Epigenetics

Question 1

Definition for epigenetic

Answer 1

1. Structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states.
2. Additional, heritable layer of information, on top of the DNA nucleotide sequence, that influences the expression level of a subset of genes in a cell.
3. Study of changes in the regulation of gene activity and expression that are not dependent on gene DNA sequence.

Question 2

Gene expression is controlled by the epigenetic mechanisms of:

Answer 2

1. Histone modification
2. DNA methylation
3. Non-coding or interfering RNA’s

Question 3

Which part of the histone can undergo modification

Answer 3

The N-terminal tails of the core histones protrude from the nucleosomes and can undergo modifications.

Question 4

What are some posttranslational covalent modifications that can happen to the histone tail.

Answer 4

1. Acetylation
2. Methylation
3. Phosphorylation
4. Ubiquitination
5. Sumoylation

Question 5

What is the the effect of acetylation on the DNA conformation.

Answer 5

Acetylation is negatively charged thus it repels the neg charge of DNA resulting in the loosening of histones and the associated DNA , accessible to the transcription factors-transcriptional activation.

Question 6

Function of histone transferase (HAT)

Answer 6

Catalyse the addition of an acetyl group at the lysine residue

Question 7

Function of histone deacetylase (HDACs)

Answer 7

Catalyse the removal of acetyl group from the lysine side chain of histone

Question 8

Function of Histone methyltransferases

Answer 8

Transfer methyl groups onto the lysine or arginine residues of the H3 and H4 histone proteins

Question 9

Where is DNA methylated?

Answer 9

Addition of a methyl group (CH3) to DNA at the site of a cytosine (C) base

Question 10

Which DNA methyltransferases is responsible for maintenance of methylation after DNA replication.

Answer 10

DNMT1

Question 11

Which DNA methyltransferases is responsible for de novo methylation.

Answer 11

DNMT3a & DNMT3b

Question 12

Explain the phenomena of agouti mice. (Blonde and brown identical mice)

Answer 12

These two mice are genetically identical and exactly the same age. Each mouses mother received a different, specialised diet, which switched on or off chemical clusters called methyl groups that reside near genes. Flipping these genetic switches created differences in size, fur colour and health in the otherwise identical offspring.

Question 13

What is the mechanism by which mature ncRNA molecules act.

Answer 13

It acts through partial complementary to one or more messenger RNA (mRNA) molecules thus preventing it from being translated into protein. Therefore it will down-regulate gene expression.

Question 14

What makes Epigenetics a suitable target for therapies.

Answer 14

1. Epigenetic marks are reversible
2. Epigenetic proteins which establish or erase marks are attractive druggable targets that can be addressed through small-molecule inhibitors.

Question 15

Epigenetic’s is crucial for which processes ?

Answer 15

1. Cellular differentiation
2. Embryonic development

Question 16

Define parthenogenesis

Answer 16

Is the development of a female gamete into a new individual without being fertilised by a male gamete.

Question 17

Can mammals go through parthenogenesis?

Answer 17

McGrath & Solter (1984) constructed fertilised zygotes that contained either two male or two female pronuclei. Development failed soon after implantation. Indicating that diploidy alone is not sufficient for normal embryonic growth and development.

Question 18

What happens to a androgenic zygote (diploid paternal)

Answer 18

1. Extra embryonic tissue (placenta) develops extensively
2. Embryo physically regarded

Question 19

What happens to a gynogenic zygote (diploid maternal)

Answer 19

1. Embryo physically well developed but small.
2. Extra embryonic tissue (placenta) poorly developed.

Question 20

What does the fact that diploidy alone is not sufficient for normal embryonic growth and development mean?

Answer 20

That the maternal and paternal genome contributions are therefore functionally non-equivalent and possession of both parental genomes are essential for development.

Question 21

Imprinting defined

Answer 21

Specific genes are inherited in such a way that one form is functional, while the other non-functional and that this is conditioned during gametogenesis

Question 22

Epimutations can lead to…..

Answer 22

Both genes being active (too much protein product) OR both genes being inactivated (too little protein product)

Question 23

What is re-established imprinting ?

Answer 23

When imprinting is wiped from the chromosome so that the correct parental Epigenetic pattern can be put in place to ensure the correct imprinting pattern is given by the female and male.

