Histone Code

Question 1

What does the histone code hypothesis state?

Answer 1

This hypothesis predicts that:
1. Distinct modifications of the histone tails induce interactions with specific chromatin-associated proteins, and

2. Modifications on the same or different histone tails may be interdependent and generate various combinations on any one nucleosome

Question 2

What does the histone code hypothesis imply?

Answer 2

The chromatin structure of euchromatic or heterochromatic domains are dependent on the combination of differentially modified nucleosomes
Modifications on the histone tails determine which proteins are bound and thus whether an active or repressive chromatin structure is generated

We are going to discuss 5 histone modifications - but only 3 are epigenetic

Question 3

What are the classes of chromatin modifying enzymes?

Answer 3

They can be classed as readers, writers or erasers
Reader - they read the signature histones - they bind to the modification
Writer - they write the signature on histones
Eraser - they erase the signature

Some marks are very transient - depending if it is an epigenetic factor

Question 4

Give an overview of histone modification?

Answer 4

They have many sites of modification
Nearly 80 different modifications on core histones alone
More modifications on H1 and on histone variants
These modifications occur in different combinations to give a very intricate code

Acetylation
Methylation
Phosphorylation
Ubiquitination etc…

Question 5

Describe the first modification - acetylation?

Answer 5

This gives active chromatin
Acetylation alters the charge on histone tails by HATs transferring an acetyl group from acetyl-CoA
It neutralises the positive charge on the histone tail
It alters histone tail interactions to give a more open chromatin structure - reducing the histone interactions
Acetylation also provides binding sites for other proteins with bromodomains

Question 6

What is some evidence for the aceytlation modification?

Answer 6

1. Gradient centrifugation shows acetylated and non-acetylated chromatin sediment differently
2. Also chemical ligation of H4K16ac prevented formation of 30 nm fibres - gradient centrifugation
3. They have increased general sensitivity to DNase I (around 3 fold)
   This correlates with increased histone acetylation, indicative of a more open chromatin structure at the chicken b-globin locus
   Elongator complex associates with the elongating RNA polymerase II - this complex has HAT activity, Elp3 in yeast

Question 7

Describe acetyltransferases?

Answer 7

They are coactivators
Examples of HATs - PCAF, p300, CBP, Gcn5
They contain bromodomains

Question 8

Give evidence for acetylation favouring the histone code hypothesis?

Answer 8

Transcription factors bring the acetyltransferases to the complex - and they acetylate the promotor (co-activators or TAF250)
Acetylated lysine’s provide binding domains for proteins with bromodomains
Bromodomain binding re-enforces the binding of activating proteins at the promoter

Question 9

What do deacetylases do?

Answer 9

They rapidly remove acetyl groups
This shows the acetylation can’t be epigenetic as deacetylases rapidly remove the acetyl groups
Deacetylases were originally identified as transcriptional repressors - they were parts of co-repressor complexes

To see the acetylation experimentally add chemicals - trichostatin a or butyric acid
They inhibit the deacetylases so the acetylation can be identified

Deacetylation of histones helps to reinforce the repression

Question 10

Give an overview of methylation of lysine?

Answer 10

It can be mono-, di- or tri-methylated
Methylation is associated with gene activity/repression, depending on which lysine is methylated

They can have both positive/negative marks - depending on where it is
Positive - lys 4 and lys 36 = gene expression
Negative - lys 9 and lys 27 (on histone tails) = heterochromatin

Question 11

Describe methylation of lysine 4 - positive mark?

Answer 11

H3K4me3 is associated with active gene promoters
This is an epigenetic modification

Set1 methyltransferase associates with Ser5 phosphorylated form of RNA polymerase II at the CTD
This methylate’s nucleosomes specifically at the promoter with H3K4me3
Set1 methyltransferase (plus MLL and hASH1) are Trithorax group proteins

Question 12

Give evidence for the histone code hypothesis - methylation of lysine 4?

Answer 12

H3K4me3 provides a binding site for proteins with a PHD (plant homeodomain) finger
Many complexes containing PHD-finger proteins are activators; their recruitment thus reinforces activation at the promoter

Question 13

What is H3K4me1?

Answer 13

H3K4me1 is a mark of active enhancers
Old dogma: H3K4me1 at repressed promoters
New finding: H3K4me1 at ACTIVE enhancers

Question 14

Describe methylation of lysine 36 - positive mark?

