Gene Regulation in Eukaryotes

1

• Chapter 17
• Gene Regulation in Eukaryotes

2

• Similarity of regulation between eukaryotes
  and prokaryote
• 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.

3

• Difference in regulation between eukaryotes and
  prokaryote
• 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.

4

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

Bacteria
Yeast
Human
Fig. 17-1
5

• 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 to ensure a linked
  promoter not responding to the activator binding.
  

6

• Topic 1
• Conserved Mechanisms of
• Transcriptional Regulation
• from Yeast
• to Mammals

7

• 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

8

• 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
9

1. Gal4 is the most studied eukaryotic activator
2. 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

Fig. 17-3 The regulatory sequences of the Yeast
GAL1 gene.
10

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

11

• 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.

12

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

13

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

14

• 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 proteins
• Recognize the DNA using the similar principles,
  with some variations in detail.
• Some form heterodimers to recognize DNA,
  extending the range of DNA-binding specificity.

15

• 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 17-5
16
Zinc containing DNA-binding domains finger
domain Zinc finger proteins (TFIIIA) and Zinc
cluster domain (Gal4)
Figure 17-6
17
Leucine Zipper Motif The Motif combines
dimerization and DNA-binding surfaces within a
single structural unit.
Figure 17-7
18
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
Figure 17-8
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22
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.
23

• 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

24

• ? Recruitment of
• Protein Complexes
• to Genes
• by
• Eukaryotic Activation

25

• Eukaryotic activators also work by
• recruiting 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.

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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
27

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

28

• Activator Bypass Experiment-Activation of
  transcription through direct tethering of
  mediator to DNA. (??????)

Directly fuse the bacterial DNA-binding protein
LexA protein to the mediator complex Gal11 to
activate GAL1 expression.
Figure 17-10
29
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
30
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

31

• 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 ?

32

• 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.

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Figure 7-39 Effect of histone tail modification
34

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

35
2-2 Action at a distance loops and insulators

• Many enkaryotic activators-particularly
• in higher eukaryotes-work from a distance.
• Why?
• Some proteins help, for example Chip protein in
  Drosophila.
• 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.

36
Specific elements called insulators control the
actions of activators, preventing the activating
the non-specific genes
37

• Insulators
• block
• activation
• by
• enhancers

Figure 17-12
38

• Transcriptional Silencing
• Silencing 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.

39

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

Figure 17-13
40

• A group of regulatory elements collectively
  called the locus control region (LCR), is found
  30-50 kb upstream of the cluster of globin genes.
  Its 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.

41

• 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.

42

• ? Signal Integration
• and
• Combinatorial
• Control

43
3-1 Activators work together synergistically
(???) to integrate signals
44

• 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

45

• a.Classical
• cooperative
• binding

b. Both proteins interacting with a third protein
d. Binding a protein unwinds the DNA from
nucleosome a little, revealing the binding site
for another protein
c. A protein recruits a remodeller to reveal a
binding site for another protein
Figure 17-14
46
3-2 Signal integration the HO gene is controlled
by two regulators one recruits nucleosome
modifiers and the other recruits mediator
47

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

alter the nucleosome
Figure 17-15
48
3-3 Signal integration Cooperative binding of
activators at the human b-interferon gene.
49

• The human ß-interferon gene is activated in
  cells upon viral infection. Infection triggers
  three activators
• NF?B, IRF,
• and Jun/ATF.
• They bind
• cooperatively
• to sites within
• an enhancer,
• form a
• structure
• called
• enhanceosome.

Figure 17-16
50
3-4 Combinatory control lies at the hear of the
complexity and diversity of eukaryotes
51

• There is extensive combinatorial control in
  eukaryotes.

Four signals
Figure 17-17
Three signals
In complex multicellular organisms, combinatorial
control involves many more regulators and genes
than shown above, and repressors as well as
activators can be involved.
52
3-5 Combinatory control of the mating-type genes
from S. cerevisiae (????)
53

• 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.

54

• 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.
• How do these regulators work together
• to keep a cell in its own type?

55

• Control of cell-type specific genes in yeast

Figure 17-18
56

• ?
• Transcriptional
• Repressors

57

• In eukaryotes, repressors dont 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 removing
• actetyl groups from the tails of histone.

58

• Ways in
• which
• eukaryotic
• repressor
• Work
• a and b

Figure 17-19
59
Ways in which eukaryotic repressor Work c and
d
Silencing
60

• A specific example Repression of the GAL1 gene
  in yeast

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.
61

• ? Signal Transduction
• and
• the Control of
• Transcriptional Regulators

62
5-1 Signals are often communicated to
transcriptional regulators through signal
transduction pathway
63

• 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.

64

• 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.

65
5-2 Signals control the activities of eukaryotic
transcriptional regulators in a variety of ways
66

• a. The STAT pathway

b. The MAP kinase pathway
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• 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

68

• Activator Gal4 is regulated by masking protein
  Gal80

69

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

70

• 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).

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72

• ? Gene Silencing
• by
• Modification of
• Histones and DNA

73

• 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.

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6-1 Silencing in yeast is mediated by
deacetylation ane 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 stand for
  silent information regulator).

Silencing at the yeast telomere
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6-1 Histone modification and the histone code
hypothesis
77

• 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.

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• 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.

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• 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.

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Figure 17-24
Switching a gene off
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• 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.

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• 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 ?lysogen, can
• be described as an example.

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Box 3

• ?lysogens and the epigentic switch

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 ?
84

• 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.

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• Patterns of DNA methylation can be maintained
  through cell division

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• ? Eukaryotic
• Gene Regulation
• at Steps
• after
• Transcription Initiation

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• 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

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HIV genome
89
?
90

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

91

• 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.

92

• 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-lethal Dpn Deadpan
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Early transcriptional regulation of Sxl in male
and female flies
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• A cascade
• of
• alternative
• splicing
• events
• determines
• the sex
• of a fly

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• 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.

96

• 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 ?

97

• high levels
• of amino
• acids
• the Gcn4
• mRNA
• is not
• translated

98

• low levels
• of amino
• acids
• the Gcn4
• mRNA is
• translated

99

• ? RNAs
• in
• Gene Regulation

100
8-1 Double-standed RNA inhibits expression of
genes homologous to that RNA 8-2 Short
interfering RNA (siRNAs) are produced from dsRNA
and direct machinery that switch off genes in
various way 8-3 MicroRNA control the expression
of some genes during development.
101

• 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.

102

• The discovery that simply introducing
• double-strand RNA (dsRNA) into a cell
• can 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 ?

103

• Dicer is an RNAse?-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 mRNA
• Inhibit translation of its mRNA
• Induce chromatin modifications within the
  promoter that silence the gene
• That machinery includes a complex called RISC
• (RNA-induced silencing complex).

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• RNA
• silencing

105

• 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.

106

• 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.

107

• 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.

108

• 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.

109

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

110

• 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.

111

• 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?