Transcript No Slide Title

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