27th Annual Conference

Epigenetics 101

or

How does Rett Syndrome arise

from the mutation

Christopher Wynder

Rett syndrome molecules

• 3 genes have been clinically implicated in

Rett syndrome: MeCP2, CDKL-5 & FoxG1

• FoxG1 is a very rare cause of Rett

syndrome (less than 1% of cases)

• 95% of all Rett syndrome cases are

caused by mutations in the MeCP2 gene

• These mutations effectively stop MeCP2

from doing its job

A step back: what is DNA and how

does it relate to genes

A gene is a DNA word

Word: a series of

letters that have a

meaning.

DNA is the

material that is

passed from

parent to child.

DNA is a 4

letter code that

provides the

parts list and

instruction

manual for life

T

AC

G T

AC

G

C G C G

P P PP P

Genes

• Genes are words.

• As with language a word by itself is not

very useful.

• However when mixed in the correct order

(grammar) they can be very powerful.

• When used inappropriately they can

change meaning or be meaningless

Gene grammar• There are approx. 15 000 genes.

• Each one has a specific use and time when it should be

used.

• In scientific parlance gene grammar is called

“Epigenetics” from the greek “epi”-above.

• In Epigenetics, we study how genes are organized and

how a cell decides which ones to use at a particular point

in time.

See Jane Stop make Run destroy brain Skinheart jeans workcells

See Jane Stop make Run destroy brain Skinheart jeans workcells

Organizing 15000 genes

Purified DNA from a cell1. Organization of the genome

1. The length of DNA from a cell is

about 2 metres (6 feet)

2. The size of a cell is about 100 mM (1

millionth of a metre)

3. The cell needs a mechanism to

package DNA to fit it into its nucleus

1. The cell packages its DNA into

organizational units

2. Each unit can be further broken

down to further organanizational

units

Compacting and organizing the genome

• Cells use special proteins called histones to compact DNA into manageable size.

• These proteins are part of the epigenetic machinery

What is MeCP2

• MeCP2: Methyl CpG binding Protein #2

What does the name tell us?

1. It’s a protein

2. Its job in the cell is to bind “stuff”

3. The thing that it binds is “methylated”

DNA

What is Methyl-CpG?

• CpG: is shorthand used to describe a

special “kind” of DNA sequence

• This DNA is a series of C’s and G’s [i.e.

CGCGCGCG]

• Methyl CpG sequences occur in discrete

“islands”

• CpG “islands” appear to be used to

regulate gene expression

C G C G

P P PP P

Grammar and Access

• To use the words (i.e. genes) the cell must

move the histones.

• There are times when certain words just

cannot be used.

• These words receive a methyl “bookmark”

• Hence methyl CpG

• MeCP2 binds to meCpG to act as a glue

to ensure that word is not used.

MeCP2 & Epigenetics

The big picture questions:

How does the cell decide who to bookmark?

Answer: Through other sets of bookmarks and readers

What does this mean for a cell?

Answer: Each cell can have a unique set of words that it uses

Epigenetics is the study of what the bookmarks are and how the cell responds

Epigenetics:so what

• Epigenetics is

responsible for the

variation seen in

nature amongst

close relations

Epigenetics also

explains how dogs

have so much

variation

Female specific Epigenetics

• Calico cats are always female.

• All cells in their fur have the same colour genes.

• But they don’t have access to all of them.

• This is because fur colour genes are on the X chromosome.

• Each female has 2 X chromosomes.

• Some genes on the X need to have low expression.

• Therefore 1 copy of the Xchromosome is turned off-“inactivated”

X inactivation and DNA methylation

• X inactivation

spreads by a

molecule known as

Xist.

• Xist acts as a guide

telling the cell

where to put

methly-CpG.

Me-CpG

MeCP2 MeCP2

MeCP2 is a X chromosome gene

Random Inactivation

2 copies of fully functional MeCP2

Doesn’t matter which X is inactive

The cell can do all of the normal

MeCP2 tasks

Random Inactivation

1 copy of fully functional MeCP2

In cells with the MeCP2 inactive

some functions are lost.

