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Slide
1

Epigenetics in Development & Disease

louise
...
ac
...
(Waddington, 1953)

Conrad Waddington’s Classical Epigenetic Landscape

Epigenetics is above- genetics
...

Aristotle used the word epigenetics first
...

The ball can represent a (pluripotent) cell and at points decisions
are made concerning the path of differentiation - this is not
reversible and the lowest point represents the differentiated cell
...


1

Slide
6

Loss of Potency

Pluripotent Cell

Shows a series of valleys, being the different cell lineages
...

Normally a cell would not switch fate- reverse back to become
more potent or switch to another cell type
...


Same genotype – different phenotypes

Slide
7

What is Epigenetics? (post-Waddington)
• A collection of biological/genetic phenomena that were hard

From Powerpoint:
Epigenetics is often described as acting as a bridge between
genotypes and phenotypes

to explain using standard Mendelian genetics

Slide
8

Position Effect Variegation (PEV) (Muller, 1930)
• X-ray mutagenesis of Drosophila
• Identification of white eye colour mutation
• Unusual patchy phenotype

Something is influencing expression of the white gene

Slide
9

Chromosomal rearrangement produces Position Effect
Variegation

Position effect variegation
...
He found mutation called the
white eye mutation: normal drosophila are red, but the mutants
have a patchy white/red phenotype, so some of the cells in the
eye are still able to make that red pigment but a lot of them are
white
...

Drosophila genetics- gene names are always named after the
mutant phenotype
...

Chromosomal rearrangement produces Position Effect
Variegation
...
So normally eye is red
...
This is how you get the patchy phenotype
...

If you sequence the white gene in both of these cases, the white
gene wouldn’t have any DNA based mutations-its all to do with
how the chromatin is packaged and its switched off the gene
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expression
...
g
...

This is oversimplified, maize genetics are complicated!
R= influency pigmentation of leaves
...
RR=green
...
So the Rst in the
homozygote state the leaves have a kind of stipled mottled
appearance whereas the RRr are just green
...
Think
about it in mendelian genetics, it would indicate st allele is
dominant
...
Even the homozygous that
before had interacted with the st allele! If you then self cross
these and take their progeny
...

They do revert occasionally
...
This mutation is very unusual- almost like th
allele interaction has imparted some information or a change in
gene expression/phenotype in the RR allele
...
Gains a pattern of methylation that’s
propagate through meiosis and mitosis
...

From Powerpoint:
R-st is able to induce a change in R-r gene expression that is
“stable”

Slide
11

Epi-mutants eg
...


with a wolf’s head”

Linnaeus (1707-1770)

peloria

Linaria

Linaria has bilateral symmetry (cut down middle and they’re
symmetrical)
...
If we
looked at that now, we might say its just a mutant that’s effected
the flowers
...
The peloria mutant of Linaria vulgaris

cf
...
Nature 401:157-161
...
Antirrhinum has this bilateral symmetry, they
identified mutant cycloidea gene which has radial symmetry and
said that peloria must be a mutant in the cycloidia gene
...
They found it was heavily DNA methylated
...

From Powerpoint:
The cycloidea gene in the peloria ‘epi-mutant’ is heavily
methylated – resulting in repression of gene expression and
thereby appearing as a loss-of-function mutation in the cycloidea
gene
...
Vol 468, Outlook supplement S20

This example relates to transgenerational stress responses
...
Also, their own offspring also suffered the same
problems
...
So what has been correlated
in these cases that theres DNA methylation changes in some key
metabolic gene –insulin growth factor is methylated
...
This can have implications for
metabolism and disease
...

Heijmans et al (2008)
...
Proc
Natl
...
;105(44):17046-9
...


DNA sequence
...
1996)
• Relates to the concept of cellular memory

Slide
15

Read slide
...


Epigenetic Mechanisms
What’s required?
• Mechanisms to create specific “expression states” that
result in differential gene expression
• Mechanisms that allow these expression states to be
maintained during cell division and development

Slide
16

Seen in previous modules
When the signal is removed, the gene expression status reverses
to its prior state (transient)
...