Question 24

What are the two events that take place during Epigenetic reprogramming

Answer 24

1. Primordial germ cells (PGCs) undergo imprinting erasure to establish correct imprinting for gametes.
2. Early embryo (32-64 cell stage) erase epimutations to return a cell to pluripotency.

Question 25

Imprinted genes are often important in ?

Answer 25

Growth and development of the foetus.

Question 26

What are some modifications that happen to human chromosome 15 to ensure imprinting ? (Closed conformation)

Answer 26

1. DNA cytosine methylation, histone H3 tail methylation at lysine 9. 2. Recruitment of histone deacetylating enzymes and deacetylayed histones.

Question 27

What are some modifications that happen to human chromosome 15 to ensure imprinting ? (Open conformation)

Answer 27

1. Absent DNA cytosine 5-methylation 2. Tails of histones H3 and H4 are lysine 4-methylated and acetylated respectively. 3. Recruitment of histone acetyltransferase (HAT) to the domain on the paternal chromosome.

Question 28

Clustering of imprinted genes

Answer 28

1. Clusters may contain 3-10 imprinted genes. 2. Human Chrs 2, 6, 7, 9, 11, 12 ,15 and 17 contain imprinted clusters.

Question 29

What are imprinting control regions (ICR)

Answer 29

When grouping of several imprinted genes within a cluster allows them to be controlled by a common regulatory elements acting cis, these regulatory region are called imprinting control regions. Example: XIC on X-chromosome which controls X-inactivation.

Question 30

Examples of imprinting disorders examples

Answer 30

1. Prayer-willi/angelman syndromes 2. Beckwith -wiedemann / Silver-Russell syndromes

Question 31

Similarities and differences between prader-willi and angelman syndromes.

Answer 31

1. Both disorders are caused by genetic deletion and uniparental disomy for a domain on chromosome 15q. 2. The difference between the two is the parental origin which leads to different clinical phenotypes.

Question 32

Spontaneous deletion of chromosome region 15q11-1 in the case of angelman syndrome.

Answer 32

1. Deletion On chromosome from mother 2. No UBESA expression 3. 65-75% of cases

Question 33

Spontaneous deletion of chromosome region 15q11-1 in the case of Prader-Willi syndrome.

Answer 33

1. Deletion on chromosome from father. 2. Thus there is no expression of SNPRN 3. (70-75% of patients)

Question 34

What is the genetic mechanism that lead to imprinting disorders.

Answer 34

1. Spontaneous ‘de novo’ deletion 2. Uniparental disomy

Question 35

UPD in 15q11-1 in the case of Prader-Willi syndrome.

Answer 35

1. Lack of paternal contribution 2. No SNRPN expression 3. (20-25%)

Question 36

UPD for 15q11-1 in the case of angelman syndrome

Answer 36

1. Lack of maternal contribution 2. No UBE3A expression 3. (2% of patients)

Question 37

Steps of Southern blot analysis

Answer 37

1. DNA is cleaved, electrophoresis is used to separate DNA 2. DNA fragments are blotted onto nitrocellulose filter 3. Filter is exposed to radioactive probe 4. Filter is exposed to photographic film, film is developed nn

Question 38

How does southern blotting work to detect imprinting disorders.

Answer 38

Restriction enzyme does not cleave when chromosome is methylated thus is UPD has occurred no cleaving will happen thus one band will be present ( different imprinting disorder. Have different bands size)

Question 39

Techniques that can be used to detect imprinting disorders

Answer 39

1. Souther blotting 2. Methylation -specific MLPA 3. Methylation-specific PCR -bisulfites conversion 4. Reduced representation bisulfite sequencing 5. ChiP-Seq (NGS)

Question 40

How does methylation -specific MLPA.

Answer 40

1. If DNA is unmethylated, RE (Hhal) will digest probe and allele will not be amplified. 2. If DNA is methylated, RE will not digest and allele will be amplified.

Question 41

ChiP-Seq process

Answer 41

1. Cell nucleus 2. Cross link Protein and shear DNA 3. Add protein specific antibody 4. Immunoprecipitation and purify complexes 5. Reverse cross links, Purify DNA and prepare for sequencing 6. Sequence DNA fragment and map to genome