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Slide 1

Dr
...
jones@york
...
uk

Epigenetics in Development & Disease
Lecture 2:

• DNA methylation (continued)
• Histone modifications

• Non-coding RNAs

Slide 2

Learning Outcomes: L2
By the end of this lecture students should be able to:
• Provide an overview of the DNA methylation profiles of different
organisms
• State what are CpG islands
• Describe current ideas regarding how histone modifications

Missed slide: have our mechanisms, they don’t act in isolation,
theres a lot of interplay between the mechanisms
...
Todaysome examples that will go the other way
...
Usually acts in an indirect way recruiting
other proteins so it can be recognised by 5-methyl-cytosine
binding proteins, you can promote a closed chromatin structure
as it’s a modification on the DNA itself-->stable through DNA replication (although we did see it was a
dynamic mark- can be lost through failure to maintain it or there
are those chemical modifications) that can result in active
demethylation
...
Its associated with both
of control of gene expression and control of genome defence
...
(2008)

Now we have capability of looking over whole genome and
saying which regions of genome are methylated
...
Different
tissue types have been compared, or different points in
development
...

Mosaic DNA methylation pattern- in fungi neurospora
...

The grey is where the DNA methylation
...

If we look at plant (Arabidopsis well studied)n it has a more
complicated mosaic pattern, virtually all its transposons are
methylated but if we look at the genes we see the mosaic
pattern quite obviously but some genes are methylated in their
gene body but some are not
...
Some ideas are that methylation in the gene body
are to do with attempts to suppress spurious types of
transcription- not much evidence, just a theory
...

Compare this to organisms that have a global methylation
pattern- eg humans
...
Most of those are methylation- almost like the
default state is methylated
...
Have gaps here, not
for all genes but for many
...
Its not
that different to the other plant Arabidopsis
...
Looks really heavily methylated even
though just as have loads of transposons
...
DNA methylation landscapes: provocative
insights from epigenomics
...
9:465-76

2

Slide 6

DNA methylation in Mammals
• Absolutely required for correct development

Focusing on mammals- when talking about development later
mainly on mammalian development
...
If can’t
methylate, is lethal! Although we’ve got global pattern there’s
tissue specific differences
...


unmethylated regions correspond to promoters of genes

Mainly there but increasing evidence for cytosine’s on a nonsymmetrical configuration so “CHH (H = any base other than G)”

...
For a lot of this we observe it but don’t understand
the significance of it
...

From powerpoint:
CpG (p = phosphodiester backbone)
CHH (H = any base other than G)
(Dec 13) paper in Nature Neuroscience by Guo et al
...
PDF in resources section on VLE
site
...
et al
...
Nature 462, 315–322 (2009)
...
If we looked in a typical gene, so
these lollypops are CpGs
...
The dense region of cpgs
are islands
...

If these become methylated this will switch a gene off
...
This event usually
associated in some key developmental genes
...
Lots of genes are turned off
during development but not all are talking about this switch
...

3

If we look at mammalian genome and think about number of cgs
would predict, we have far less than you’d predict based on the
actual numbers
...
This might be important in terms
of which organisms have kept this and which have not
...
So if this is methyl cytosine,
when it gets de-aminated it basically goes to thymine
...

But methylcytosine thymine isnt necessarily recognised
(although is as a mismatch) but its not recognised, it can
become fixed in a genome
...
So if
most of our CpGs are methylated, over time they become
changed to thymine
...
So in areas where most are
unmethylated, they’re kept unmethylated by another (not very
well understood) mechanism, these are not mutagenic
...

Doesn’t explain if we’ve got a default state of methylation of why
these stay unmethylated
...
Lots of CpG islands of key regulators of
development are methylated during development
...

Slide 8
Control of CpG island methylation
• Some CpG islands remain unmethylated throughout
development
...
What directs these changes?
• Aberrant CpG methylation can lead to disease

•Some CpG islands remain unmethylated throughout
development
...
And there’s evidence that are active demethylation
pathways that are active at these places
...