OFF
signal

signal
OFF

ON

ON
signal

Slide
17

“Epigenetic” re-programming of gene expression
signal
ON

OFF

Whereas epigenetics reprogramming requires once the signal is
removed, the signal pattern expressions stay on or off
...


ON
signal

signal
ON

OFF

OFF
signal

Slide
18
The Players

Whats involved in these mechanisms? DNA methylation- the
classic epigenetic mark
...
The
complication in all of this is they don’t act in isolation
...
Appreciate interplay between different
marks
...


5

Slide
19

Need to consider:
• How the epigenetic mechanisms influence gene

expression
• Whether the epigenetic marks are heritable (mitotic

How mechanisms influence gene expression
...

Added notes in red
...

In plants, more survive (more developmentally flexible) but
profoundly developmentally compromised
...

Methylations two main roles: control gene expression and
defence against invasive nucleic acids eg transposons
...


Slide
21

Evolution of Eukaryotic DNA Methylation Patterns

The fungi who do methylate (not all), do it to suppress
transposons- not to control gene expression
...

Plants/organisms are the most heavily reliant on methylation
...


6

Slide
22

The reaction that adds the methyl group to cytosine uses SAM as
the donor for lots of methyl transferase reactions in cells, not just
the donor for DNA
...

Sam adds methyl group to the 5’ of the carbon- 5-methylcytosine
...

From Powerpoint:
SAM acts as a methyl donor in many cellular reactions
...
This is important in early mammalian
development
...
Ive said that where the
methyl group is on the DNA is accessible to DNA binding protein
so perhaps what the methylated cytosine is doing is blocking the
binding of the TF
...
This is the minor route, of how it blocks transcription
...

From Powerpoint:
Direct blocking of transcription factors is thought to be the less
common mechanism

7

Slide
25

Kass et al
...
DNA
methylation directs a time-dependent repression of
transcription initiation
ptk

CAT

So we’re going to look at the experiments that went to show to
demonstrate that this is the case: linking methylation with change
in chromatin
...
So what they used in their
experiments was xenopus oocyte as their expression system
...

Look at chimeric constructs, what happens if the promotor of the
constructs is methylated or not
...
And then they’ve taken a promotor- all the regulatory
regions where RNA polymerase would be binding and they’ve
taken this from the herpes simplex virus, HSVtk driving CAT
...


Slide
26

tk promoter methylation reduces transcription…
...
1997

What they’ve got here is a time course- 30minutes to 12 hours
...
What
they’ve also injected in the experiments is a control piece of DNA
that’s not been methylated that’s the same in all samples- the
CMV is the control, unmethylated DNA
...

What you see here is an RNA blot- measuring expression
...
Whereas the unmethylated construct expresses very
nicely
...
So what does that implicate
(that it occurs but not immediately)?
Went back to slide 24 one thing it tells you is its not a direct
block- if you you’d expect to see that immediately that there was
a direct difference
...

From Powerpoint:
Experiment is measuring RNA levels of the CAT gene under
control of the tk promoter (HSVtk) and using the CMV construct
8

as a control
...
So they saw this
repressive effect tk promoter DNA methylation
...


plus

competitor DNA
or

either unmethylated or

mmmmmmmmmmmmmm

methylated

Slide
28

Repressive effect of tk promoter DNA methylation can be
removed by adding methylated competitor DNA

Kass et al
...
Can you compete out this repressive effect
that they’d seen in the first experiment?
Shows they’ve got increasing amounts of competitor DNA (016ng), have original injector pairings (methylated or non),
competitor DNA (methylated or not)
...
If the competitor DNA is unmethylated Competitor
experiment by adding competitor ‘sequestering’ this takes time
to happen, needs something else eg proteins transacting factor
...
Therefore, what
even required is in limited quantities
...
Then they did the same experiment, but extract DNA
out, looking at structure of DNA (not expression, but how packed
etc)
...

i
...
1997

The nuclease treatment is nonspecific, if in more compact
structure, finds harder to cut, cuts loose DNA easily
...

