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

EarlyLate

T-Ag +

Enhancer

wt

Enh-

pos

orien

T-Ag +

T-Ag +T-Ag -

SV40:

Enhancers also regulate cellular genes

Enhancer contains multiple binding sites for transcriptional activators

EarlyLate

high level

Enhancer

wt

SV40:

A C B

low leveldeletion

revertant

T-Ag

C B

C BC high level

An enhanson

Enhancers can occur in a variety of positions with respect to genes

Transcription unitP

Ex1 Ex2

EnhancerEnhancer

Adjacent

Downstream

Internal

Distal

Upstream

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

N C

1 98 148 196 768 881

Activation ActivationDNAbinding

DimerizationGAL80binding

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