Chromatin remodelling: Histone modifications

Question 1

What is chromatin?

Answer 1

The protein-DNA complex of which chromosomes are composed. It condenses 2m of DNA into the nucleus.

Question 2

What is the nucleosome composed of?

Answer 2

2H3-H4 dimers and 2H2A-H2B dimers. These have 147bp of DNA wrapped around 1.7 turns.

Question 3

How far apart are nucleosomes spaced?

Answer 3

35bp

Question 4

In what form of chromatin is transcription activated?

Answer 4

Euchromatin (open)

Question 5

In what form of chromatin is transcription not active?

Answer 5

Heterochromatin (closed)

Question 6

Are epigenetic changes inherited into daughter cells and what is the importance of this?

Answer 6

Yes, this maintains the pattern of gene activity and therefore cell identity in the daughter cell.

Question 7

How is the white eye phenotype seen in drosophila?

Answer 7

A chromosome inversion causes the insulator element that blocks the spread of heterochromatin to be lost, and the white gene becomes adjacent to the heterochromatin that then silences it. This is passed on to daughter cells giving a white phenotype

Question 8

What are the three main ways in which chromatin can be modified?

Answer 8

1) DNA methylation - methyl added to cytosine in CpG dense regions represses transcription.
2) Histone modification- Lysine residues in histone tails are methylated, creating binding sites for regulatory proteins.
3) Nucleosome remodelling - Nucleosomes can be removed, slid up or down, or different histones inserted.

Question 9

How is nucleosome remodelling achieved?

Answer 9

A remodelling protein disaggregates nucleosomes to make space for TF and transcription complex proteins to bind. A second remodelling protein can then bind to the nucleosome to allow transcription without disaggregation of RNA pol II.

Question 10

What is the writer protein associated with H3K4?

Answer 10

MLL - methylation of K leads to transcriptional initiation

Question 11

What is the reader protein associated with genetic repression on H3K27?

Answer 11

Polycomb

Question 12

How does acetylation of histones reduce the interaction of histone tails and DNA?

Answer 12

The lysine groups that become acetylated are positively charged, so will be attracted to DNA and fold onto it which blocks access to the DNA. Acetylation neutralises the lysine’s charge which allows for nucleosome motility and creates access for bromodomain containing proteins.

Question 13

What bromodomain containing proteins interact with acetylated H3K9/14?

Answer 13

SWI/SNF - a nucleosome remodeller

BRD4, which in turn recruits P-TEFb (transcription elongation factor)

Question 14

What enzymes are required for the addition and removal of acetyl groups onto histone tails?

Answer 14

histone acetyl transferase (HAT) required for addition of acetyl groups. 
Histone deacetylases (HDACs) required for the removal of acetyl groups.

Question 15

How can lysines be methylated?

Answer 15

mono, di, or tri-methylated.

Question 16

What reader proteins are able to recognise methylated mysine residues>

Answer 16

Proteins containing chromodomains or WD-40 domains.

Question 17

Are all methylation events activatory?

Answer 17

No, depending on the lysine residue, some can be repressive.

Question 18

Is H3K4 methylation activatory or repressive?

Answer 18

Activatory - associated with transcriptional initiation.

Question 19

Are H3K36 tri-methylation and H3K79 di-methylation activatory or repressive>

Answer 19

Activatory - both are associated with transcriptional elongation

Question 20

Is H3K27 tri-methylation activatory or repressive?

Answer 20

Repressive - associated with transcriptional repression.

Question 21

What is the normal role of MLL? How is it recruited?

Answer 21

Duringg transcription, RNA pol II is blocked by nucleosomes positioned in the gene. PAF1 complex binds to RNA pol II and recruits MLL which methylates histone H3K4. H3K4me3 is recognised by NURF chromatin remodelling complex which moves the histone to allow Pol II to pass.

Question 22

What is the reader protein that recognises tri-methylated H3K4 and what is the outcome of this?

Answer 22

NURF chromatin remodelling complex. It moves histones to allow RNA pol II to pass, allowing for transcriptio

Question 23

How is MLL normally mutated in mixed lineage leukaemia?

Answer 23

The MLL gene translocates. There are over 50 fusion partners but most frequently it fuses to the super elongation complex components AF4 or ENL?

Question 24

What are AF4 and ENL

Answer 24

Different components of the super elongation complex>

Question 25

What is the effect of the MLL/ENL or MLL/AF4 fusion protein?

Answer 25

MLL is required for transcription at the beginning of the gene. The super elongation complex that it is now fused to MLL recruits DOTL1 which catalyses the methylation of H3K79. Also recruits RNA pol II kinase P-TEF (cyclinT1-CDK9). Both of these are required for transcriptional elongation. The fusion protein therefore hyperactively initiates and allows for elongation.

Question 26

What genes are bound by the MLL/AF4 fusion proteins.

Answer 26

Developmentally regulatory genes not normally bound by MLL/AF4. Results in their active expression.

Question 27

What interaction does MLL-AF4 have with BRD4?

Answer 27

It interacts with BRD4 inappropriately. BRD4 then recruits P-TEFb which induces transcriptional elongation.

Question 28

Can BRD4 be targetted for cancer MLL treatment?

Answer 28

Yes - BRD4 inhibitors repress the growth of leukaemic cells in vitro and in mice. (I-BET-151 and JQ1)

Question 29

What protein is found at high concentrations at the IgH-MYC super-enhancer and how is it targetted?

Answer 29

BRD4, addition of JQ1 reduces the growth of MYC-driven multiple myeloma.

Question 30

Which PRC complex is responsible for the methylation of H3K27 and what is the result of this event?

Answer 30

PRC2 methylates H3K27, this methylation recruits PRC1

Question 31

Which H3 lysine reidue is methylated by the PRC2 complex?

Answer 31

H3K27

Question 32

What is the role of polycomb proteins

Answer 32

H3K27 methylation by PRC2 blocks H3K27 acetylation which is associated with transcriptional initiation. PRC1 ubiquitinates H2A, which blocks transcriptional elongation by RAN pol II transcription. PRC1 oligomerises, compressing chromatin. This represses transcription.

Question 33

What is EZH2?

Answer 33

Functional enzymatic component of PRC2 (histone methyltransferase).

Question 34

What mutations in EZH2 are cancerous and why is this the case>

Answer 34

EZH2 overexpression, gain of function mutations. Both are associated with the suppression of cell cycle inhibitors p16INK4A and p15INK4B and the apoptosis regulator p14ARF. It also blocks the expression of differentiation associated genes. The cell therefore remains cycling in an undifferentiated state.

Question 35

How has PRC2 been targeted in therapeutics?

Answer 35

EZH2 inhibitors

Question 36

What histone mutation has been found in glioblastoma?

Answer 36

Histone H3K27 mutates to become a methionine (H3K27M)

Question 37

How is the H3K27M mutant cancerous>

Answer 37

It prevents the methylation of H3K27 which causes reactivation of genes that would normally be repressed by PRC2.

Question 38

How can the H3K27M mutant be targeted therapeutically and why is this the case?

Answer 38

Loss of H3K27 methylation causes an increase in H3K27 acetylation which in turn can be targeted using BET inhibitors