lies at the heart of the complexity and diversity of eukaryotes, in
which Both activators and repressors work together.

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Review

Page 499 & Slide 47 in Ch 16

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

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

Four signals

Three signals

Figure 17-18

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

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5. An example: combinatory control of the mating-type genes from S. cerevisiae ( )

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The yeast S. cerevisiae exists in three forms: ---two haploid cells ( ) of different mating types a and . ---the diploid cells ( ) (a/) formed when an a and an cell mate and fuse. Cells of the two mating types (a and ) differ because they express different sets of genes: a specific genes and specific genes. genes
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a cells make the regulatory protein a1, cells make the protein 1 and 2. Both cell types express the fourth regulator protein Mcm1 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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CHAPTER 17 Gene Regulation in eukaryotes

Signal transduction---A life science frontier Signal transduction---A life science frontier centered on the eukaryotic transcriptional centered on the eukaryotic transcriptional regulation. regulation.

Topic 5: Signal Transduction ( ) and the Control of Transcriptional Regulators

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Topic 4: Signal Transduction and the Control of Transcriptional Regulators

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

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Environmental Signals/Information ( ) 1. Small molecules such as sugar, histamine ( ). 2. Proteins released by one cell and received by another. In eukaryotic cells, most signals are communicated to genes through signal transduction pathway (indirect), in which (indirect) the initiating ligand is detected by a specific cell surface receptor. What about in bacteria?
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Signal transduction pathway 1. The initial ligand (signal) binds to an extracellular domain of a specific cell surface receptor 2. The signal is thus communicated to the intracellular domain of receptor (via an allosteric change or dimerization ) 3*. The signal is then relayed ( ) to the relevant transcriptional regulator. 4. The transcriptional regulator control the target gene 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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Topic 4: Signal Transduction and the Control of Transcriptional Regulators

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

In eukaryotes, a signal can be communicated, directly or indirectly, to a transcriptional regulator. regulator

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

(1) (2)

(3)

A conformational change to reveal the previously buried activating region. Releasing of the previously bound masking protein. Example: the activator Gal4 is controlled by the masking Gal80) (Fig.1723). 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 82 expression.

Activator Gal4 is regulated by a masking protein Gal80

Gal4

Figure 17-23

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Mechanism 2: Transport into and out 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: A cascade of kinases that ultimately cause the phosphorylation of regulator in nucleus (new) (Fig.19-4a). (new

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

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CHAPTER 17 Gene Regulation in eukaryotes

Topic 6: Gene Silencing by Modification of Histones and DNA

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

The most common form of silencing is associated with a dense form of chromatin called heterochromatin. Heterochromatin is frequently associated with particular regions of the chromosome, notably the telomeres, and the centromeres. In mammalian cells, about 50% of the genome is estimated to be in some form of heterochromatin.

Transcriptional silencing is associated with Modification of nucleosomes that alters the accessibility of a gene to the transcriptional machinery and other regulatory proteins. The modification enzymes for silencing include deacetylases, DNA methylases.

Topic 6: Gene Silencing by Modification of Histones and DNA

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

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The telomeres, the silent mating-type locus, and the rDNA genes are all silent regions in S. cerevisiae. Three genes encoding regulators of silencing, SIR2, 3, and 4 have been found (SIR stands for Silent Information Regulator).
Rap1 recruits Sir complex to the temomere. Sir2 deacetylates nearby nucleosome.

Fig. 17-24. Silencing at the yeast telomere

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

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Transcription can also be silenced by methylation of DNA by histone methyltransferase (H3 and H4, Chapter 7). This enzyme have been recently found in yeast, but is common in mammalian cells. Its function is better understood in higher eukaryotes. In higher eukaryotes, silencing is typically associated with chromatin containing histones that both deacetylated and methylated.

Topic 6: Gene Silencing by Modification of Histones and DNA

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

HP1

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HP1 protein is a component of heterochromatin in Drosophila that binds to methylated residue in histone H3. Such a histone modification is produced by Su(Var)3-9. Different types of modification at the histones can be involved in distinct geene regulation. What will happen when multiple forms of modification occur? ---A Histone Code hypothesis.

According to this idea, different patterns of modification on histone tails can be read to mean different things. The meaning would be the result of the direct effects of these modifications on chromatin density and form. But in addition, the particular pattern of modifications at any given location would recruit specific proteins.

Box 17-4 Is there a histone code?

Topic 6: Gene Silencing by Modification of Histones and DNA

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

DNA DNA Some mammalian genes are kept silent by methylation of nearby DNA sequences.
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Large regions of mammalian genome are marked by methylation of DNA sequences, which is often seen in heterochromatic regions. [Why?] The methylated DNA sequences are often recognized by DNA-binding proteins (such as MeCP2) that recruit histone decetylases and histone methylases, which then modify nearby chromatin. Thus, methylation of DNA can mark sites where heterochromatin subsequently forms (Fig. 17-25).

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

DNA methylation lies at the heart of Imprinting

Imprinting- in a diploid cell, one copy of a gene from the father or mother is expressed while the other copy is silenced. Two well-studied examples: human H19 and insulin-like growth factor 2 (Igf2) genes.

Enhancer: activate both gene transcription ICR: an insulator binds CTCF protein and blocks the activity of the enhancer on Igf2.Methylation of ICR allows the enhancer to activate Igf2. H19 repression is mediated by DNA methylation and the subsequent MeCP2 binding to the methylated ICR

Figure 17-26 Imprinting

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CHAPTER 17 Gene Regulation in eukaryotes

Topic 7: Epigenetic Regulation

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Patterns of gene expression must sometimes be inherited.

After the expression of specific genes in a set of cells are switched on by a signal, these genes may have to remain switched on for many cell generations, even if the signal that induced them is present only fleetingly ( ).

The inheritance of gene expression patterns, in the absence of both mutation and the initiating signal, is called epigenetic regulation.

Topic 7: Epigenetic Regulation

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

The maintenance of a bacteriophage lysogen is an example of epigenetic regulation. [Figure 17-27]

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stablishment of lysogeny: synthesis of the

essential lysogenic repressor is established by transcription from one promoter and then maintained by transcription from another one.

DNA methylation provides a mechanism of epigenetic regulation. DNA methylation is reliably inherited throughout cell division [Fig. 17-28]. Certain DNA methylases can methylate, at low frequency, previously unmodified DNA; but far more efficiently, the socalled maintenance methylases modify hemimethylated DNA the very substrate provided by replication of fully methylated DNA.
[A link with reprogramming of somatic cells into Embryonic Stem-like cells. Box 17-6]

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

Key points of the chapter

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 twohybrid and ChIP).
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