Genome organisation and function

Question 1

What are TADs?

Answer 1

Topologically associated domains - Regions of high interaction frequency due to chromatin loops held together by a shared trunk. This brings distal regions of chromatin into a close 3D proximal space.

Question 2

What lies between TADs and why is this the case?

Answer 2

Insulator regions (CRE) which ensures that there are no interTAD associations.

Question 3

How can intraTAD interactions cause a cell to differentiate?

Answer 3

Changes within a TAD could change the interactions taking place between intra-TAD loops. This could prevent the interaction of an enhancer with a promoter that maintained an undifferentiated cell fate. By removing this interaction the cell can differentiate.

Question 4

What is CTCF?

Answer 4

An insulator that acts to form a barrier between TADs, creating insulation to TAD contacts

Question 5

What is cohesin?

Answer 5

A multi-protein complex that’s formed of Smc1a, Smc3, Rad21 and Stag1/2/3.

Question 6

What functions does cohesin have?

Answer 6

Sister chromatid cohesion, genome organisation, DNA replication, Double strand break repair.

Question 7

How do CTCF and cohesin interact on DNA?

Answer 7

Stag interacts with the C-terminal of CTCF to physically bind cohesin and hold it in place.

Question 8

Why is the orientation in which CTCF binds to DNA important?

Answer 8

One CTCF binds facing one way, with another facing the opposite way. These convergent sites mean a loop can form between the two CTCF binding sites.

Question 9

What protein is responsible for the loading of cohesin onto chromatin?

Answer 9

Nipbl - loads cohesin onto chromatin. It initiates loop extrusion through the cohesin loop.

Question 10

What happens following loop extrusion?

Answer 10

Loop extrusion continues until a boundary where a protein too big to pass through the cohesin loop is found. (CTCF). At this point cohesin and CTCF hold the conformation in place and the TAD is formed.

Question 11

How is cohesin released from chromatin?

Answer 11

WAPL

Question 12

Why is mutant STAG2 more likely to induce a cancerous phenotype then STAG 1

Answer 12

Because STAG2 is X-linked so there is only one copy of the gene. Therefore, a mutation in STAG2 will automatically act in a dominant fashion.

Question 13

STAG mutations show clonal heterogeneity. True or false?

Answer 13

False, lack of clonal heterogeneity suggests early hit/trunk mutation .

Question 14

What effect do cohesin mutations have on a cell?

Answer 14

Depending on the cell type, it might experience aneuploidy, gene expression changes, unfaithful DNA replication and genome instability.

Question 15

What effect can STAG2 mutations have on the protein that is effected as a result?

Answer 15

May cause a loss of expression or have a dominant negative effect, preventing WT STAG1 from performing as usual.

Question 16

Is STAG-1 able to compensate for STAG-2 mutations?

Answer 16

No.

Question 17

How do STAG-2 mutations induce AML in mice?

Answer 17

STAG-2 KO haematopoeitic stem cells have an increased self renewal capacity. STAG-2 loss at the Ebf-1 locus, STAG-1 is unable to compensate. Cohesin complex cannot form with CTCF so a TAD required for the expression of Ebf-1 won’t form. Ebf-1 required for B cell differentiation so without it AML develops.

Question 18

How can STAG-2 deficient cancers be targeted therapeutically?

Answer 18

In cancer cells, there is an oncogenic addiction to STAG1. In normal cells there is WT STAG-1 and STAG-2. Therefore if STAG-1 is inhibited normal cells can undergo faithful sister chromatid cohesion using STAG-2. In STAG-2 deficient cancer cells, loss of STAG-1 will lead to mitotic catastrophe.