Epigenome

Question 1

What does DNAseI do?

Answer 1

DNAseI cleaves DNA.

Question 2

What percentage of mammalian cytosines are methylated?

Answer 2

2 - 7% of mammalian cytosines are methylated.

Question 3

Where are 90% of methylated cytosines found?

Answer 3

90% are in 5′ - CG - 3′ dinucleotide.

Question 4

What is the effect of increased DNA methylation on gene activity?

Answer 4

Increased DNA methylation leads to inactive genes.

Question 5

What does Mspl do?

Answer 5

Will cleave DNA regardless of methylation.

Question 6

What is the function of HpaII?

Answer 6

Will only cleave unmethylated DNA.

Question 7

What is the recognition site for Isoschizomers?

Answer 7

CCGG.

Question 8

What does Sodium Bisulfite do?

Answer 8

Converts unmethylated cytosines to uracil. No impact on methylated cytosines.

Question 9

How is DNA methylation detected?

Answer 9

Southern blotting or DNA sequencing is used to detect if DNA was methylated or not methylated.

Question 10

What is an example of DNA methylation effect on chromatin?

Answer 10

Methylated globin gene introduced into cells adopts compact conformation and is not expressed; non-methylated globin gene is expressed.

Question 11

What is 5-azacytidine treatment?

Answer 11

5-azacytidine is an analog of cytidine that cannot be methylated and results in reactivation of regulatory regions previously switched off.

Question 12

Why are DNA methylation patterns important?

Answer 12

Methylation patterns are crucial for regulating gene expression and can be propagated through cell division cycles.

Question 13

What enzyme is responsible for methylating DNA?

Answer 13

DNA methyl transferase (Dnmt).

Question 14

What is the function of Dnmt 3a/3b?

Answer 14

De novo methylation during embryonic development.

Question 15

What is the role of Dnmt1 in DNA methylation?

Answer 15

Maintenance methylation at hemi-methylated sites.

Question 16

What are the two ways loss of methylation sites can occur?

Answer 16

Active demethylation and passive demethylation.

Question 17

What enzyme is involved in active demethylation?

Answer 17

Demethylation enzyme.

Question 18

What happens during passive demethylation?

Answer 18

Maintenance methylase is inhibited.

Question 19

What is required for the maintenance of stem cell lines?

Answer 19

Demethylation may be required.

Question 20

What occurs during cell division involving stem cells?

Answer 20

Production of one differentiated and one de-differentiated daughter cell.

Question 21

What can Ten eleven translocase (TET) enzymes do?

Answer 21

Oxidize 5-methylcytosine into 5-hydroxymethylcytosine.

Question 22

How do TET enzymes impact DNMT1?

Answer 22

DNMT1 cannot recognize 5-hydroxymethylcytosine.

Question 23

What happens to carboxylcytosine during DNA repair?

Answer 23

Cleaved by DNA repair enzymes and replaced with unmodified cytosine.

Question 24

What do inhibitory proteins recognize and bind to?

Answer 24

Methylated DNA, not unmethylated DNA.

Question 25

What is the mechanism of MeCP2 protein?

Answer 25

It binds to methylated CG (meCG) and recruits HDAC complex, causing closed chromatin conformation. It can also recruit histone methyltransferases

Question 26

What happens with a mutation in MeCP2?

Answer 26

Humans with mutations in MeCP2 exhibit Rett syndrome, a developmental disorder causing mental retardation.

Question 27

What is the effect of acetylation as a histone modifier?

Answer 27

Acetylation loosens histone-DNA interaction and improves access to activators.

Question 28

What recognizes acetylated histones?

Answer 28

Bromodomains.

Question 29

What is the structure and mechanism of Polycomb proteins?

Answer 29

Contain histone methyltransferases that methylate histone 3, lysine 9, causing closed chromatin structure.

Question 30

What do Trithorax proteins do?

Answer 30

Demethylate H3K9 and H3K27, leading to loose chromatin conformation.

Question 31

How do polycomb proteins cause closed chromatin?

Answer 31

HP1 binds to methylated H3K9 and recruits HMT that methylates H3 on adjacent nucleosome, spreading tightly packed structure.

Question 32

How do histone proteins interact with each other?

Answer 32

Single protein can recognize multiple histone modifications, and multiple proteins can bind to single histone modifications.

Question 33

What do regulatory proteins recognize in the histone code?

Answer 33

A pattern of histone modifications rather than a single modification at a site.

Question 34

What is mono-allelic gene expression?

Answer 34

Occurs during embryogenesis with exclusive expression of certain genes from one parent only.

Question 35

How do histone modifications impact DNA modifications?

Answer 35

Some regions contain methylated H3K4, which protects the DNA from being methylated.

Question 36

What is the role of regulatory RNA and PRC2?

Answer 36

lncRNA recruits polycomb repressor complex 2 (PRC2) which methylates histone and results in DNA methylation. -lnRNA also recruits dnmt1 or dnmt3a/3b which results in DNA methylation and closed chromatin conformation

Question 37

What is the effect of regulatory RNA on the CEBPA gene?

Answer 37

lncRNA binds and sequesters Dnmt1, preventing access to the CEBPA gene and promoting open chromatin structure.

Question 38

What is the effect of X-inactivation on chromatin structure?

Answer 38

Inactive X-chromosome is highly condensed and exhibits histone methylation, resulting in a Barr Body. -60 of 61 cpgs are methylated in pkg1 gene -inactive X gene exhibits histone methylation of H3K9 and H3K27 as well as ubiquination of H2A and reduced acetylation

Question 39

What is the role of regulatory RNA in X-inactivation?

Answer 39

lncRNA Xist binds and spreads across the inactive X chromosome, recruiting PRC2 and converting X to heterochromatin. -It also associates with the nuclear scaffolding protein HNRNPU ( which attaches chromatin to nuclear structural components)

Question 40

DNAsel technique

Answer 40

blotting experiment Chromatin from two tissue types Two genes probed – one active in each tissue Active genes digested first Methylated DNA associated with closed chromatin conformation