What directs these changes?
• Aberrant CpG methylation can lead to disease
From powerpoint:
All three of these points will be revisited in later lectures
...

Techniques that are valid but not high resolution is Using
Methylation-Sensitive restriction enzymes (combined with PCR,
or Southern blotting for example)
...
So you can
use techniques- the enzymes linked with techniques such as
PCR or southern blotting
...
But you’re limited to the
restriction enzyme site, which is valid but you can’t get a whole
genome view
...
Have antibodies that will specifically
recognise those modifications
...
So once pulled down-immunoprecipitated DNA
...

Or you can use NGS, sequence all the DNA you’ve pulled down
with the antibodies- which regions of the genome have been
enriched with the antibodies
...

You’d just know in that region theres cytosines that have that
mark
...

From powerpoint:
Next Generation sequencing: Illumina (or similar) highthroughput methods that are applicable to large and complex
genomes such as those found in vertebrates and plants
...


5

Slide
10

Bisulfite sequencing
• Treatment by sodium bisulfite followed by desulfonation
• Cytosine converts to uracil
• 5-methylcytosine is unaffected
• Sequencing will reveal C-to-T transitions for unmethylated cytosines

Does not distinguish between 5-methylcytosine & 5hydroxymethylcytosine

Way to get single base resolution: bisulfite sequencing
This is really common
...

Its treatment of sodium bilsulfide followed by desulfonation
...

It’s a sulfonation and deamination and then deamination
...
CT transition
If you’ve got 5’methyl cytosine, its resistant to that, it stays a
cytosine
...

This technique has been used for a long time-issues recently:
before had no idea of 5-hydroxy-methyl-cytosine and bisulfite
doesn’t distinguish between the two so if we have a base that’s
actually 5-hydroxy-methyl-cytosine it will also be read as
cytosine in the end
...
The 5
hydorxy is on the route to that
...
The problem is it looks like one is an
active mark and one is an inactive mark
...

From powerpoint:
The importance of 5-hydroxymethylcytosine is increasingly being
recognized and recently methods to distinguish between 5hmC
and 5mC have been developed
...
Theres a couple of different
techniques: one is TAB seq
...

So in the standard treatment the end would be red as this would
be converted
...
(2012) Cell 149, 1368-1380

The difference in the TAB seq is the pre treatment first
...
So
the methyl cytosine isnt labelled in that case
...

So the treatment: the hydroxy-methyl-cytosine that’s got the
glucose on it is unaffected by that
...
And once its done that and
then you do the bisulfite treatment, this is converted to uracil and
read as T, but other stays as cytosine
...

The unmethylated cytosine is always converted to thymine but
you really have to compare: the ones that stay the cytosine are
genuinely the hydroxy methyl cytosine whereas for the other you
see the difference
...
(2012) Base-Resolution Analysis of 5Hydroxymethylcytosine in the Mammalian Genome
...


HKMT: Histone
lysine

Methyltransferase

Sin3a

HDAC

Inhibition of transcription by DNA methylation

HKMT

Slide
13

Recruiting proteins are incorporated into the complex: histone
modifying enzymes
...


MeCP2

Region of
methylated DNA
MeCP2 recruits the Sin3a co-repressor and a Histone
deacetylase (HDAC)
...


7

Slide
14

Chromatin Revisited
• Basic unit of packaging is the nucleosome
• Octamer of 4 core histone

Got basic unit of packaging: nucleosome
...
Added notes to
slide
...

Can divide genomes into regions of heterochromatin (dense)
which can be constitutive or facultative
...

This is important- almost like structural features
...

Also have eurchromatin contains most of our active/inactive
genes
...
In terms of nucleosome
structure you see a very different arrangement in an inactive and
active region in terms of how those are spaced
...
There are modifications
that are associated with that transition
...

From powerpoint:
DNA packaging has a major influence on gene expression in
eukaryotes
...
Also within euchromatin – histone
modifications vary between active and inactive genes
...


