30th Jan - Epigenetic Regulation

Question 1

What is epigenetics?

Answer 1

The study of modifications that alter the phenotype of the cell without altering the genotype. heritable traits over rounds of cell division and sometimes transgenerationally

Question 2

What is the function of DNA methylation?

Answer 2

Long term Inhibition of transcription, mainly to keep the junh (transposons, repetitive elements and endogenous retroviruses) quiet

Question 3

Which base is methylated in eukaryotes?

Answer 3

Cytosine

Question 4

Which base is methylated in prokaryotes?

Answer 4

Adenine

Question 5

Where does methylation occur in eukaryotes?

Answer 5

At CpG dinucleotides

Question 6

When is methylation reversible in a eukaryote?

Answer 6

During embryogenesis

Question 7

Give three examples of DNA methyltransferases

Answer 7

Dnmt1, Dnmt 3a and Dnmt 3b

Question 8

Outline the function of Dnmt1

Answer 8

It methylates hemi-methyalted DNA after replication therefore copying the epigenetic code from the template strand thus is replication dependent

Question 9

Outline the function of Dnmt 3a and 3b

Answer 9

It methylates DNA de novo i.e. it methylates one strand of DNA, Dnmt1 then methylates the other strand

Question 10

What Is the evidence for DNA methylation maintaining genomic instability?

Answer 10

5-azacytidine

• -Cytosine analogue that can’t be methylated
• -When added to the cells it dilutes DNA methylation through each round of cell division
• -Loss of DNA methylation leads to reactivation of endogenous retroviruses

Dnmt1 KO mice

• -Retain only 10% of normal activity
• -Develop aggresive T cell tumours due to trisomy of chromosome 15

hypomethylation is a common trait in cancer

Question 11

How was it demonstrated that coat colour controlled in mice is controlled by DNA methylation?

Answer 11

Agouti = yellow pigment
Used a series of genetically identical mice, and added an endogenous retrovirus which inserts above the Agouti gene.
In yellow mice most CpG islands in Agouti are unmethylated
In pseudoagouti mice every CpG in the Agouti gene is methylated –> brown mice
Gradient of colours dependent upon the level of methylation

Question 12

What percentage of CpG dinucleotides are methylated in the mammalian genome?

Answer 12

> 50%

Question 13

Why are cytodines under-represented in the mammalian genome?

Answer 13

Over time cytodines can be converted to thymidines. Most of these are repaired however some will escape over the course of evolution –> loss of approx 3/4 of CpG dinucleotides

Question 14

Where are CpG dinucleotides most common?

Answer 14

Around 5’ end of active genes

Question 15

What are CpG islands?

Answer 15

Regions with >50% CpG content about 1-2kb in length usually located at the 5’ end of the genome, and are resistant to DNA methylation

Question 16

How does DNA methylation lead to long term silencing?

Answer 16

If Methylated CpG > unmethylated CpG –> Gene off
As the methylation is recognised by methylated CpG binding proteins that act as transcriptional repressors by recruiting histone modifying enzymes

Question 17

Give an example of a methyl CpG binding domain

Answer 17

Mecp2
MBD2
MBD1

Question 18

Give an example of a Zn-finger protein which is a methyl CpG binding protein

Answer 18

Kaiso
ZBTB4
ZBTB38

Question 19

What is the function of NuRD?

Answer 19

The Nucleosome remodelling and deacetylase complex integrates DNA methylation and histone modification activities

Question 20

What are the constituent proteins of NuRD?

Answer 20

the histone deacetylase core proteins HDAC1 and HDAC2,
the histone-binding proteins RbAp46 and RbAp48,
the metastasis-associated proteins MTA1 (or MTA2 / MTA3),
the methyl-CpG-binding domain protein MBD3 (or MBD2)
the chromodomain-helicase-DNA-binding protein CHD3 (aka Mi-2alpha) or CHD4 (aka Mi-2beta).

Question 21

What are the two histone modifying enzymes in the NuRD complex?

Answer 21

HDAC 1/2

Mi2beta twists nuclesomes either further apart or closer together

Question 22

In which cells does hydroxymethylcytosine predominantly occur?

Answer 22

Brain and stem cells

Question 23

What is hydroxymethylcytosine?

Answer 23

A stable intermediate in the demethylation process

Question 24

What is the function of TET proteins?

Answer 24

Ten-eleven translocation proteins plays a role in the active demethylation of cytidine

Question 25

How does histone lysine acetylation –> gene activation?

Answer 25

The lysines are positively charged thus by adding an acetyl group it masks the positive charge therefore reducing interaction with the DNA backbone

Question 26

Does H3K27me3 activate or repress genes?

Answer 26

Repress

Question 27

Does H4K20me3 activate or repress genes?

Answer 27

Repress

Question 28

Does H3K4me3 activate or repress genes?

Answer 28

Activate

Question 29

How is Oct 4 inactivated during stem cell differentiation?

Answer 29

1. Repressors bind RA receptor element. Recruiting G9a which recruits HDAC
2. HP1 binds (perhaps through g9a) to H3K9me3
3. Dnmt3a catalyses the methylation of DNA

Question 30

How can epigenetic modifications be monitored?

Answer 30

ChIP
MeDIP
ChIP-Seq

Question 31

How can ChIP be used to identify epigenetic modifications?

Answer 31

Use antibodies targeted to histones, modified histones, TFs, histone modifying enzymes or chromatin remodelling proteins

Question 32

What is MeDIP?

Answer 32

Methylated DNA immunoprecipitation

Question 33

Outline the common epigenetic changes in cancer

Answer 33

Transcriptional silencing of tumour suppressor genes e.g. pINK4a
General global hypomethylation
Overall loss of H4K16Ac and H4K20me3