Chapter 17

Gene Regulation in Eukaryotes

Similarity of regulation between eukaryotes and prokaryote

1. Principles are the same: signals, activators and repressors, recruitment and allostery, cooperative binding

2. Expression of a gene can be regulated at the similar steps, and the initiation of transcription is the most pervasively regulated step.

Difference in regulation between eukaryotes and prokaryote

1. Pre-mRNA splicing adds an important step for regulation.

2. The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart.

3. Nucleosomes and their modifiers influence access to genes.

4. Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are bacterial genes.

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

Fig. 17-1

Bacteria

Yeast

Human

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 ( 绝缘体 ) or boundary elements are regulatory sequences to ensure a linked promoter not responding to the activator binding.

Topic 1Conserved Mechanisms of Transcriptional Regulation

from Yeast to Mammals

Topic 1Conserved Mechanisms of Transcriptional Regulation

from Yeast to Mammals

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

1-1 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

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

1.Gal4 is the most studied eukaryotic activator2.Gal4 activates transcription of the galactose

genes in the yeast S. cerevisae.3.Gal4 binds to four sites upstream of GAL1,

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

The separate DNA binding and activating domains of Gal4 were revealed in two complementary experiments

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 experiment

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.

Box 1 The two hybrid Assay The two hybrid Assay is used to is used to identify proteins identify proteins interactinginteracting with each with each other.other.

1-2 Eukaryotic regulators use a range of DNA binding domains, but DNA recognition involves the same principles same found in bacteria

Homeodomain proteinsZinc containing DNA-binding domain: zinc finger and zinc clusterLeucine zipper motifHelix-Loop-Helix proteins : basic zipper and HLH proteins

Bactrial 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. Some form heterodimers to recognize DNA, extending the range of DNA-binding specificity.

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

Figure 17-5Figure 17-5

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

Figure 17-6Figure 17-6

Leucine Zipper Motif: The Motif combines dimerization and DNA-binding surfaces within a single structural unit.

Figure 17-7Figure 17-7

Dimerization is mediated by hydrophobic interactions between the appropriately-spaced leucine to form a coiled coil structure

Dimerization is mediated by hydrophobic interactions between the appropriately-spaced leucine to form a coiled coil structure

Helix-Loop-Helix motif:

Figure 17-8Figure 17-8

Because the region of the a-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.

Because the region of the a-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.

1-3 Activating regions are not well-defined structures

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

• Acidic activation domains• Glutamine-rich domains• Proline-rich domains

Ⅱ Recruitment of Protein Complexes

to Genes by

Eukaryotic Activation

Eukaryotic activators also work byrecruiting as in bacteria, but recruit polymerase indirectly in two ways :

2-1 Interacting with parts of the transcription machinery.

2-2 Recruiting nucleosome modifiers that alter chromatin in the vicinity of a gene.

The eukaryotic 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 17-9Figure 17-9

Box 2 Chromatin Immuno-precipitation (ChIP) to Chromatin Immuno-precipitation (ChIP) to visualize the recruitment (visualize the recruitment (WhereWhere a given a given protein is bound in the genome of a living protein is bound in the genome of a living cell.)cell.)

Activator Bypass Experiment-Activation of transcription through direct tethering of mediator to DNA. ( 很有应用价值 )

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

Figure 17-10Figure 17-10

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

2-2 Activators also recruit modifiers that help the transcription machinery bind at the promoter

1. Modifiers direct recruitment of the transcriptional machinery

2. Modifiers help activate a gene inaccessibly packed within chromatin

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

How do these modification help activate

a gene ?

Two basic models for how thesemodification help activate a gene

:Remodeling and certain modification can uncover DNA-binding sites that would otherwise remain inaccessible within the nucleosome.By adding acetyl groups, it creates specific binding sites on nucleosomes for proteins bearing so-called bromodomains.

Figure 7-39 Effect of histone tail modificationFigure 7-39 Effect of histone tail modification

Fig 17-11 Local alterations in chromatin

directed by activators

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

distance. Why?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.

