Lecture 2 Flashcards

1
Q

Where does DNA methylation happen in mammals?

A

In all tissues - but methylation profile is tissue specific
Mainly on CG dinucleotides (CpG) but also CHH (where H= anything except G) in some tissues (brain, placenta, embryonic SC)

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2
Q

What are CpG islands?

A

Region (300-3000bp) in promoters with higher than average CpG content, found in 60% of genes, and are usually un-methylated, and hence the gene is active.

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3
Q

Why are methyl-cytosines considered more mutagenic?

A

SPpontaneous Deamination of 5-mC will lead to thymine which if not repaired will lead to a mutation. Whereas spontaneous deamination of cytosine leads to uracil which will be repaired by mismatch repair

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4
Q

What type of genes have their CpG island methylated during development?

A

Stem cell master regulators when becoming differentiated cells that need to lose their pluripotency

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5
Q

How do the different techniques for assessing methylation profiles differ?

A

Their resolution

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6
Q

What techniques can be used to assess DNA methylation profiles?

A

1) Using methylation-sensitive restriction enzymes and then PCR with primers for the intact product ; only tells you about that site
2) Using anti-methylcytosine and/or anti-hydroxymethylcytosine antibodies to immunoprecipitate methylated DNA followed by next generation sequencing; only know that some site within that fragment is methylated
3) Bisulphide sequencing; Treat with sodium bisulphite and then use desulphonation (Deaminated, sulphinated and desulphinated). Cytosine converted to uracil and methyl-cytosine not affected. Sequencing will reveal C-to-T transition for un-methylated cytosines. Cannot distinguish between 5-mC and 5-hmC

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7
Q

How can you distinguish between 5-mC and 5-hmC in bisulphite sequencing?

A
TET-assisted bisulphite sequencing 
Glucosylate the 5-hmC 
TET oxidation of mC to caC
5-mC read as T whereas 5-hmC read as C
Combine TET-assisted bisulphite sequencing and bisulphite sequencing to discover which are 5-hmC and which are 5-mC
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8
Q

What are core histones?

A

Four small and basic particles (H2A, H2B, H3 and H4), with C-terminus core with DNA wrapped round and N-terminus tails

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9
Q

What post-translation modifications can occur to histones?

A

over 100 exist however not all effect DNA expression

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10
Q

What effect does methylation of histone 3 have on gene expression?

A

Can be both repressive, e.g. Lys4, and activating, e.g. Lys 9.

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11
Q

How can post-translation histone modification affect chromatin structure?

A

Two possibilities

1) Disrupting contacts between nucleosomes in order to unravel the chromatin, by lessening the interaction between the histone and the DNA - e.g. acetylation of Lys neutralises the basic (positive) charge
2) Recruitment of non-histone proteins (Histone code hypothesis)

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12
Q

How does Epigenetics effect Honeybee Queens?

A

Queen larvae gets fed royal jelly throughout development
Royal jelly contains phenyl butyrate (a histone de-actetylase inhibitor)
Leads to chromatin being more open, loss of DNA methylation and more active genes
Can be mimicked by giving larvae methylation inhibitors

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13
Q

Why does opening the chromosome decrease methylation?

A

Activating the gene expression causes other machinery to bind blocking access to methyltransferases
Chromatin more open for demethylating enzymes to act.

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14
Q

Give some example proteins that can recognise different modifications to histone tails according to the histone code hypothesis

A

Methylation: Chromo, Tudor, PHD and MBT
Acetylation: Bromo
Phosphorylation: 14-3-3

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15
Q

What effect does methylation have on the histones?

A

Does not affect charge
Recruits proteins to affect chromatin structure
Associated with gene activation (H3K4me and H3K36me) and repression (H3K9me and H3K27me)

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16
Q

Which protein recognises H3K9me?
What is it involved in?
What do its domains do?

A

HP1 protein
Required for position effect variegation (drosophila eye)
Required for heterochromatin formation
Two domains: Chromodomain and chromoshadow
Chromodomain recognises H3K9me
Chromoshadow domain recruits methylase to methylate other H3K9 sites - how heterochromatin spreads
Chromodomains and shadow chromodomains dimerize with adjacent HP1 proteins for heterochromatin formation

17
Q

How is heterochromatin spreading usual stopped?

A

Boundary element separates the hetero- and eu-chromatin

As seen in the drosophila - which is lost due to the inversion

18
Q

How are the nucleosomes dealt with for DNA replication?

A

The nucleosomes are spread between the new daughter strands roughly equally

19
Q

What happens to histone modification after replication?

A

Two schools of thoughts:

1) Modifications are partitioned and the gaps are filled in by methyltransferase complex - which binds to remaining marks and fills in gaps
2) Modification is lost after replication but the methyltransferase stays associated and re-methylates nucleosomes after

20
Q

What are non-coding RNA’s?

A

RNA’s that do not code for proteins
Small (~21-24 nts) and long (200bp+)
Act to direct epigenetic change - recruit DNA methyltransferases and/or histone modifying enzymes

21
Q

Outline the core RNA silencing pathway

A

dsRNA cut by Dicer-like (RNAsell enzyme)

sRNA produced which interacts with Argonaute(AGO) proteins

22
Q

How does the RNA silencing pathway direct DNA methylation in plants?

A

sRNA+Argonaute can recruit DRM2 (a de novo DNA methyltransferase)
The sRNA then directs sequence specific DNA methylation with the aid of PolV, by binding to the PolV product and bringing the DRM2 close to the DNA
The DNA is then transcribed by PolIV and RNA-dependent RNA polymerase 2 (RDR2) forms dsDNA which makes more sRNA for this region of DNA (+ve feedback loop)

23
Q

What previous example is explained by the RNA silencing pathway for DNA methylation?

A

The paramutation is Maize

24
Q

What role do long non-coding RNAs play in chromatin remodelling?

A

As they are longer can form complex secondary structures and act as scaffolds for chromatin remodelling complexes
e.g. HOTAIR - repressing gene expression; PRC2 binds and adds H3K27me (repressive) and LSD1/CoREST binds and removes the H3K4me (activating)

25
Q

What types of methylation pattern can be seen? and in which organisms?

A

Mosaic; fungi for transposons, plants for transposons and genes, animals for mainly genes
Global; animals and plants: with the promoters being the regions that are un-methylated