The Epigenome

Question 1

As a recap, describe genome organisation.

Answer 1

The genome exists in a cell as an organised structure made up of a number of macromolecules with DNA as the primary building block

Histones and other proteins provide a support around which the DNA is wound

These structures are then organised in 3D to form fibres and ultimately, chromosomes

So they’re organised in nucleosomes, fibres and, ultimately, chromosomes.

Question 2

Describe the nucleosome.

Answer 2

It is the first level of packing.

It has a histone core (the octamer with the DNA wrapped around it] and H1 in between the cores.
It regulates gene expression.

Question 3

Define euchromatin and heterochromatin.

Answer 3

EUCHROMATIN: gene-rich, typically unique sequences within them and have lots of space between nucleosomes.

HETEROCHROMATIN: gene-poor, less transcription and little space between the nucleosomes of typically repetitive DNA.

Question 4

Describe the packing solution of DNA (i.e. how DNA is so compact).

Answer 4

• Nucleosomes are wound up to form 30nm fibres
• Fibres are then wound up further with scaffold proteins to generate higher-order structures
• Chromosomes are the most densely packed form of genomic DNA.
  (the higher order structures aren’t really understood, which is why they are simply called higher order structures)

Chromosomes are least accessible by transcription machinery - essentially, heterochromatic.

Question 5

Define the epigenome.

Answer 5

It is the sum of all the (heritable) changes in the genome that do not occur in the primary DNA sequence and that affect gene expression

An epigenetic change results in “a change in phenotype but not in genotype”.

[note: these are very broad definitions as they have to take into account different things]

Epigenetic marks allow for the regulation of the accessibility of DNA.

Question 6

What are imprinted genes?

Answer 6

Imprinted genes are genes whose expression is determined by the parent that contributed them. These genes appear to be heritable, but they’re a small part of the genome.

Question 7

List some epigenetic mechanisms.

Answer 7

• DNA methylation
• histone modification
• X-inactivation
• genomic imprinting

Question 8

Describe DNA methylation.

Answer 8

DNA methylation in humans is the addition of a methyl group in the 5’ position of cytosine.
This is catalysed by DNA methyltransferase enzymes (DNMT1, DNMT3a and DNMT3b).

It requires S-Adenosyl Methionine to provide the methyl group. In differentiated cells, it occurs in CpG dinucleotides.

Question 9

Describe DNA demethylation.

Answer 9

It was thought to be a passive process, but TET (ten-eleven translocation enzymes) were discovered.

BER (base excision repair) alters the base even further, and its (the methyl group) removed completely to replace a normal carbon.

We are still unsure if the intermediates are important in epigenetics.

Question 10

What does DNA methylation have to do with gene expression?

Answer 10

In general, DNA methylation turns transcription off by preventing the binding of transcription factors.

DNA methylation patterns change during development and are an important mechanism for controlling gene expression.

They can be unmethylated - when observing gene expression, we expect them to be unmethylated. They’re methylated to stop gene expression; the idea is to block factors that could bind and enhance gene expression.

Question 11

Describe histone modification.

Answer 11

This is the addition of chemical groups to the proteins that make up the nucleosome. There are a large number of known histone modifications (>100), many of which the function is still unknown.
Common modifications include acetylation and methylation. A large range of enzymes catalyse the modifications.

The modifications are named based on which histone is modified, the amino acid and them the modification that took place.

Question 12

List some histone modifiers.

Answer 12

WRITERS:

• Histone Acetyltransferase - HAT1
• Histone Methyltransferase - EHMT1

ERASERS:

• Histone Deacetylase - HDAC1
• Histone Demethylase - KDM1

READERS:

• Bromodomain and extra0terminal (BET) proteins - BRD2
• Chromodomain proteins - CBX1

Question 13

What are the role of histone modification?

Answer 13

Histone acetylation at Lysine residues relaxes the chromatin structure and makes it accessible for transcription factors.

Histone methylation is more complex and can either repress or activate transcription depending on where it occurs, thus it’s hard to look at the roles in isolation.

Histone modifications can occur simultaneously and so their effects can interact or modify each other.

Question 14

What is X-inactivation?

Answer 14

This is the inactivation of one of the two X chromosomes in every somatic cell in females.

This is needed as the Y chromosome has virtually no genes, so there is only one copy of each X chromosome gene in males (hemizygosity).

X-inactivation ensures that every somatic cell in all humans has the same number of active copies of every gene.

Question 15

Describe the process of X-inactivation.

Answer 15

The Xist gene is transcribed as a long noncoding RNA (lncRNA) from the X-inactivation centre (Xic) and binds all over the X-chromosome.

Histone acetylation is removed and histone and DNA methylation occurs; this represses gene expression. The inactive X-chromosome is heterochromatic – also known as a Barr body.

Tsix is derived by transcription in the opposite direction and antagonises Xist RNA to keep one X active.

Question 16

Give an example of how X-inactivation affects organism phenotype.

Answer 16

All tortoiseshell cats are female. Tortoiseshell cats have one X with an orange fur allele and one X with a black fur allele

Random X-inactivation results in patches of orange and black fur.

Question 17

What is imprinting?

Answer 17

Imprinting is the selective expression of genes related to the parental origin of the gene copy. Imprinted genes tend to be found in clusters, probably because they need to be regulated together, we’re still not too sure.

Every autosomal gene has one paternal and one maternal copy. Most genes have no difference between the paternal and maternal copies.

There are very few imprinted genes (~250).

Question 18

What is the significance of imprinted genes, and how is imprinting mediated?

Answer 18

Imprinting is mediated by imprinting control regions (ICRs). ICRs are similar to the targets that exist on the X chromosome.

We have one copy (either maternal or paternal). This one copy is silenced by DNA methylation catalysed by DMNT3a and histone methylation, leading to inactivation

LncRNAs are essential to the process, because when their functions are damaged or they do not work properly, we get drastic effects either on the whole organism or person, or you get fairly well-known syndromes.

Imprinting patterns are reset during gamete formation. We are still unsure as to why imprinting happens and why we need it.

Question 19

Can we treat cancer by targeting epigenetic enzymes?

Answer 19

Global DNA methylation has long been known to be altered in tumour cells:

• Hypermethylation of tumour suppressor genes
• Hypomethylation of tumour activating genes

Epigenetic enzymes are often mutated in tumour cells:

• DNMT3A and TET1/2
• Histone Acetyltransferases
• Histone Methyltransferases
• Histone Kinases
• Histone Readers (acetyl/methyl/phosphoryl)
• Histone Demethylases

The enzymes are all involved in different stages in epigenetic regulation of gene expression.
The tumour is trying to grow as fast as it can so there is clearly something that is inhibiting the process that would normally stop that from happening.

Question 20

List some examples of pharmacoepigenetic drugs.

Answer 20

DNA Methyl Transferase Inhibitors:

• 5-Azacytidine (Vidaza)
• Myelodysplastic syndrome

Histone Deacetylase Inhibitors:

• Romidepsin (Istodax)
• Cutaneous T-cell lymphoma

These are successful drugs for what we have so far gained in knowledge of epigenetics.

All the effects of gene expression might be directly affected by the environment. So, the sequence is not changing but epigenetic signals might change because of the chemicals you are exposed to which changes the gene expression, which might make you more resistant or susceptible to a disease.