2-2 Action at a distance: loops and insulators

Specific elements called insulators control the actions of activators, preventing the activating the non-specific genes

Insulators

block

activation

by

enhancers

Figure 17-12Figure 17-12

Transcriptional SilencingSilencing is a specializes 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.So a gene inserted at random into the mammalian genome is often “silenced”.

2-4 Appropriate regulation of some groups of genes requires locus control region (LCR).

Figure 17-13Figure 17-13

A group of regulatory elements collectively called the locus control region (LCR), is found 30-50 kb upstream of the cluster of globin genes. It’s made up of multiple-sequence elements : something like enhancers, insulators or promoters.

It binds regulatory proteins that cause the chromatin structure to “open up”, allowing access to the array of regulators.

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

Ⅲ Signal Integration and

Combinatorial Control

3-1 Activators work together synergistically ( 协同的 ) to integrate signals

In eukaryotic cells, numerous signals are often required to switch a gene on. So at many genes multiple activators must work together.

They do these by working synergistically: two activators working together is greater than the sum of each of them working alone.

Three strategies of synergy :Two activators recruit a single complex Activators help each other binding cooperativity One activator recruit something that helps the second activator bind

a.“Classical” cooperative

binding

b. Both proteins

interacting with a third

protein

c. A protein recruits a remodeller to reveal a binding site for another protein

d. Binding a protein

unwinds the DNA from

nucleosome a little,

revealing the binding site for another

protein

Figure 17-14Figure 17-14

3-2 Signal integration: the HO gene is controlled by two regulators; one recruits nucleosome modifiers and the other recruits mediator

The HO gene is involved in the budding of yeast.It has two activators : SWI5 and SBF.

alter the nucleoso

me

Figure 17-15Figure 17-15

3-3 Signal integration: Cooperative binding of activators at the human -interferon gene.

The human β-interferon gene is activated in cells upon viral infection. Infection triggers three activators :

NFκB, IRF, and Jun/ATF. They bind cooperatively to sites within an enhancer, form a structure called enhanceosome.

Figure 17-16Figure 17-16

3-4 Combinatory control lies at the hear of the complexity and diversity of eukaryotes

There is extensive combinatorial control in eukaryotes.

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

Four signal

s

Three signal

s

Figure 17-17Figure 17-17

3-5 Combinatory control of the mating-type genes from S. cerevisiae ( 啤酒酵母 )

The yeast S.cerevisiae exists in three forms: two haploid cells of different mating types － aand － and the

diploid

formed when an a and an cell mate

and fuse.Cells of the two mating types differ because they express different sets of

genes : a specific genes and specific

genes.

a cell make the regulatory protein a1,

cell make the protein 1 and 2.

A fourth regulator protein Mcm1 is also involved in regulatory the mating- type specific genes and is present in both cell types.

How do these regulators work togetherto keep a cell in its own type?

Control of cell-type specific genes in yeast

Figure 17-18Figure 17-18

Ⅳ Transcriptional

Repressors

In eukaryotes, repressors don’t work by binding to sites that overlap the promoter and thus block binding of polymerase, but most common work

by recruiting nucleosome modifiers.

For example, histone deacetylases repress transcription by removingactetyl groups from the tails of

histone.

Ways in which

eukaryotic

repressor Work

a and b

Figure 17-19Figure 17-19

Ways in which

eukaryotic

repressor

Workc and d

Silencing

In the presence of glucose, Mig1 binds a site between the USAG and the GAL1 promoter. By recruiting the Tup1 repressing complex, Mig1 represses expression of GAL1.

A specific example: Repression of the GAL1 gene in yeast

Ⅴ Signal Transduction and

the Control of Transcriptional

Regulators

5-1 Signals are often communicated to transcriptional regulators through signal transduction pathway

Signals refers to initiating ligand (can be sugar or protein or others), or just refers to “information”.There are various ways that signals are detected by a cell and communicated to a gene. But they are often communicated to transcriptional regulators through signal

transduction Pathway, in which the initiating ligand

is detected by a specific cell surface receptor.

In a signal transduction pathway:initiating ligand binds to an extracellular domain of a specific

cell surface receptor this binding bring an allosteric change in the intracellular domain of receptor the signal is relayed to the relevanttranscriptional regulator often through a cascade of kinases.