Slide
16

Missing slide
Last week talked about position effect variegation– how
chromatin structure/packaging could influence expression of a
gene
...
These four histone particles,
if we look at how they’re arranged- they’re small so they're basic
which is good for packaging an acidic molecule like DNA
...


You get this core which is where the DNA is wrapped around
and have the tails- N termini of the histone proteins
...

Is where most of the critical modifications are happening (on the
tails), they’re positioned so proteins can recognise them
...


• acetylation (eg
...
The modifications are
dynamic, there’s enzymes that add them and theres enzymes
that remove them
...


• methylation (eg
...
Serine, S-ph;
Threonine, T-ph)

Modifications are DYNAMIC

Slide
19

Dynamic Histone Modifications

Histone Acetyltransferase
(HAT)

Ac

Histone Deacetylase
(HDAC)

Read slide
...
Dynamic
process
...

Doesn’t mean its the most important
...
Once you get that you get an escalation
of amount of information there
...

Look at N terminal tails, theres modifications known across
them
...
The methylation ones- there are active
methylation marks and repressive ones
...
In terms of an epigenetic code theres things
that read that code, interpret them and can distinguish
modifications that are very close on the proteins
...


Slide
21

Impact of covalent histone modifications on transcription
H3K4

mono-methylation

activate

tri-methylation

activate

H3K27

H3K79

activate

activate

repress

repress

activate

repress

repress

repress

activate

di-methylation

H3K9

acetylation

Introducing some nonclamenture and highlight some
modifications: often written like this, H3 is histone 3, K is lysine
in the 4 position
...


activate

nomenclature you may see in papers:
eg
...
A lot of
specificity here!
Don’t have to remember as table, but will know at end of module
whats active/repressive

Slide
22

How do histone modifications affect chromatin structure?
• Disrupting contacts between nucleosomes in order to
unravel chromatin
• e
...
acetylation of lysines neutralizes the basic charge

How do these marks result in a change in chromatin structure or
impact on gene expression
...

In terms of direct effect, one idea is the modifications are
disrupting the contact between the nucleosomes or between the
nucleosomes and DNA to unravel the chromatin
...
This is in terms of acetylation of lysines
...


Slide
23

Histone Lysine Acetylation
Lysine
N-C-C-

N-C-C-

CH2
CH2

CH2

CH2
acetylation
deacetylation

CH2

CH2

CH2

CH2

NH3 +

Have lysine, is normally positively charged
...
This is a dynamic change
...
Think about
this in terms of the packaging of DNA- histones are basic, but
here reducing basic charge, means DNA can’t wrap as tightly
around the nucleosomes, don’t have as strong attraction
...
People have engineered histone
proteins to reduce charge and even reducing single charged aa
has an effect!
Another thing to point out is the actylation is not on a single
lysine, but on multiple- not talking normally of a single change in
charge
...
(We don’t know where tails are really
...
g
...
The idea is the
modification of the histone tails are providing a code that’s read
by the other proteins
...
Theres various classes of proteins that will recognise
specific modifications- recognising methylation theres Chromo,
Tudor, PHD, MBT etc
...
And phosphates recognise 14-3-3 proteins
...

They’ve got to be proteins, not enough to read methylation mark
but have to be able to distinguish it at a certain point in the DNA
and see if its mono,di,tri etc
...

From powerpoint:
‘The histone code hypothesis predicts that the post-translational
modifications of histones, alone or in combination, function to
direct specific and distinct DNA-templated programs
...
’
Slide
25

Histone Methylation

Just having proteins there doesn’t tell you why it impacts
expression etc
...

Gonna focus on H3K9me typically on heterochromatin
...


11

Slide
26

HP1 and heterochromatin formation
• HP1 identified as being associated with heterochromatin

• Required for Position Effect Variegation
• Required for heterochromatin formation

• Recognizes H3K9me

Reading mark on heterochromatin is HP1
...
Its identified as being required for
heterochromatin
...
Identified
as being required for heterochromatin formation
...
Also has chromo shadow domain
involved in protein protein interactions
...

