Epigenetics

Question 1

What is the heritability of epigenetic changes?

Answer 1

Still up for discussion

There is proof to show either side of the coin

Question 2

What is epigenetics?

Answer 2

Broad definition

Includes any modification that changes how the DNA is used, switched on or off and changes in different tissues

Question 3

What is the nature of epigenetics like?

Answer 3

Dynamic

Change during healthy development and ageing

Can be disturbed in certain diseases like cancer

Change due to environmental factors

Question 4

Examples of environmental factors that affect epigenetic profiles

Answer 4

Drug exposure

Excercise

Medicine

Question 5

What are the two main types of epigenetic changes?

Answer 5

Histone modification

DNA mehtylation

Question 6

Mechanisms of histone modification

Answer 6

Acetylation

Methylation

Phosphorylation

Ubiquination

Question 7

What does histone acetylation determine?

Answer 7

How strongly wrapped the DNA is bound around the histones

Adding an acetyl group = more expressed since the TF can get to the genes

Removing an acetyl group = less expressed since the TF can no longer access it

Question 8

What does euchromatin represent?

Answer 8

A more loosely packed DNA, since it wraps around histone proteins less strongly

Question 9

What does heterochromatin represent?

Answer 9

A more tightly packed DNA, since it wraps around proteins more strongly

Question 10

What are enhancers?

Answer 10

Activate the transcription of a certain gene sequence

Can influence the expression of 100s of different genes along the chromosome

Question 11

What is the difference between enhancers and promoters?

Answer 11

Enhancers are found on locations far away from where the DNA is transcribed

Promoters are normally found right next to the gene sequence

Question 12

What is histone methylation?

Answer 12

Addition of methyl groups to the amino acids making up the histones

Question 13

Are the effects of histone methylation always the same?

Answer 13

No

Depends on the amino acid they methylate

Methylation events that weaken chemical attractions between histone tails and DNA increase transcription

Question 14

What sites on a histone protein does methylation take place?

Answer 14

Lysine

Arginine

Question 15

Do histone modifications always cause the same changes?

Answer 15

No

Location and type of modification determines the effect on gene activation and repression

Question 16

What is DNA methylation?

Answer 16

Methylation of CpG islands in the DNA

Question 17

What are CpG regions?

Answer 17

Areas in the DNA where cytosine is found next to guanine

These are sites of methylation

Question 18

Is the effect of methylation on gene expression always the same?

Answer 18

No

Depends on the location of methylation

Question 19

Methylation in which location is associated with up-regulation of genes?

Answer 19

Gene body methylation

Question 20

Methylation in which location is associated with down-regulation of genes?

Answer 20

Promoter methylation

Question 21

Why does promoter methylation lead to down-regulation of genes?

Answer 21

Promoter region is bound by methyl groups

So the promoters cannot bind to the DNA sequences

Question 22

Is methylation symmetrical?

Answer 22

Yes

Question 23

What does the fact that methylation is symmetrical mean?

Answer 23

Methylation patterns are heritable from cell to cell following semi-conservative replication

Question 24

Why have more studies been conducted on DNA methylation patterns compared to histone modifications?

Answer 24

Freezing samples destroys the histones but not the methylation patterns of the sample

Question 25

What can we predict looking at the methylation patterns?

Answer 25

Ageing Call type Whether the individual is a smoker or not

Question 26

Why can we predict age by looking at methylation patterns?

Answer 26

Methylation patterns change in precise DNA segments

Question 27

Why can we predict cell type by looking at methylation patterns?

Answer 27

Each cell has distinct methylation patterns Some are unique to the cell

Question 28

Why can we predict if a person is a smoker by lookng at the methylation patterns?

Answer 28

The epigenomic profule of a specific segment of DNA is particularly affected by smoking

Question 29

Why is looking at the histone modifications of a sample not practical?