5-2 Signals control the activities of eukaryotic transcriptional regulators in a variety of ways

a. The STAT pathway

b. The MAP kinase pathway

Once a signal has been communicated, directly or indirectly, to a

transcriptional regulator, how does it control the activity of that regulator ?In eukaryotes, transcriptional

regulators are not typically controlled at the level of DNA binding. They are usually controlled in one of two basic ways :

Unmasking an activating region Transport in or out of the nucleus

Activator Gal4 is regulated by masking protein Gal80

The signalling ligand causes activators (or repressors) to move to the nucleus where they act from cytoplasm.

5-3 Activators and repressors sometimes come in pieces.

For example, the DNA binding domain and activating region can be on different polypeptides. same of an activator

In addition, the nature of the protein complexes forming on DNA determines whether the DNA-binding protein activates or represses nearby genes. For example, the glucocorticoid receptor (GR).

Ⅵ Gene “Silencing” by

Modification of Histones and DNA

Gene “silencing” is a position effect － a gene is silenced because of where it is located, not in response to a specific environmental signal.

The most common form of silencing is associated with a dense form of chromatin called heterochromatin. It is frequently associated with particular regions of the chromosome, notably the telomeres, and the centromeres.

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

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 stand for silent information regulator).

Silencing at the yeast telomere

6-1 Histone modification and the histone code hypothesis

A histone code exists ? 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.

Transcription can also be silenced by methylation of DNA by enzymes called DNA methylases.

This kind of silencing is not found in yeast but is common in mammalian cells.

Methylation of DNA sequence can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression.

Switching a gene off :

A mammalian gene marked by methylation

of nearby DNA sequence recognized by DNA-binding proteinsrecruit histone decetylases and histone methylasesmodify nearby chromatinThis gene is completely off.

Switching a gene off

Figure 17-24

DNA methylation lies at the heart of a phenomenon called imprinting. Two examples :Human H19 and Igf2 genes.Here an enhancer and an insulator are critical.

Patterns of gene expression must sometimes be inherited. These may remain for many cell generations, even if the signal that induced them is present only fleetingly. This inheritance of gene expression patterns is called epigenetic regulation.

Maintenance of a phage λlysogen, can be described as an example.

λlysogens and the epigentic switchλlysogens and the epigentic switchBox 3

Lysogenic gene expression is established in an infected cell in response to poor growth conditions. Then the lysogenic state will remain through cell division in both cells. This is resulted from a two-step strategy for repressor synthesis.How ?

Nucleosome and DNA modifications can provide the basis for epigenetic inheritance.DNA methylation is even more reliably inherited, but far more efficiently is the so-called maintenance methylases modify hemimethylated DNA － the

very substrate provided by replication of fully methylated DNA.

Patterns of DNA methylation can be maintained through cell division

Ⅶ Eukaryotic Gene Regulation

at Steps after

Transcription Initiation

In eukaryotic cells, some regulational proteins aim at elongation.

At some genes there are sequence downstream of the promoter that cause pausing or stalling of the polymerase soon after initiation.

At those genes, the presence or absence of certain elongation factors greatly influences the level at which the gene is expressed.

Two examples : HSP70 gene and HIV

HIV genome

Many individual eukaryotic genes have exons interrupted by introns. So when the whole gene is transcribed, mRNA need to be spliced.

In some cases a given precursor mRNA can be spliced in alternative ways to produce different mRNAs that encode different protein products.

The regulation of alternative splicing works in a manner reminisencent : the splicing machinery binds to splice sites and carries out the splicing reaction.

The sex of a fly is determined by the ratio of X chromosomes to autosomes. This ratio is initially measured at the level of transcription using two activators SisA and SisB. The genes encoding these regulators are both on the X chromosome.

Sxl : Sex-lethalDpn :Deadpan

Early transcriptional regulation of Sxl in male and female flies

A cascade of

alternative splicingevents

determines

the sex of a fly

Gcn4 is a yeast transcriptional activator

that regulates the expression of genes

encoding enzymes that direct amino acid

biosynthesis.

