Regulation of eukaryotic genes

1
Regulation of eukaryotic genes

• Gene silencing
• Enhancers
• Activators
• Functional domains of activators

2
Idea for another extra credit project

• Explore DNA binding domains of proteins.
• Go to a web site with a Chime tutorial, e.g. GAL4
  or Cro
• Or use Kinemages
• Write a roughly 2 page report on how a particular
  protein recognizes a DNA sequence

3
States of eukaryotic genes

• Inactive
• Closed chromatin
• Open chromatin, but repressors or lack of
  activators keep frequency of initiation low.
• Open chromatin, transcription has initiated, but
  polymerases will not elongate.
• Active
• Open chromatin, basal transcription requires
  TATA Inr
• Open chromatin, activated transcription requires
  enhancer or upstream activator sequences

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Silent and open chromatin
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Transcription initiation and pausing
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Basal and activated transcription
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Silencing Mechanism
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Silencer

• Cis-acting sequences that cause a decrease in
  gene expression
• Similar to enhancer but has an opposite effect on
  gene expression
• Gene repression - inactive chromatin structure
  (heterochromatin)
• Examples
• Telomeric silencing
• a or ? genes - silent loci of mating type
  switching in yeast

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Silencer binding proteins

• Silencer binding protein serve as anchors for
  expansion of repressed chromatin
• Rap1 protein binds to silencer elements
• SIR proteins (Silent Information Regulators)
• Nucleates assembly of multi-protein complex
• hypoacetylated N-terminal tails of histones H3
  and H4
• methylated N-terminal tail of H3 (Lys 9)
• Experiments Condensed chromatin
• Resistant to DNaseI digestion
• Delete silencer - genes are derepressed

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Gene Silencing
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Silencing Mechanism
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Enhancers

• Cis-acting sequences that cause an increase in
  expression of a gene
• Act independently of position and orientation
  with respect to the gene.
• Can act to
• Increase the rate of initiation at a promoter
• Increase the fraction of cells in which a
  promoter is active

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SV40 Control region

• Origin of replication
• Promoter and upstream activator sequences for
  early transcription
• Promoter for late transcription
• Enhancer

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SV40 map
15
Many regulatory DNA sequences in SV40 control
region
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Stimulation of transcription by enhancer is
independent of orientation and position
SV40
Early
Late
T-Ag
pos
T-Ag
wt
Enhancer
orien
T-Ag
Enh-
T-Ag -
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Enhancers also regulate cellular genes
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Enhancer contains multiple binding sites for
transcriptional activators
SV40
Early
Late
Enhancer
T-Ag
high level
A
C
B
wt
low level
C
B
deletion
high level
revertant
C
B
C
An enhanson
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Enhancers can occur in a variety of positions
with respect to genes
Transcription unit
P
Enhancer
Enhancer
Upstream
Adjacent
Downstream
Internal
Distal
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Activator proteins
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Modular nature of activator proteins

• DNA binding domain recognition and binding to
  specific DNA sequences
• Multimerization domain allows formation of homo-
  or hetero-multimers
• Activation domain
• Needed for increase in expression of responding
  gene
• Targets are still under investigation
• General transcription factors
• Histone modifying enzymes
• Nucleosome remodeling complexes, etc

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Modular structure of GAL4
148
1
98
196
768
881
N
C
Activation
Activation
DNA binding
GAL80 binding
Dimerization
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Induction by galactose exposes an activation
surface

• In the presence of galactose, GAL4 activates
  several genes whose products are required for
  galactose metabolism.
• GAL4 binds to a DNA sequence called UASG.
• In the absence of galactose, GAL80 blocks GAL4
  activation.
• Binding of the sugar causes GAL80 to move.
• This exposes the activation domain of GAL4.

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Induction of GAL4
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Domain swap experiments show the domains are
interchangeable

• Fuse an DNA-binding domain (DBD) from one
  transcription factor to the activation domain
  (AD) of a different one.
• DBD from LexA (E. coli)
• AD from GAL4 (yeast)
• Now a target gene can be placed under control of
  the DNA binding site for the first factor
• GAL1 gene with oLex (LexA binding sites) can be
  activated by the fusion protein.
• Basis for 2-hybrid screen for any interacting
  proteins

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Domain swap experiments Diagram 1
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Domain swap experiments Diagram 2
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Two Hybrid Screens (Interaction Cloning), part 1
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Two Hybrid Screens (Interaction Cloning), part 2