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CMB2001 - Control of Eukaryotic Gene Expression > L4 - Chromatin remodelling & transcriptional repression > Flashcards

L4 - Chromatin remodelling & transcriptional repression Flashcards

1
Q

Post translational modifications of histones

A

Acetylation
Methylation
Ubiquitination
Phosphorylation

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

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2
Q

ATP-dependent chromatin remodelling

A

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

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3
Q

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

A 
Sliding 
Unwrapping 
Eviction 
Spacing 
Histone variant exchange
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4
Q

How does SWI/SNF remodel chromatin?

A

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)

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5
Q

How does Snf2 unwind DNA?

A

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

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6
Q

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

A

Human SWI/SNF hBRM

Human SWI/SNF BRG1

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7
Q

SWI/SNF & cancer

A

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

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8
Q

How do ATP-dependent & HAT complexes work together?

A

Synergistically

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9
Q

How do ATP-dependent & HAT complexes work synergistically?

A

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

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10
Q

How does the transcription factor Tra1 work in yeast?

A

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

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11
Q

Repression of transcription

A

Cells commonly exploit chromatin structure to bring about transcriptional repression

Mediated by the recruitment of chromatin modifying factors

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12
Q

What are some chromatin modifying factors?

A

Histone Deacetylases (HDACs)

ATP-dependant Remodellers

Histone Methylases (heterochromatin)

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13
Q

Where is the genome hyperacetylated & where is it hypoacetylated?

A

Where ACTIVE regions of the genome are HYPERACETYLATED

REPRESSED regions are HYPOACETYLATED

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14
Q

Histone deacetylation

A

Deacetylation is mediated by HISTONE DEACETYLASES (HDACs)

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

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15
Q

What are the 4 groups of HDACs?

A

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

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16
Q

HDAC co-repressor complexes

A

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

17
Q

ATP-dependent remodelling complexes + an example

A

Some ATP-dependent remodellers commonly mediate repression

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

18
Q

NuRD complex

A

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

19
Q

What are the 2 types of chromatin?

A

Euchromatin

Heterochromatin

20
Q

Whats the difference between euchromatin & heterochromatin?

A

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

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

21
Q

Examples of heterochromatin

A

Centromeres
Telomeres
X-chromosome inactivation

22
Q

Biochemical features of heterochromatin

A

Hypoacetylation

Specific histone H3 methylation (Lys9 & Lys27)

Association of specific silencing factors

23
Q

The assembly of (H3Lys9me) Heterochromatin

A
  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
24
Q

What is HP1?

A

Heterochromatin protein 1

It is a chromodomain protein

The chromodomain of HP1 is specific for H3 Lys9me

25
Q

What do chromodomains do?

A

Chromodomains often recognise & bind to methylated lysine residues

26
Q

Analysis of heterochromatin using reporter silencing assays

A

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

27
Q

Analysis of heterochromatin using reporter silencing assays

What happens if you get a mutation in a gene encoding component of heterochromatin?

A

Then you get pink/white colonies

Tells you you have a mutation in an important component of heterochromatin

28
Q

Heterochromatin: X-chromosome inactivation

A

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

29
Q

What is the formation of Barr bodies controlled by?

A

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

30
Q

How does X-chromosome inactivation occur?

A
  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

CMB2001 - Control of Eukaryotic Gene Expression (10 decks)

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