The mRNA encoding the Gcn4 protein

contains four small open reading frames

(called uORFs) upstream of the coding

sequence for Gcn4.

Although it is a activator, Gcn4 is itself

regulated at the level of translation. In the presence of low levels of

amino acids, the Gcn4 mRNA is translated (and so the biosynthetic are expressed).

In the presence of high levels, it is not translated.

How is this regulation achieved ?

high levels

of amino acids :the Gcn4 mRNA is not translated

low levels of amino acids :the Gcn4 mRNA is translate

d

Ⅷ RNAs in

Gene Regulation

8-1 Double-standed RNA inhibits expression of genes homologous to that RNA8-2 Short interfering RNA (siRNAs) are produced from dsRNA and direct machinery that switch off genes in various way8-3 MicroRNA control the expression of some genes during development.

Short RNAs can direct repression of genes with homology to those short RNAs. This repression, called RNA interference (RNAi), can manifest as translational inhibition of the

mRNA, destruction of the mRNA or transcriptional silencing of the promoter that directs expression of that mRNA.

The discovery that simply introducing double-strand RNA (dsRNA) into a cellcan repress genes containing

sequence identical to (or very similar to) that dsRNA was remarkable in 1998 when it was reported.A similar effect is seen in many other organisms in both animals and plants.How dsRNA can switch off expression of a gene ?

Dicer is an RNAseⅢ-like enzyme that recognizes and digests long dsRNA. The products are short double-stranded fragments.These short RNAs (or short interfering RNAs, siRNAs) inhibit expression of a homologous

gene in three ways :

Trigger destruction of its mRNAInhibit translation of its mRNAInduce chromatin modifications within the promoter that silence the gene

That machinery includes a complex called RISC (RNA-induced silencing complex).

RNA silencin

g

RNAi silencing is extreme efficiency.Very small amounts of dsRNA are enough to induce complete shutdown of target genes.

Why the effect is so strong ?It might involve an RNA-dependent RNA polymerase which is required in many cases of RNAi.

There is another class of naturally occurring RNAs, called microRNAs (miRNAs), that direct repression of genes in plants and worms.

Often these miRNAs are expressed in developmentally regulated patterns.

The mechanism of RNAi may have evolved originally to protect cells from any infectious, or otherwise disruptive, element that employs a dsRNA intermediate in its replicative cycle.

Now RNAi has been adapted for use as a powerful experimental technique allowing specific genes to be switched off in any of many organisms.

Summary :

There are several complexities in the organization and transcription of

eukaryotic genes not found in bacteria :

Nucleosomes and their modification

Many regulators and larger distances

The elaborate transcriptional machinery

Pay attention to these differences, and tell

them in details by yourself.

Do you know these conceptions ? Promoter Regulator binding site Regulatory sequence Enhancer Insulator Reporter gene Gene silencing

Critical Thinking Exercises 1 .Compare the mechanisms used by zinc-binding, leucine zipper,

and HLH motifs to bind DNA. What is the role of zinc in the zinc-binding domain?

2 .Describe the physical characteristics of a typical transcriptional activation region. How are these characteristics thought to reflect the mechanism of activation? If a novel transcription factor is identified, and you would like to locate the domain within the protein that is responsible for activation, how would you do this?

3 .You transform a population of cells with a transgene, and isolate a cell line that has integrated the gene into its genome, but in which the gene is not expressed. Speculate as to what may be preventing the gene from being expressed. Describe two ways to test this possibility.

4 .To determine how an activator contributes to the formation of the holoenzyme at your favorite promoter, design an experimental strategy to tell you which components are directly recruited by the activator, and which of these activator-mediated recruitment steps are required for transcriptional activation. How could you test whether the role of the activator stops with the holoenzyme assembly, or if it also induces allosteric changes within the DNA or the holoenzyme?

 5 .You isolate a mutant strain of mice that grow at an unusually fast rate, perform a blood test on the mice, and find that they have elevated levels of insulin-like growth factor. The phenotype is the result of a mutation in a region of the genome containing a gene encoding a DNA methylase. What kind of mutation is causing the rapid growth of these mice?