Gene Expression 5 -

Epigenetics

Isobel Gowers
Post-doctoral Research Associate in Molecular
Medicine
 2

Learning Objectives
After this lecture you should be able to explain the
following terms/procedures:
• Epigenetics
• Heterochromatin
• Euchromatin
• Histone acetylation
• Histone methylation
• DNA methylation
• ChIP assay
• Bisulphite modification

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 3

Genetics

• The study of heredity and the

variation of inherited characteristics

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 4

Epigenetics

• The study of changes in gene

function that occur without a change
in the sequence of DNA

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 5

The importance of
epigenetics
• X-inactivation
• Imprinted genes
• Cell differentation
• Complex diseases

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

Chromatin and it’s Role
in Gene Regulation
 7

Heterochromatin and
Euchromatin
• Heterochromatin - condensed chromatin
containing mostly inactive genes
• Constitutive - fixed and irreversible in form
• Facultative - has the ability to return to
euchromatic state
• Euchromatin - genetically active portion
of chromatin, uncoiled and dispersed
during interphase

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 8

Chromatin Structure

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 9

Nucleosome

Nucleosome contains 4 heterodimers of histone core

proteins. Histone tails have been removed and
heterodimers differentially coloured for clarity
11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5
 10

Histone Tails
• 25 amino acids at amino-terminal of
each of the 8 core histones
• Highly conserved
• Positively charged
• Subject to wide variety of enzyme
catalysed post-translational
modifications
11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5
 11

Histone Modifications
• Acetylation
• Methylation
• Phosphorylation
• ADP-ribosylation
• Ubiquitination
• Isomerisation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 12

Histone Acetylation
• Ubiquitous post-translational
modification
• Acetylation of -amino group of
specific lysines
• Acetate group from acetyl-
CoenzymeA

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 13

Histone Acetylation
• Reversible
• Histone Acetyl Transferases (HATs)
• Histone Deacetylases (HDACs)

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 14

Histone Acetylation

HDAC

HAT

Histone octomer Acetyl group

DNA
11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5
 15

Histone methylation
• Methylation also occurs on -amino
group of lysine
• Does not remove positive charge
• 1,2 or 3 methyl (CH3) groups can be
added to each -amino group
• The methyl donor is S-adenosyl
methionine

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 16

Histone methylation
• Methyl transferases have been identified that
add methyl groups at specific residues, i.e.
Set9 H3 Lys4, Suv39h H3 Lys 9
• Histone demethylases remove methyl group
• Histone methylation can be a mark of both
active and inactive chromatin (Lys4 active,
Lys9 inactive)

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 17

Histone Phosphorylation
• Histones are phosphorylated at selected serine
and threonine residues
• Most extensively phosphorylated in H1
• Ser 10 phosphorylation is widespread at mitosis
(associated with condensation of chromatin-
heterochromatin)
• On stimulation with growth factors increased
Ser10 phosphorylation at promoters of genes
activated (in this case associated with acetylation
and active chromatin-euchromatin)

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 18

H3 modifications in
mammals

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 19

Heterochromatin
• Absence of acetyl groups at the amino
termini of histones H3 and H4
• High density of methylated CpG
dinucleotides
• Dimethylated or trimethylated lysine 9
residues on histone H3
• Methylation of H3 lysine 9 provides a
binding site for members of
heterochromatin protein 1 (HP1) family
11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5
 20

Active genes
• Presense of acetyl groups at the amino
termini of histones H3 and H4
• Dimethylated or trimethylated lysine 4 and
36 residues on histone H3
• Phosphorylation of H3 serine 10 at the
promoter of the active gene

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 21

Rheumatoid arthritis (RA)

• Inflammatory disease
• Genetic factor
contribute around
50% of excess risk
• Complex diseases
cannot be explained
by Mendalian
genetics

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 22

Hypothesis
• TNF along with other inflammatory
cytokines are involved in RA
• Dysregulation of TNF during RA is
important in the pathology of the disease
• We hypothesize that TNF is epigenetically
regulated and that epigenetic
mechanisms have a role in the
pathogenesis of RA

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

The role of histone
modifications in the
regulation of TNF
expression
 24

Overview
• Chromatin
Immunoprecipitation(ChIP) assay is
being used to monitor histone
acetylation in:
• synovial fibroblasts - to see if incubation
with cytokines affects acetylation of
histones

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 25

ChIP assay (1)

Adapted from Lodish et al,

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5 2000
 26

Optimisation of sonication

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 27

Immunoprecipitation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 28

Results
Following reversal of the cross-links,
DNA that has been immunoprecipitated
is examined using PCR and is
normalised to input DNA

This can be done using:

• Standard PCR
• Real time PCR

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 29

TNF promoter
ChIP
primers

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 30

Acetylation at Histone H3 Lys 9 in

the TNF promoter
6

Control
3
0.1ng/ml TNF

Fold
2 induction

0
2 8 24
Hours

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 31

Summary
• Formaldehyde cross-links DNA and histones
• Sonication shears DNA to ~500bp (3
histones)
• Immunoprecipitate with antibody to histone
modification, DNA associated with
modification forms complex with antibody,
other DNA is removed during washes
• Reverse cross-links and analyse by PCR

