Regulation of eukaryotic genes Gene silencing Enhancers Activators

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. GAL 4 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 • Rap 1 protein binds to silencer elements • SIR proteins (Silent Information Regulators) • Nucleates assembly of multi-protein complex – hypoacetylated N-terminal tails of histones H 3 and H 4 – methylated N-terminal tail of H 3 (Lys 9) • Experiments: Condensed chromatin – Resistant to DNase. I 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

SV 40 Control region • Origin of replication • Promoter and upstream activator sequences for early transcription • Promoter for late transcription • Enhancer

SV 40 map

Many regulatory DNA sequences in SV 40 control region

Stimulation of transcription by enhancer is independent of orientation and position SV 40: 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 SV 40: 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 P Transcription unit Upstream Adjacent Downstream Internal Distal Ex 1 Ex 2

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 GAL 4 1 98 148 196 N DNA Activation binding Dimerization 768 881 C Activation GAL 80 binding

Induction by galactose exposes an activation surface • In the presence of galactose, GAL 4 activates several genes whose products are required for galactose metabolism. • GAL 4 binds to a DNA sequence called UASG. • In the absence of galactose, GAL 80 blocks GAL 4 activation. • Binding of the sugar causes GAL 80 to move. • This exposes the activation domain of GAL 4.

Induction of GAL 4

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 Lex. A (E. coli) – AD from GAL 4 (yeast) • Now a target gene can be placed under control of the DNA binding site for the first factor – GAL 1 gene with o. Lex (Lex. A 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