Slide
27

Mode of action of HP1 in heterochromatin formation

• HP1 interacts with a histone
methylase SU(VAR)3-9 and with
H3K9me

• in vitro studies suggest that HP1 molecules
dimerize via their chromodomains (CD) to
recognize H3K9me3 and contact adjacent
dimers via the chromoshadow domain (CSD)

If this is our nucleosome, and we’ve got our tail sticking outh3k9
...
The
chromoshadow domain interacts with histone-methylase
...

The other thing it can do is when the chromo domain is binding
the histone, the chromoshadow domain is also able to cause
dimerisation- can dimerise via that
...
A physical
brining together via chromo shadow domain which can then add
methylation marks
...

So it methylates and is compacting and does that until it reaches
the boundary element which blocks it
...

This is whats gone wrong in this Position Effect Variegation
...


Chromosomal rearrangement produces Position Effect
Variegation
boundary element

Lecture 1

In the invertion that’s happened (due to xray mutagenesis) the
boundary has been moved
...


12

A lot of these are HP1, they were looking for suppressors of this
heterophenotype
...


Slide
30
Reversal of crosslinking & purify DNA

Sequence
ChIP-seq

If we wanted to look at those histone marks either on a single
region of a genome or over a whole genome, what techniques
do we have available? We need to link the histones with a
particular region of the genome- requires us to do chromatin
immunoprecipitation
...
What will happen is we need to crosslink the histone with
DNA- formaldehyde, then shear the DNA (to make it possible to
do the immunoprecipitation) and we have antibodies against a
lot of the histone marks
...
And then we can use techniques to reverse the
cross linking once we’ve immunoprecipitated
...
Controls requirednot using the antibody or using it against something else
...

Probably less common is also to take the DNA amplify it and do
some hybridisation array so you can get a CHIP with all the
regions of genome on it and do a hybridisation
...


Slide
31

Histone modifications and Epigenetics

Read slide
...


• Strong evidence that histone modifications affect
gene expression
• Can patterns of histone modifications be
maintained through cell division? i
...


Histone modifying enzymes (HME) must be directed to

particular nucleosomes
...
Can be recruited by non
coding RNAs but can also be recruited by interaction with TFs
...
?

Diagram shows nucleosomes and histone modifying enzyme
...
Recruit the histone modifying
enzymes that will then have enzymatic activity on nucleosomes
closeby
...
But epigenetics requires a triggering event and
for that to be maintainable even in the absence of whatever
firwst triggered it
...
Here in
lies the controversy
...

Methyltransferase
complex is able to bind
to the remaining
modifications and
methylate adjacent
nucleosomes

Modification is lost after
DNA replication but the
methyltransferase
remains associated and
re-methylates
nucleosomes

Comment by Abmayr & Workman (2012) Cell, 150:875-877

Models for how we get modifications to be maintain in the
absence of whatever first triggered it, lots of events in early
development where there’s a transcription factor set up this
event but is only there very early in dveelopment but the actual
cell lineages stay on the right track (even in absence of initiating
event)
...
In this
case we’ve got a methyl transferase, gonna add methylate
histones
...
So what
happens after replication fork passes? We think: nucleosomes
get partioned roughly equally and then there’s mechanisms to
replace the gaps in the nucleosomes
...

There’s evidence that the enzymes that add methyl mark
14

(methyl transferases) is able to recognise the methylation mark
and methylate adjacent nucleosomes
...
This allows it to be
propagatable as long as the enzyme itself is able to recognise
that
...

The alternative is that when the replication fork passes, the
nucleosomes get partioned but the modification is lost
...
As long
as the enzyme stays and gets partioned its able to replace that
mark
...

First sounds more logical- why would it be removed to be
replaced? There’s evidence the enzymes will bind the marks
...

Good evidence that the enzymes stay associated with
nucleosomes, and the modifications are lost to be replaced
...

Slide
34

Epigenetics in action: The making of a Queen Bee
• Honeybee Queen and
workers are genetically

Going to illustrate example of how histone modifications can
effect DNA methylation
...


identical

• Queen larvae get fed royal
jelly throughout larval
development and into

Bees are an interesting model organism
...
Plos Biology 8, 1-4

All get royal jelly to an extent during early development but the
queen gets much more
...