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

DNA methylation - a
method for repressing
transcription
 33

DNA Methylation
• Methylation of C5 position of cytosine residues by
DNA methyltransferases (Dnmt)
• In eukaryotes this only occurs at the sequence
CpG
• CpG methylation is low when gene is
transcriptionally active and high when gene is
silent active
CpG
methyl silent

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 34

DNA methylation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 35

DNA Methyltransferases
• DNA methyltransferases (Dnmts)
• Dnmt 1 responsible for maintenance
methylation
• Dnmt3a and Dnmt3b involved in de
novo methylation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 36

CpG methylation
• In organisms that support CpG methylation,
including mammals, methylation is essential
for maintaining gene silencing
• Absence of methylation in some eukaryotes
including S.cerevisiae, S.pombe and D.
melanogaster suggest methylation-
independent mechanisms for maintaining
gene silence must exist

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 37

CpG Methylation 3
• Parental strand of DNA is methylated, progeny
strand is initially unmethylated
• Resulting hemimethylated DNA duplex is recognised
by maintenance DNA methyltransferase Dnmt1
• Adds methyl groups to CpG dinucleotides on progeny
strand
• The fully methylated CpG dinucleotides recruit a
family of proteins that selectively bind CpG
dinucleotides known as methyl-CpG binding-domain
(MBD) proteins

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 38

Maintenance methylation
Dnmt 1

Methyl group

Parental Strand

Progeny Strand

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 39

DNA methylation and histone

modifications-Methyl Binding
Domain proteins
• Some MBDs are associated with HDACs possibly
responsible for initiating reassembly of silent
chromatin or protecting silent chromatin which has
been assembled using a MBD-independent pathway
• Evidence for MBD-independent pathways include
the association of HDAC2 with Dnmt1, therefore
recognition of hemimethylated DNA by Dnmt1
might directly promote histone deactylation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 40

DNA methylation and

histone modifications
• Evidence that DNA methylation is
dominant to histone acetylation
• HDAC inhibitor only increased
expression after removal of
cytosine methylation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

The Role of DNA
methylation in
regulating TNF-
expression
 42

TNF promoter

700 600 500 400 300 200 100 0

B2 SP1 Ets AP2 EGR-1 B3 AP2 TATA

CRE
CpG SNPs C-jun
AP-1/AF

Only SP1 and EGR1 contain CpG’s within their

binding sites. However, a number of CpG’s are
adjacent to binding sites including B2, AP-2 and
Ets.
11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5
 43

cytosine C5-methyl-cytosine

Cytosines are sulfonated Sodium bisulphite C5-methyl-cytosines

and deaminated are not modified

uracil sulphonate

Desulphonation
Basic pH

uracil

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 44

Bisulphite sequencing
• Primers are used that do not contain CpG’s
• Use PCR to amplify fragments of
approximately 400 base pairs and clone
PCR product
• Sequence multiple clones to calculate the
percentage of C’s that are methylated at
each site

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 45

Other methods
• 5-aza-2’-Deoxycytidine (5-aza-CdR) is a
compound that is incorporated into DNA on
replication instead of cytidine but cannot be
methylated
• Treatment with 5-aza-CdR cause global
demethylation of DNA over a number of
replications and results in derepression of
transcription

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 46

Fibroblast-like synovial cells

• From RA patients
• Taken from patient before
and 16 weeks into anti-TNF
treatment
• Used between passage 3
and 6
• Takes 6 to 8 weeks from
biopsy to passage 3
Rohini Raman, 2006

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 47

5-aza-CdR enhances TNF- gene expression

in FLS
8000

7000

6000

5000

4000

%3000
control

2000

1000

0
0 10 100 0 10 100
5-Aza-CdR (µM) 0.1ng/ml TNF-

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 48

Macrophages
• From control blood
• Leukocytes purified on Ficoll gradient
• 7 day adhesion step then non-
adherant cell are removed

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 49

LPS-induced TNF in macrophages

1000

100 75 fold

10
5 fold
1

0.1

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 50

Methylation of TNF promoter - all

clones <5 fold 103 clones from 9
90
donors
1 >100 fold 92 clones from 9
80 donors
2 1.) 2=5.087, P = 0.0121
70 2.) 2=7.755, P = 0.0027

60

50
<5 fold
>100 fold
40
% Methylation
30

20

10

0
-304 -245 -239 -170 -164 -162 -147 -120 -73 -50 -39 10
Position of cytosine of CpG dinucleotide

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 51

TNF promoter methylation - by

donor -304 -245 -239 -170 -164 -162 -147 -120 -73 -50 -39 +10

0 - 29% methylated

30 - 69% methylated

70-100% methylated

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 52

Conclusions
• Reduced methylation at -304 and -
245 in macrophages that produce
greatest amount of TNF mRNA
transcript

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 53

Summary
• DNA methylation is a mechanism for
silencing genes and is indicative of
heterochromatin
• Sodium bisulphite is used to convert
unmethylated cytosine to uracil
• Sequencing can then be used to determine
DNA methylation status at individual CpG
dinucleotides

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5

 54

Conclusions
You should now know the following:
• Which histone modifictions are associated
with heterochromatin
• Which histone modifications are indicative
of active chromatin
• How the ChIP assay works
• How bisulphite modification is used to
analyse DNA methylation

11/10/06 Isobel Gowers - Molecular Medicine - Gene Expression 5