Answer 14

H3K36me3 is associated with actively transcribed genes - involved in resetting chromatin structure

Set2 methyltransferase associates with Ser2 phosphorylated form of RNA polymerase II at the CTD
This methylate’s nucleosomes in the transcribed portion of genes with H3K36me3

Question 15

Give evidence for the histone code hypothesis - methylation of lysine 36?

Answer 15

H3K36me3 is involved in the “re-setting” of transcribed chromatin
Histone acetyltransferases (HATs) associated with pol II, acetylate nucleosomes to help their displacement for transcription
H3K36me3 recruits histone deacetylases (HDACs) to remove this acetylation and re-set this chromatin mark
As polymerases could bind and transcribe anywhere it can bind in this open region - illegitimate transcription

H3K36me3 favours the histone code hypothesis by recruiting a HDAC complex

Question 16

Describe methylation of lysine 9- negative mark?

Answer 16

Epigenetic = inherited from generation to generation

H3K9me3 is bound by the heterochromatin protein, HP1 via its chromodomain

Question 17

Give evidence for the histone code hypothesis - methylation of lysine 9?

Answer 17

The histone methyltransferase, methylate’s H3 at lysine 9 via its SET domain
This creates a binding site for HP1
Interaction of HP1 (via its chromodomain) with H3K9me3 recruits the HAT to allow methylation of adjacent nucleosomes and the spreading of heterochromatin

Question 18

Describe the spreading of heterochromatin?

Answer 18

This can lead to position effect variegation (PEV)
Genes that lie adjacent to heterochromatin can become silenced if the heterochromatin spreads into that gene
Once the gene is silenced, then this repressed state is inherited epigenetically

Heterochromatin propagates along a chromosome
The variable amount of heterochromatin spreading leads to variable gene expression
Insulators can stop this spread of heterochromatin

Question 19

Describe methylation of lysine 27 - negative mark?

Answer 19

Epigenetic = inherited from generation to generation
It is a repressive mark - bound by the polycomb chromodomain

Polycomb - they maintain repressive chromatin
Even when the repressor is lost, repression continues
HP1 and polycomb group proteins are bound at a large fraction of heterochromatin sites
Polycomb and Trithorax proteins in cellular memory

Question 20

Describe the polycomb group complexes?

Answer 20

Polycomb binds to PRE

PRC1 - binds the mark = heterochromatin
Pc – chromodomain – binds H3K27me3
Ph – zinc finger
Psc – zinc finger
dRING – RING zinc finger

PRC2 - adds the methylation mark
Esc (Eed) – WD40 repeats
E(z) (Ezh2) – SET domain – H3K27 methyltransferase
Su(z)12 (mSU(Z)12 – zinc finger
P55 (RbAp48/RbAp4) – histone binding domain

Polycomb proteins repress via H3K27me3

Question 21

Give evidence for the histone code hypothesis - methylation of lysine 27?

Answer 21

Polycomb repressor complex 2 methylate’s H3K27 via Ezh2 – a SET domain protein
H3K27me3 provides a binding site for polycomb via its chromodomain
Interaction between PRC1 and PRC2 allows spreading of the repressive heterochromatin signal

Question 22

Give a summary of these histone modifications?

Answer 22

Epigenetic

• *H3K4me3 – active mark – bound by PHD fingers
• *H3K9me3 – repressive mark – bound by HP1 chromodomain
• *H3K27me3 – repressive mark – bound by Polycomb chromodomain

Normal modifications
H3K14Ac – active mark – bound by bromodomain proteins
H3K36me3 – active mark – bound by HDAC complex to re-set transcribed chromatin

Methylation of lysine’s carried out by SET domain proteins
Acetylation of lysine’s carried out by histone acetyltransferases (HATs)

Question 23

How are epigenetic chromatin modifications maintained at replication?

Answer 23

For a chromatin mark to carry epigenetic information, it must be stably heritable
Need to have a mechanism to “inherit” the chromatin marks

More difficult with histones as histone tetramers are randomly deposited as the replication fork moves through during transcription
Newly synthesized histones are deposited to fill the “gaps”
The chromatin assembly Complex CAF1 deposits newly synthesized H3/H4 to fill the gaps
Asf1 is a H3/H4 chaperone (to prevent charged proteins from sticking)

Current thinking: The “old” histones provide a template to recruit modifying enzymes and re-establish the histone modifications within a chromatin domain