Answer 29

Requires fresh samples Less stable than DNA methylation

Question 30

Practicalities of DNA methylation

Answer 30

More stable Can be performed on frozen DNA

Question 31

Factors that change methylation profiles of DNA

Answer 31

Environment Ageing Disease

Question 32

Environmental factors that change DA methylation profiles

Answer 32

Smoking Excercise Diet Medication

Question 33

Describe the characteristic changes in DNA methylation profiles of cancer cells

Answer 33

Localised hypermethylation in promoters of tumour suppressor genes = suppression of tumour suppressor genes Global hypomethylation leads to genomic instability and contributes to cell transformation

Question 34

Name 4 technologies used for investigating DNA methylation

Answer 34

Enzyme based methods Methylation microarrays Targeted sequencing techniques Whole genome bisulfite sequencing

Question 35

How are enzyme based methods often used for investigating DNA methylation?

Answer 35

Crude information Gives a good review of methylation

Question 36

Examples of enzyme based methods to look at DNA methylation

Answer 36

ELISA Methylation specidic PCR Methylation sensitive restriction enzymes

Question 37

Examples of methylation microarrays

Answer 37

MethyLight

Question 38

Explain how MethyLight works

Answer 38

Looks at one CpG at a time so has little coverage But cost means it is used a lot in the clinic

Question 39

Examples of targeted sequencing techniques

Answer 39

Sanger bisulfite sequencing MethyLight qPCR Reduced representation bisulphite sequencing

Question 40

What promises to be the future of methylation analysis?

Answer 40

Nanopores

Question 41

What is the underlying mechanism in Bisulfite conversion?

Answer 41

Conversion of an epigenetic signature into a genetic signature

Question 42

What does Bisulfite conversion tell you?

Answer 42

The proportion of the DNA that is methylated vs the proportion that is not No information of where the methylation takes place

Question 43

What happens if the bisulfite is added for too long?

Answer 43

Both the methylated and unmethylated cytosines will be deaminated into uracil

Question 44

How can we check that there hasn't been too much bisulfite added to the mixture?

Answer 44

Check the cytosines not next to guanine Make sure there have not been converted to thymine

Question 45

What technologies can be used to look at epigenetic modifications?

Answer 45

Microarrays Sequencing

Question 46

Information obtained from microarrays?

Answer 46

Differentially methylated positions Differentially methylated regions Copy number variations Epigenetic age estimation Smoking prediction

Question 47

Advantages of sequencing

Answer 47

Greater coverage across the genome Can identify novel gene variants and structural variations Unbiased

Question 48

Disadvantages of sequencing

Answer 48

Expensive Very complex analysis

Question 49

Advantages of microarrays

Answer 49

Cheap Well validated analysis Low amount of DNA

Question 50

Disadvnatages of microarrays

Answer 50

Limited to a certain number of known loci Depend on prior sequence knowledge Biased

Question 51

What is important to consider in epigenomic studies?

Answer 51

Confounders

Question 52

What are confounders?

Answer 52

Change the results through a separate mechanism different from what we are looking for Provide false positive/negatives

Question 53

What is the problem with epigenomic studies?

Answer 53

A lot of factors can affect the genome Designing a study can be very difficult

Question 54

What are the two types of confounders?

Answer 54

Technical Biological

Question 55

Examples of technical confounders

Answer 55

DNA quality affected by storage technique Bisulfite conversion efficiency Batch effects - different lab technicians prepare the samples slightly differently and introduce errors

Question 56

Examples of biological confounders

Answer 56

Genetics - female X chromosomes are silences to prevent the effect of gender bias Different cell types have different methylation profiles Environmental exposures Therapies - cancer therapies are known to alter methylation profiles

Question 57

Real life example of effect of confounders on results

Answer 57

Paper published that epigenetic changes were observed between older and younger patients In reality, the epigenetic changes were caused by different cell populations in older populations Older people = more monocytes So the changes in epigenomic profiles were caused in reality by cellular heterogeneity

Question 58

Methods to minimise technical confounding

Answer 58

Rigorous quality control Randomise samples Normalisation

Question 59

Methods of reducing biological confounding

Answer 59

Important to select correct cell you want to use since cellular heterogeneity increases genetic heterogeneity Match populations for gender, age, environmental exposures and therapy

Question 60

What is the most ideal study cohort?

Answer 60

Longitudinal analysis of epigenetic changes in monozygotic twins