L4 - Chromatin remodelling & transcriptional repression

Question 1

Post translational modifications of histones

Answer 1

Acetylation
Methylation
Ubiquitination
Phosphorylation

Enzymes that mediate these modifications are writers
Enzymes that remove them are erasers

Question 2

ATP-dependent chromatin remodelling

Answer 2

Cells have multiple remodelling complexes – most are subunit complexes

Can be divided into 4 distinct subgroups
• They all have a catalytic subunit associated with the yeast SNF2 ATPase
• Form part of a bigger family called the ‘helicase & NTP driven nucleic acid translocase’
• Differ in other types of protein domains

Use the energy from ATP hydrolysis to remodel chromatin

Question 3

What reactions can be catalysed by ATP-dependent chromatin remodelling?

Answer 3

Sliding 
Unwrapping 
Eviction 
Spacing 
Histone variant exchange

Question 4

How does SWI/SNF remodel chromatin?

Answer 4

The catalytic subunit of SWI/SNF is called Snf2

It hydrolyses 1000 ATP molecules per minute in the presence of DNA or nucleosomes

Snf2 is related to DNA helicases (unwind DNA)

Question 5

How does Snf2 unwind DNA?

Answer 5

Cant fully unwind it so causes torsional stress (stress due to twisting)

Thought to be a molecular motor that uses the energy from ATP hydrolysis to track along the DNA & cause torsion

This results in the disruption of histone-DNA interactions & movement of the nucleosome
– DNA binding site moves relative to the nucleosome

Question 6

Humans have 2 distinct but related complexes to yeast SWI/SNF
What are they?

Answer 6

Human SWI/SNF hBRM

Human SWI/SNF BRG1

Question 7

SWI/SNF & cancer

Answer 7

Human SWI/SNF ATP-dependent remodelling complexes implicated as a potent tumour suppressor

One or more subunits mutated in 20% of all human cancers

Mutations in multiple subunits prevalent in some cancers

Question 8

How do ATP-dependent & HAT complexes work together?

Answer 8

Synergistically

Question 9

How do ATP-dependent & HAT complexes work synergistically?

Answer 9

SWI/SNF & GCN5 regulate the same genes in yeast

SWI/SNF complex is a good reader complex of modifications put down by GCN5 histone acetylase

Commonly HATs & ATP-dependent remodellers are recruited to the same promotors

You get a transcription factor that intersects with histone acetyl complex for a specific subunit – eg. Tra1

Once recruited the HAT will acetylate histone tails which drives recruitment of more HATs as they have subunits with bromodomains

Question 10

How does the transcription factor Tra1 work in yeast?

Answer 10

Recruits histone acetylase complex to the chromatin which when acetylates neighbouring nucleosomes

Can drive the recruitment of SWI/SNF complexes

SWI/SNF recruited by the binding of acetylated lysines on histone tails

Question 11

Repression of transcription

Answer 11

Cells commonly exploit chromatin structure to bring about transcriptional repression

Mediated by the recruitment of chromatin modifying factors

Question 12

What are some chromatin modifying factors?

Answer 12

Histone Deacetylases (HDACs)

ATP-dependant Remodellers

Histone Methylases (heterochromatin)

Question 13

Where is the genome hyperacetylated & where is it hypoacetylated?

Answer 13

Where ACTIVE regions of the genome are HYPERACETYLATED

REPRESSED regions are HYPOACETYLATED

Question 14

Histone deacetylation

Answer 14

Deacetylation is mediated by HISTONE DEACETYLASES (HDACs)

Just as HATs were shown to be transcriptional co-activators HDACs function as co-repressors

Question 15

What are the 4 groups of HDACs?

Answer 15

Class I
Class II
Class IV
All classical HDACs - zinc dependent enzymes

Class III
Sir2 family - require NAD as a co-factor
• Not structurally related
• Sir2 is important in aging

Question 16

HDAC co-repressor complexes

Answer 16

HDACs commonly function in the context of large multi-subunit complexes

Eg. SIN3 CO-REPRESSOR complexes, which are conserved from yeast to mammals

Recruited to promotors by interaction with site-specific DNA binding proteins – don’t bind DNA directly

Question 17

ATP-dependent remodelling complexes + an example

Answer 17

Some ATP-dependent remodellers commonly mediate repression

Eg. the NuRD complex which belongs to the Mi2/CHD family

Question 18

NuRD complex

Answer 18

Highly conserved in plants & animals, it is broadly expressed in most tissues & plays roles in normal differentiation & tumourogenesis

Several known oncogenic transcription factors have been shown to recruit NuRD complex to suppress the transcription of tumour suppressor genes

It is a multi-subunit complex that also contains HDACs

Good at regularly spacing nucleosomes

Question 19

What are the 2 types of chromatin?

Answer 19

Euchromatin

Heterochromatin

Question 20

Whats the difference between euchromatin & heterochromatin?

Answer 20

Euchromatin - lightly stained
• Gene rich
• Potential to be transcribed

Heterochromatin - darkly stained
• Gene poor
• Repetitive regions
• Transcriptional silencing

Question 21

Examples of heterochromatin

Answer 21

Centromeres
Telomeres
X-chromosome inactivation

Question 22

Biochemical features of heterochromatin

Answer 22

Hypoacetylation

Specific histone H3 methylation (Lys9 & Lys27)

Association of specific silencing factors

Question 23

The assembly of (H3Lys9me) Heterochromatin

Answer 23

1. H3 globular domain
2. Removal of the acetyl group by HDACs
3. Residue recognised by Suvar39 which then methylates the H3
4. Methylated lysine9 acts as a specific recruitment beacon for the Hp1 class of proteins – directs its assembly

Question 24

What is HP1?

Answer 24

Heterochromatin protein 1

It is a chromodomain protein

The chromodomain of HP1 is specific for H3 Lys9me

Question 25

What do chromodomains do?

Answer 25

Chromodomains often recognise & bind to methylated lysine residues

Question 26

Analysis of heterochromatin using reporter silencing assays

Answer 26

You use a reporter gene (ade6 gene) Normally it would be expressed & would form white colonies If you put in heterochromatic repeats, heterochromatin will spread & silence the ade6 gene – no longer expressed – Get the build-up of a red pigment Easy mechanism to determine whether a gene is required for heterochromatin

Question 27

Analysis of heterochromatin using reporter silencing assays What happens if you get a mutation in a gene encoding component of heterochromatin?

Answer 27

Then you get pink/white colonies Tells you you have a mutation in an important component of heterochromatin

Question 28

Heterochromatin: X-chromosome inactivation

Answer 28

Females have 2 X chromosomes one of which is inactivated This equalises the number of X-linked genes expressed in males & females The inactivated X-chromosome is seen in the nucleus as a condensed structure (Barr body) that is assembled into a specific form of heterochromatin

Question 29

What is the formation of Barr bodies controlled by?

Answer 29

Controlled by non-coding RNAs Xist & Tsix – very complex mechanism – not templates for translation

Question 30

How does X-chromosome inactivation occur?

Answer 30

1. Early development – both X chromosomes will express the Tsix RNA 2. Tsix RNA represses the expression of Xist – the DNA is in the opposite orientation 3. One of the chromosomes makes the decision to stop expression of Tsix & instead express Xist 4. The 1 that expresses Xist is targeted for silencing 5. Silencing spreads over the chromosome