This cell specific

brainlung

Heart

MeCP2

In the Brain

During development

Brain

So what: What does MeCP2 do

in a cell?

Controls access to the instructions to different tissues

Neuron

(brain cell)

So what: What does MeCP2 do

in a cell?

Tu parle

francais?Do you

speak

english?

Yes

MeCP2 turns genes off

DNA in the cell is ALWAYS found in

association with histones.

The cell takes advantage of this to

set bookmarks and reminders

(e.g. methyl CpG)

There are another whole set of

bookmarks that occur on the

histone proteins themselves.

These bookmarks are put onto

place by enzymes. These enzymes

are the writers and erasers of

epigenetic regulation.

They are also a source of possible

therapeutic targets

(making a gene)

Example of how Histone modifications

relate to MeCP2

HAT

HDM

MeCP2

• Writers:

1. HAT

2. HMT

• Erasers:

1. HDAC*

2. HDM

• Readers:

1. HP1

2. MeCP2

Why target these proteins?

HDAC inhibitors- stops inappropriate bookmarking of needed genes

(already FDA approved for cancer, 1 is a anti-epileptic)

HDM inhibitors-stops inappropriate use of specific words and chapters

The biology suggests that these may be able to “replace” MeCP2 lost function

Certain HDM inhibitors mimic

MeCP2 function

meH3K4

meH3K27

Requires a HDM 1st

Then a HMTinhibitor HDM

Normally blocking this

HDM is MeCP2’s job

Another strategy is to “turbocharge” HDMs that remove meH3K4

Why is RTT mainly the brain?

• MeCP2 is part of a family.

• This whole family does the same job.

• Each family works in a specific tissue.

• Similar mutations in the other family members cause specific types of cancer or “wasting” disorders not related to Rett syndrome.

How does MeCP2 effect the brain

function?

• Through it’s job as a reader of

epigenetic bookmarks

• The wide array of functions that

MeCP2 performs ALL

contribute to Rett syndrome.

• The different mutations have

different effects on the

presentation of the disease.

• In addition since each person is

different based on their

personal epigenetics, the

disease will be individual as

well.

Change its ability to “see” meCpG

Genes do not get turned off

appropriately

Change its ability to interact

with the writers and erasers

Genes do not stay off

MeCP2 has multiple jobs in the

brain

Undecided

cell

During development, MeCP2

has role in what kind of neuronsIn the brain, MeCP2 has role in

how neurons communicate with

each other

Each different neuron uses different genes

To define themselves.

In the absence of MeCP2 individual

neurons “talk” with a different language

than their neighbours

How do we “know” these things?

• Mouse work and genetic sequencing by

pioneers such as Adrian Bird and Huda

Zoghbi i.e. “in vivo” models

• Recently a new model has surfaced:

iPSCs-induced pluripotent stem cells. An

in vitro model

In vivo- used to describe research done in a whole organism

(e.g. in a mouse mutant)

In vitro- any other model including cells in a dish.

What are Stem cells?

• Stem cells have the ability to differentiate into all cell lineages and self renew

• Recent advances have allowed us to convert skin into stem cells

• Since Rett syndrome mutations happen in all cells we can take skin and make stem cells by epigenetically re-programming the skin

• iPS can be used as a model system to monitor neural differentiation to test where the errors are and possibly what effect therapeutics have on these errors

Endoderm

Mesoderm

EctodermNeurons

RBC

Skeletal system

Pancreaticcells

Skin cells

Stem cell

Stomach cell

Modeling Rett syndrome in human

cells

In vitro model confirms mouse

models

Funded by IRSF

Current therapeutic models

Epigenetics and RTT

• MeCP2 is a epigenetic regulator

• It has diverse jobs in brain cells

• Some of these jobs involve other

epigenetic regulators

• New models based on new technologies

are showing a possible role for novel

therapeutics based on epigenetics