Read slide
...


Slide
35

• Royal jelly contains phenyl butyrate, a histone deacetylase

inhibitor
• Inhibiting DNA methylation in larvae mimmicks the effects
of royal jelly

15

Slide
36

Come back to looking at lysine actylation
...


Histone Lysine Acetylation

N-C-C-

N-C-C-

CH2

CH2
acetylation

CH2
CH2

CH2
CH2

deacetylation

CH2
NH3 +

CH2
NH2 COCH3
• Open chromatin configuraion

- Repression of
transcription

• Loss of DNA methylation

• Activation of gene expression
inhibition of histone deacetylation by phenyl butyrate

Slide
37

Non-coding (nc)RNAs in Epigenetic Regulation
• both small (~21-24nts) and long (200 bp+) non-coding
RNAs have been associated with various epigenetic
phenomena
• Act to direct epigenetic change eg
...

Theres a link between effecting actylations status and this loss
of DNA methylation that results in difference in active gene
expression that will out a larvae on the path to being a queen or
not- due to extent of inhibition
...

Have small and long non coding RNAs
...

They can form fantastic secondary structures- so proteins can
not only recognise in a sequence specific way but in a structural
way
...

Small RNAs and how these can cause epigenetic change
...
Small RNAs come from dsRNA
...
These small RNAS form effector complexes- complex
with protein from class of proteins: argonautes
...


Slide
39

In plants RNA silencing pathways can direct DNA
methylation
dsRNA
Dicer-like (RNAseIII enzyme)
small RNAs

DRM2

AGO

Small RNAs form argonaute effector complex which is able to
recruit a DNA methyl transferase of this de novo class- one that
will methylate unmethylated targets
...
Going to go
through pathway
...
This longer
...

It will transcribe the region that needs to be methylated, and
provides a scaffold transcript
...

Have RNA RNA interaction and RNA protein interaction that’s
bringing in this complex
...
Not on CG
configurations, on any cytosines
...

In plants theres another plant specific polymerase- polymerase 4
which is also involved in transcribing this region
...
A self maintaining loop of small RNA production and
methylation
...
In regions where we think this should be
transcriptionally inactive
...

There is a change in chromatin structure but it stil allows these
polymerases to transcribe that region so it seems to produce a
structure that’s recalcitrant to transcription by polymerase 2
...
But this goes on! Theres
an equivalent in our germ cells, keeping transposable elements
off in our germ cells
...

This pathway is required for paramutation in maize that was
described in lecture 1
...

This secondary structure that can be created: theres examples
of different structures being recruited: the DNA methyl
transferases that are going to come in and methylate specific
residues, examples of repressive marks being put down
recruiting repressive complexes
Examples: hot air where it actually recruits two different
complexes, one that puts down repressive marks and one that
blocks active marks- two pronged approach
...
The ability of RNAs to
interact with both DNA and proteins and form extensive
secondary structure makes them ideal molecules to recruit the
enzymes and proteins that will trigger changes in chromatin
structure

Slide
42

Long ncRNAs can act in cis

Come back to when will give these examples
...
HOTAIR ncRNA that is
required for repression of HOX
gene targets
...


organisms
• State what are CpG islands
• Describe current ideas regarding how histone modifications

Lecture 2: Reading List

influence chromatin structure

• Kouzarides, T
...
Cell 128,

• Discuss whether histone modifications are heritable

693-705

• Describe how non-coding RNAs can direct epigenetic change

• Zentner & Henikoff (2013) Regulation of nucleosome dynamics by histone
modifications
...
20, 259-266

• Understand the basis of techniques that can be used to assess
epigenetic marks of single genes and whole genomes

• Fatica & Bozzoni (2014) Long non-coding RNAs: new players in cell
differentiation and development
...
15, 7-21

• Sabin et al
...
Molecular Cell
...
Genes &
Dev

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