Accomplish the gene regulation of prokaryotes, we comeback to the eukaryotes.

You well exclaim with it’s complication and accuration.

Chapter 17

Gene Regulation in Eukaryotes

Similarity And Difference of

regulation between eukaryotes and

prokaryote

Similarity :

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

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

Difference:

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

• 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.

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.

The typical eukaryotic activators works in a manner similar to the simplest bacterial case.

Repressors work in a variety of ways

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.

DNA-binding domains and activating regions are separable:

1. Activator produces a protein bound to the DNA normally but did not activate transcription.

2. Fusion of the C-terminal region of the activator to the DNA binding domain of a bacterial repressor, LexA activates the transcription of the reporter gene. Domain swap experiment

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

Homeodomain proteinsZinc containing DNA-binding domain

Leucine zipper motifHelix-Loop-Helix proteins

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

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

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 a 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

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.

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

2-1 Interacting with parts of the transcription machinery.

Some activators not only recruit parts of the transcriptional machinery, they also induce allosteric changes in them

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

Figure 17-9Figure 17-9

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 nucleosome 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

Two basic models for how these

modification 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.

Fig 17-11 Local alterations in chromatin directed by activators

Many enkaryotic activators －particularly

in higher eukaryotes－ work from a distance.

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.

2-3 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.

E.P: A gene inserted at random into the mammalian genome is often “silenced” because it becomes incorporated into a particularly dense form of chromatin called heterochromatin .But if insulators are placed up-and downstream of that gene they protect it from silencing.

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 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

SBF cannot bind its sites unaided; their disposition within chromatin prohibits it. SWI5 can bind to its sites unaided but cannot, from that distance, activate the HO gene. SWI5 can, however, recruit nucleosome modifiers. These act on nucleosomes over the SBF sites

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

The yeast S.cerevisiae exists in three forms: two haploid cells of different mating types － a and a － and the diploid formed when an a and an a cell mate and fuse.Cells of the two mating types differ because they express different sets of genes : a specific genes and a specific

genes.

a cell make the regulatory protein a1,a cell make the protein a1 and a2.

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

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.

Ways in

which

eukaryotic

repressor

Work

a and b

Figure 17-19Figure 17-19

Ways in

which

eukaryotic

repressor

Work

c 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

For example, histone deacetylases repress transcription by removing actetyl groups from the tails of histone.Other enzymes add methyl groups to histone tails, and this frequently represses transcription.

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 relevant transcriptional 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,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

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 proteins

recruit histone decetylases and histone methylases

modify nearby chromatin

This 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.

Nucleosome and DNA odifications

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 ethylated DNA.

Patterns of DNA methylation can be maintained through cell division

Ⅶ Eukaryotic Gene Regulation

at Steps after

Transcription Initiation

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 :

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 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.

High levels

of amino

acids :

the Gcn4

mRNA is not

translated

Low levels

of amino

acids :

the Gcn4

mRNA is

translated

Ⅷ RNAs in

Gene Regulation

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.

RNA silencin

g

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

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

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.