Epigenetics

Question 1

What is epigenetics?

Answer 1

Regulation of gene expression mediated through changes in DNA modifications, histone modifications and chromatin structure. These changes are reversible. Does not involve changes of the DNA sequence. Does not change the mRNA sequences or proteins produced, but does change how much of each mRNA is synthesised, and where and when it is synthesised.

Question 2

Describe the mechanism of DNA methylation

Answer 2

DNA methyl transferase (DNMT) transfers a methyl group onto carbon 5 of a cytosine residue. The methyl group is taken from S-adenosyl methionine (SAM), leaving behind S-adenosyl homocysteine

Question 3

Where in the genome does DNA methylation occur?

Answer 3

On cytosine residues in a 5’CG3’ position (CpG). Sites where there are lots of CpG sequences are known as CpG islands. CpG islands present in 50% of promoter regions.

Question 4

What is the result of DNA methylation?

Answer 4

Inhibition of transcription factor binding, either directly or via altered histone acetylation.
Also attracts methyl binding proteins.

Both lead to transcriptional silencing.

Question 5

Describe the group of enzymes that carry out DNA methylation

Answer 5

DNMT1 - maintenance methylase, carries out methylation following mitosis using the hemi-methylated DNA strand as a substrate
DNMT2
DNMT3a and DNMT3b - responsible for de novo methylation during ageing and disease processes
DNMT3L

Question 6

Describe the mechanism of active DNA demethylation

Answer 6

5mc undergoes hydroxylation by TET enzymes. A hydroxyl group is added onto the methyl group, forming 5hmc. Then undergoes further oxidation forming a carboxylic acid group which is eventually removed.

Question 7

What is the role of 5hmc?

Answer 7

Blocks the recruitment of repressive proteins that might otherwise have bee recruited to 5mc. Promotes gene expression, during active demethylation.

Question 8

Where is 5hmc most frequently found?

Answer 8

In embryonic stem cells and the brain

In promoter regions, gene bodies and intergenic regions

Question 9

What are the two methods by which DNA can become demethylated?

Answer 9

Active demethylation

Passive demethylation - methylation pattern lost following cell division due to errors

Question 10

Name 5 functions of DNA methylation

Answer 10

Long term gene silencing for cell differentiation
Silencing of repetitive DNA elements such as transposons
X chromosome inactivation
Establishment and maintenance of gene imprinting
Suppression of viral genes that have incorporated into the host genome

Question 11

Describe the structure of a nucleosome

Answer 11

Negatively charged DNA wraps around positively charged histone proteins
8 histones in a nucleosome
2 x (H2A, H2B, H3, H4)
Histone H1 connects nucleosomes to the linker DNA

Question 12

What are the two states that chromatin can be in?

Answer 12

Euchromatin = transcriptionally active
Heterochromatin = tightly compact, transcriptionally inactive

Question 13

What are the possible histone post translational modifications?

Answer 13

Methylation
Acetylation
Phosphorylation
Ubiquitylation
Biotinylation

Question 14

How do histone modifications affect gene expression?

Answer 14

1. Histone tails form hydrogen bonds with histone tails on adjacent nucleosomes. Modification of the tails affects how well they can interact, so how tightly the chromatin condenses.
2. Affects the charge of the histone and therefore how tightly DNA binds to it
3. Recruitment of transcription factors
4. Recruitment of histone modification enzymes (positive feedback mechanism)

Question 15

What are epigenetic regulators?

Answer 15

Proteins that ‘read’ and interpret the histone modifications, and put mechanisms in place to either facilitate or block gene transcription

Question 16

What is the histone code?

Answer 16

Histone modifications can occur in many combinations, each combination has a different meaning and is read by specific protein complexes

Question 17

On which amino acids can acetylation, methylation and phosphorylation occur?

Answer 17

Acetylation - Lysine
Methylation - Arginine (mono or di-methylated) or Lysine (mono, di- or tri-methylated)
Phosphorylation - Serine

Question 18

How does histone acetylation lead to gene expression?

Answer 18

1. Reduced affinity of histone tail for adjacent nucleosomes
2. Removal of histone positive charge, DNA doesn’t bind as tightly
3. Recruitment of proteins with bromodomains, such as transcription factors and HAT

Question 19

How does histone methylation lead to gene suppression?

Answer 19

Recruitment of silencing or regulatory proteins

Recruitment of proteins with chromodomains

Question 20

What is a topologically associating domain?

Answer 20

A genomic region containing DNA sequences that frequently physically interact with eachother, due to the 3D looping nature of chromatin

Question 21

What proteins control chromatin loop structures?

Answer 21

CTCF proteins

Cohesion proteins

Question 22

What are the two types of interfering RNAs?

Answer 22

Micro RNA (miRNA)
Small interfering RNA (siRNA)

Question 23

Describe the process of micro RNA synthesis

Answer 23

Pri-miRNA transcribed from DNA
Processed by drosha into pre-miRNA
Exported into the cytoplasm by exportin-5
Processed by dicer
Mature miRNA forms RISC complex

Question 24

How does miRNA regulate gene expression?

Answer 24

Complementary base pairing to specific mRNA
Hybridises to a sequence in the 3’UTR
The target mRNA is then destroyed

Question 25

What are some of the functions of epigenetic modifications?

