Regulation of eukaryotic genes
Gene silencing
Enhancers
Activators
Functional domains of activators
Idea for another extra credit project
Explore DNA binding domains of proteins.
1. Go to a web site with a Chime tutorial, e.g.
GAL4 or Cro
2. Or use Kinemages
3. Write a roughly 2 page report on how a
particular protein recognizes a DNA sequence
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
Silent and open chromatin
Transcription initiation and pausing
Basal and activated transcription
Silencing Mechanism
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
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
Gene Silencing
Silencing Mechanism
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
SV40 Control region
• Origin of replication
• Promoter and upstream activator sequences
for early transcription
• Promoter for late transcription
• Enhancer
SV40 map
Many regulatory DNA sequences in SV40
control region
Stimulation of transcription by enhancer is
independent of orientation and position
SV40:
Early
Late
wt
T-Ag
+
pos
T-Ag
+
orien
T-Ag
+
Enhancer
Enh-
T-Ag
-
Enhancers also regulate cellular genes
Enhancer contains multiple binding sites
for transcriptional activators
SV40:
Early
Late
Enhancer
T-Ag
wt
A C B
high level
deletion
C B
low level
revertant
C C B
high level
An enhanson
Enhancers can occur in a variety of
positions with respect to genes
Enhancer
Upstream
Enhancer
P Transcription unit
Adjacent
Downstream
Internal
Distal
Ex1
Ex2
Activator proteins
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
Modular structure of GAL4
1
98 148 196
N
DNA
Activation
binding
Dimerization
768
881
C
Activation
GAL80
binding
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.
Induction of GAL4
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
Domain swap experiments: Diagram 1
Domain swap experiments: Diagram 2
Two Hybrid Screens (Interaction Cloning), part 1
Two Hybrid Screens (Interaction Cloning), part 2