Lecture 30 - Epigenetics

Question 1

Differentiate between Heterochromatin and Euchromatin

Answer 1

Heterochromatin:
• Dense chromatin
• Inactive regions

Euchromatin:
• Open chromatin
• Active transcription

Question 2

What can DNA double stranded breaks lead to?

Answer 2

These are lethal lesions

If they are not properly repaired, it leads to genomic integrity and carcinogenesis

Question 3

What is the structure of a nucleosome?

Answer 3

146 bp of DNA wrapped around octamer core

Octamer:
2 of each of the following:
 • H2A
 • H2B
 • H3
 • H4

Question 4

What are the following associated with:
• H3K4me3
• H3K9me3

Answer 4

H3K4me3: Transcriptionally active genes

H3K9me3: transcriptionally inactive genes

Take home messages:
Very minor differences can have very different consequences

Question 5

Where does DNA methylation occur?

Answer 5

Methyl group at 5th position on the cytosine

Typically in the CpG islands

Question 6

What is seen epigenetically in cancer?

Compare this with normal situations

Answer 6

Normal:
Global methylation

Cancer:

• • Aberrant DNA methylation –
• • Aberrant histone acetylation –
• Global demethylation → increased transcription
• Focal CpG hypermethylation → transcriptional repression (of TSGs in particular)

Question 7

What are DNMT inhibitors used for?

Answer 7

Anti-cancer agents

DNA methylation inhibitors

No methylation of the tumour suppressor genes → removal of one of the ‘hits’

Question 8

What are the enzymes associated with the following:
• Histone tail methylation
• Histone tail acetylation

Answer 8

• Histone methyltransferases
• Histone demethylases
• Histone acetyltransferases (HAT)
• Histone deacetylases (HDAC)

Question 9

Describe the regulation of histone acetylation

Answer 9

Regulated by the opposing actions of HAT and HDAC

HAT: histone acetylases (writer)

HDAC: histone de-acetylase (eraser)

Question 10

What is the definition of epigenetics?

Answer 10

“Heritable changes in gene expression and cellular phenotype that are independent of changes in the underlying DNA sequence”

Question 11

How many genes in the human genome?

Answer 11

20,000 - 25,000

Question 12

How much of the genome is genes that encode protein?

What is the importance of the rest?

Answer 12

1-2%

The vast remainder of the sequences in the human genome are yet to be fully understood

Non-coding DNA:
• Previously considered “Junk DNA”
• Regulatory sequences - critical biochemical functions (such as miRNA)

Question 13

Describe what was observed in identical twin studies

Answer 13

Identical twins are born with the same epigenome

Differences increase in monozygotic twins as they age

Epigenetic profiles begin to diverge as they age: epigenetic marks are:

1. Removed at random
2. Changed through environmental influences

Question 14

Describe the effect of diet on epigenetics and disease

Answer 14

Apis mellifera study (honey bee)

3 types of bee:
• Worker bee (female)
• Drone (male)
• Queen bee (female)

Very different size and life span between the different bees

The only thing that determines whether a bee will become a worker or a queen is differential feeding during development
Royal jelly consumption throughout development → queen bee development

Question 15

Describe the Agouti mouse study

Answer 15

Genetically identical mice

Agouti gene: gives the mouse the typical colour

Two groups of mice with different nutrition:

1. Normal diet + high in methyl donors:
   • Methyl donors: Folic acid, vitamin Bs
2. Normal diet

Both groups of mice fed toxic bisphenylate (found in plastics)

Outcomes:
1. Methyl donor group
 • Methylation of Agouti gene
 • Despite bisphenylate
 → Thin, healthy mouse, brown coat colour

2. Normal group
   • No methylation
   • Bisphenylate brings about toxicity
   → Obese, diabetic mouse, like coat colour

Question 16

How is all the DNA in cells organised?

Answer 16

Chromatin: complex structure made up of nucleosomes

Nucleosomes are separated by 100 bp linker

Further compacted into 30 nm fibres

Question 17

What is the Epi-Twin Study?

Answer 17

Big study going on in currently the UK

Study many many identical twins

Sequencing of the entire methylome: every methylation mark in genome

They will track the changes in the methylome over time

Question 18

Describe the role of histones in DNA double stranded breaks

Answer 18

After a double stranded break, HSA.X is rapidly phosphoryated

Heterochromatin is much less susceptible to double stranded breaks, because it is so compacted

That is one way it protects itself

Question 19

Is euchromatin or heterochromatin more susceptible to double stranded breaks?

Answer 19

Euchromatin is more susceptible, as it is less compacted

Question 20

Describe methylation

Answer 20

Once there is methylation of the DNA strand at the CpG islands, a methyl-binding protein will come and bind to it

This adds bulk to the DNA

Transcription factors cannot access the DNA

No DNA transcription

Question 21

In which regions are CpG found?

Answer 21

Promoters

Question 22

Why would epigenetic changes be more favourable for tumour cells, as opposed to mutations in DNA?

Answer 22

Epigenetic changes are reversible, compared to DNA mutations, which aren’t

This gives the cancer cell a lot of flexibility

e.g. Epigenitic changes allow it to leave tissue and metastasise

Once it gets to a new location, it can rapidly undo these changes so it can colonise

This is not possible with changes to the DNA

Question 23

Describe the various histone modifications

Answer 23

Histone acetylation
• More open chromatin
• Transcriptionally active

Histone methylation
• Transcriptionally inactive

Question 24

What is the clinical application of HDACs?

Answer 24

HDACi’s have been developed and are being using clinically

Example:
1. Sodium butyrate (an HDACi)
Leads to cell death specifically in cancer cells

2. Vorinostat
   Selective death in cancer cells

Question 25

What is the effect of the Agouti gene in mice?

Answer 25

Homozygous Agouti: Lethal

Heterozygous: Obese, cancer, diabetes

Question 26

In classical epigenetic studies, how could the Agouti gene be ‘over-ridden’?

Answer 26

When the mother was fed a diet rich in methyl group donors (e.g. folate), the Agouti gene was methylated, and thus silenced

The phenotype was a normal healthy mouse