In the methylated sample you get higher weight bands, the size
of DNA wrapped around nucleosome, so see mononucleosomes,
shifted
...

Links DNA methylation with changes into DNA structure
...
Its activity will be affected if access to the DNA is
restricted – ie if the DNA is tightly packaged within chromatin
...

6 obvious methylation binding proteins
...

They generally have methylation binding domain
...


MBD: Methylcytosine-binding domain, TRD: Transcriptional Repressor Domain

MeCP2, Sin3 and histone deacetylase activity co-purify
Fractionation of Xenopus oocytes by ion exchange chromatography

MeCP2
Sin3

histone deacetylase activity

Slide
32

Jones et al
...

Nature Genetics
...

MeCP2- identified as likely DNA methylation binding protein
...

Another activity known to be involved in repression is histone
deacetylase activity (HDA)
...

Take xenopy, fractionate cell, extract by ion exchange
chromatography
...

From Powerpoint:
Sin3 was already known as being involved in repression of gene
expression as was histone deacetylase activity
...
Pull out sin3 from MCP2
...


(TAS) relieves transcriptional

immunoprecpitate

repression

Jones et al
...


They used chemical TAS (an inhibitor of deacetylase activity
...


HKMT: Histone
lysine

Methyltransferase

Sin3a

HDAC

Inhibition of transcription by DNA methylation

HKMT

Slide
34

MeCP2 recognises methylated DNA and recruits the
sin3A/HDAC (usually active genes are acetylated) these
modifications can be effected, effecting chromatin structure
...
Results in a change in chromatin structure

Effecting methylation impacts chromatin structure, actylation is
important for gene expression
...
Also recruits histone methyl transferase- (adds
repressive marks)
From Powerpoint:
MeCP2 is a complex protein
...
It is highly abundant in neurons
(at levels approaching that of histones)

Slide
35

Rett Syndrome

Is an autistic spectrum disorder but very serious
...
bio
...
ac
...
The link has
videos of his MeCP2 knock-out mouse in which theRett
Syndrome disease phenotype can be reversed
...
The
asymmetrical site
...


How are DNA methylation patterns inherited?

*

---------CG------------------GC----------

*

DNA replication

*

---------CG------------------GC---------+

Hemi-methylated

---------CG------------------GC----------

*

Slide
37

Get hemimethylation state, enzymes recognise this as substrate
...

See echo/see previous slide
...

Proofreading mechanisms to replace methylation marks when
they’ve been missed
...


Requires the action of a de novo DNA
methyltransferase to re-establish methylation pattern
...


Slide
40

Two waves of epigenetic re-programming during
Mammalian development

paternal
maternal

imprinted
genes

If we follow what happens to DNA during development
...

Have maternal and paternal and the imprinted genes
...
It’s a very dynamic process, genome
demethylated in active fashion
...

4- during development, germ cells
...

Maintenance- once established cell division requires
...
They can oxidise 5ce-tetize to 5 hydroxy cytosine

5-hydroxymethylcytosine

• Ten-eleven translocation (TET) enzymes oxidize 5mC to 5hydroxymethylcytosine (5hmC)

From Powerpoint:
Enzymes discovered in 2009 and has been an area of active
interest since
...


5-hydroxymethyluracil

Base excision repair route
...


Deamination

5-formylcytosine

5-carboxylcytosine

Williams et al
...
13, 28-35

Slide
44

From Powerpoint:
5hmC abundance is highest in the brain and central nervous
system
...
Suggestion that it is
an active mark
...

Song & He (2013) Potential functional roles of DNA
demethylation intermediates
...
sciencemag
...
Nature 447, 396-398
• Law & Jacobsen (2010) Establishing, maintaining and modifying DNA
methylation patterns in plants and animals
...
Proc
...
Acad
...
USA 110,
7101-7103

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