Lecture 6: Gene regulation

Question 1

How do steroids bind to nucleosomal DNA? Draw a diagram.

Answer 1

Steroid-thyroid hormone receptors bind to specific sequences in nucleosomal DNA.
• The nucleosome must be remodelled. This is done with ATPases.
• Glucocorticoid receptor, ligand and ATPase cycles on a reconstituted chromatin template in vitro.
• SWI/SNF ATPase binds and transiently remodels the nucleosomes so the GR and ligand can bind to DNA. The remodelled state collapses back to the ground state and the GR and ligand are kicked off.

Question 2

How is chromatin remodelling dynamic?

Answer 2

It was previously though that chromatin remained the same. The mammalian pS2 receptor is a good example to show that it isn’t.
• Binds oestrogen receptor at ERE (oestrogen response element).
• Both the ERE and TATA box are wrapped in nucleosomes NucE and NucT.
• The oestrogen receptor (ER) and ligand is associated with its binding site in a periodic (every 40 minutes) and transient (interaction lasts about 20 minutes).
• The TATA binding protein is shown to also cycle, but with a different periodicity.
• This periodicity is accomplished through modification of histones and mobilisation of nucleosomes.
• The changes are based on change in acetylation status and nucleosome conformation. Histone deacetylases close the chromatin. HATs and ATPases add acetyl groups and cause DNA to open and allow protein binding.
• The cyclical association of receptor and TBP allows the response to oestrogen to be rapidly altered if conditions changed. pS2 expression can be rapidly shut off if oestrogen is withdrawn.
• Cycling of TBP requires the NuRD complex (Mi2 ATPase + HDACs). It is correlated with loss of R3 methylation of the H4 tail (deimidation). This suggest that there are at least two distinct cycles.

Question 3

What is chromosome conformation capture?

Answer 3

• Chromosome conformation capture allows us to look at the spatial organisation of chromatin in a cell.
• The genomes are first cross-linked with formaldehyde.
• The DNA is then digested with a restriction endonuclease.
• Next random ligation occurs using T4 DNA ligase. Ligating of cross-linked fragments is favoured.
• The DNA can then be amplified and sequenced.

Question 4

How do TAD boundaries change?

Answer 4

TADs divide the genome into distinct globules that are related to whether genes have the potential to be expressed or not and show distinct chromatin modifications.
• TAD boundaries are not fixed. They can change between cell types and in disease.
• Regulated genes in clusters form domains which are flaked by a boundary.
• For example, the β-globin cluster is only acetylated (and activated) in erythroid lineage cells. The domain becomes more sensitive to DNase I.

Question 5

What are locus control regions?

Answer 5

• Locus control regions are clusters of enhancers which can control a cluster of genes.
• They can control chromatin opening (acetylation and DNase sensitivity), timing of DNA replication and expression of individual genes.
• Mutations around the LCR define distinct functions.