Lecture 2: Promoters, Enhancers and TFs

Question 1

What factors influence the ability of a transcription factor to bind?

Answer 1

• Different binding specificities.
• TF-TF interactions.
• TF-cofactor interactions.
• DNA modifications.
• Shape of the DNA.
• Genome context, what is next to the binding site.
• Coding and noncoding variation.

Question 2

What are fos and jun?

Answer 2

Fos and jun are AP1 family transcription factors which bind to DNA as dimers.
• They can form in different combinations: fos and jun heterodimers, jun homodimers or jun heterodimers (with other TFs).
• Different combos have different AP1 affinities.
• Jun and fos together bind 30 times stronger than a jun homodimer.

Question 3

How do fos and jun bind to each other?

Answer 3

Both proteins use leucine zipper domains to bind.
• The leucine zipper is a coiled coil with amphipathic characteristics.
• The helix uses lots of leucine and other hydrophobic residues to create a hydrophobic face for binding.
• A basic domain is used for DNA binding.
• Specificity of interaction is mediated by the residues flanking the hydrophobic region.
• Fos homodimers cannot form because of similarly charged residues flanking the leucine zipper.
• The combination of the basic regions of fos and jun can bind more strongly to the AP1 site.

Question 4

What is EMSA?

Answer 4

EMSA stands for electrophoretic mobility shift assay. It is used to detect DNA-protein interaction.
• A polyacrylamide or agarose gel is run with probes for DNA/RNA (or radioactively labelled DNA/RNA).
• Different lanes will have different proteins.
• If a protein binds, then the DNA will not move as far because it is slowed down. The band will shift upwards.

Question 5

How can we modify EMSA to confirm our hypotheses?

Answer 5

• Antibodies raised against the protein can confirm that it is there. This leads to a super shift due to the presence of the antibody.
• Protein concentration can determine the bound to free ratio.
• We can also add unlabelled, specific DNA to demonstrate that specific binding is occurring. This can be compared to a control of random DNA (e.g. salmon sperm).
• We can also use mutated DNA sequences as competitors to find which residues are the most important for TF binding.

Question 6

How does cooperativity of transcription factors play a role in expression?

Answer 6

There are so many genes in humans that they cannot be controlled by different regulators.
• Cooperativity allows different combos of TFs to achieve regulation. It also increases specificity.
• Sox2, Oct4, cMyc and Klf4 (SOCK) are required to reprogram somatic cells. They interact collaboratively.

Question 7

Why is DNA binding not sufficient for gene activation? How is this shown?

Answer 7

A TF cannot just bind DNA to activate it.
• Domain swapping demonstrates that both activation and DNA binding domains are required.
• In Gal4p, the DNA binding region will not bind by itself. However, a fusion of another DNA binding region and the Gal4p activating region will.
• This idea can be used in yeast two hybrid. By adding interactions between a bait domain (DNA binding) and a prey domain (activation).