Answer 25

``` Gene imprinting Disorders caused by uniparental disomy X inactivation Parental conflict theory Response to environmental pressures without changing DNA sequence ```

Question 26

What is gene imprinting and how is it carried out?

Answer 26

Certain genes will always be switched off or left switched on depending on if they are inherited maternally or paternally. Means you only have one active allele for that gene. The gene is turned off by DNA methylation in the parental germline, by DNMT1.

Question 27

What is uniparental disomy?

Answer 27

Both chromosomes in a homologous chromosome pair are inherited from one parent. If any of the genes on that chromosome are imprinted, you will get either two active or two inactive alleles when there should be one active allele.

Question 28

Describe the process of X inactivation

Answer 28

Females have two X chromosomes. At the blastocyst stage either the paternal or maternal X chromosome becomes randomly inactivated in each cell. Inactivation carried out by the Xist gene which initiates histone modifications.

Question 29

What is the parental conflict theory of gene imprinting?

Answer 29

Paternally inherited alleles endeavour to use up the mothers resources, as future children may not have his genes (IGF2 gene active, IGF2R gene inactive). Maternally inherited alleles endeavour to limit resource use, to conserve resources for future children (IGF2 gene inactive, IGF2R gene active). Ligers grow very large due to incompatible gene imprinting patterns from the lion father and tiger mother.

Question 30

How can environmental pressures lead to long term changes in gene expression?

Answer 30

DNA methylation affected by environment Highly sensitive during embryogenesis and perinatal period Early epigenetic modifications inherited mitotically down somatic cell lineages

Question 31

Name some environmental factors that can have epigenetic effects

Answer 31

``` Diet Medications Recreational drugs Toxins Radiation exposure Climate Hormones ```

Question 32

How can epigenetic processes be involved in cancer?

Answer 32

Mutations in epigenetic writers, readers and erasers - particularly DNMT3a and TET are mutated in blood cancers Mutations in proteins in the chromatin remodelling complex

Question 33

What are the implications of mutations in epigenetic proteins?

Answer 33

Global DNA hypomethylation can lead to activation of proto-oncogenes and genomic instability Promoter hypermethylation can lead to silencing of tumour suppressor genes Methylated cytosines can become deaminated into guanine - major DNA sequence change

Question 34

Describe an example of where mutations in an epigenetic protein is implicated in cancer

Answer 34

Mutations in DNMT3a can lead to AML. Mutated DNMT3a acts dominantly over the wildtype gene allele. Reduced de novo methylation by methyltransferase.

Question 35

How can non coding RNAs be implicated in cancer?

Answer 35

``` Suppression of tumour suppressor gene mRNA Promotion of metastasis Used in cancer diagnosis Confer susceptibility to cancer Regulation of epigenetic proteins ```

Question 36

What is a CpG island methylator phenotype?

Answer 36

Cancers with high degrees of methylation. Hypermethylation sillences tumour suppressor genes, leading to accumulation of more mutations. Documented in glioma, breast cancer and leukaemia.

Question 37

Why is DNA methylation a good biomarker of cancer?

Answer 37

Very commonly accompanies carcinogenesis Can be detected non invasively (cell free circulation DNA in body fluid samples) Can be detected to a high degree of sensitivity More stable than RNA or protein based markers

Question 38

Why is DNA methylation not a good biomarker of cancer?

Answer 38

DNA methylation of a certain gene is not specific to one cancer type, cannot be used to diagnose what type of cancer Assays for DNA methylation sometimes have low sensitivity

Question 39

Name two types of epigenetic therapy that may be used to treat cancer

Answer 39

DNA hypomethylating agents (DNMTi's), such as 5-azacytidine | Histone deacetylase inhibitors (HDACi's), such as Vorinostat

Question 40

Describe how DNA hypomethylating agents work

Answer 40

They are nucleotide analogues that become incorporated into the DNA during DNA replication, and irreversibly bind DNMT1/DNMT3a/DNMT3b to inhibit their activity in further rounds of DNA replication. Can increase gene transcription of genome protective genes such as p15 and p16, leads to cell cycle arrest and apoptosis of cancer cells

Question 41

What are the limitations of DNA hypomethylating agents

Answer 41

Lack of specificity for which genes they target Development of resistance is common Not effective at treating solid tumours

Question 42

Describe how histone deacetylase inhibitors work

Answer 42

Prevention of histone deacetylation leads to increased transcription of genome protective genes such as p21 and p53, leads to cell cycle arrest and apoptosis of cancer cells

Question 43

Name some of the lab techniques that are used to study epigenetic modifications

Answer 43

Methylated DNA immuno precipitation Sodium bisulfite conversion to detect DNA methylation Pyrosequencing and Whole genome bisulfite sequencing to analyse DNA produced by sodium bisulfite conversion Chromatin remodelling assays such as ATAC-SEQ

Question 44

What is methylated DNA immunoprecipitation? Name 1 pro and 1 con

Answer 44

Antibodies used to detect presence of 5mc The method can be adapted to be specific for different residues (e.g. 5hmc) No base-pair resolution - cannot tell specifically which base pair is methylated

Question 45

How does sodium bisulfite conversion work?

Answer 45

Bisulfite applied to DNA Cytosine residues change into uracil but ones 'protected' by a methyl group do not Following PCR, uracil residues become thymine Levels of cytosine conversion to thymine inversely correlate with levels of methylated cytosines

Question 46

What is pyrosequencing? Name 1 pro and 1 con

Answer 46

Sequences DNA by addition of nucleotides, as they become incorporated there is release of pyrophosphate which initiates an enzyme substrate cascade and emits light Quick and cost effective, but low throughput and can only be used to analyse short fragments

Question 47

Name 1 pro and 1 con of whole genome bisulfite sequencing

Answer 47

Can sequence long DNA sequences Produces lots of redundant information if you are only interested in one area Very costly