Download Chromatin DNA Methylayion

The bilateral interrelationship
between chromatin and DNA
methylation and its impact on cancer
The bilateral interrelationship of
chromatin and DNA methylation
•
DNA methylation is a reversible reaction, the DNA methylation pattern is a
balance of methylation and demethylation.
•
Active demethylation is directed by chromatin structure
•
Proteins that inhibit histone acetylation inhibit demethylation, a mechanism for
regional hypermethylation in cancer.
•
MBD2/demethylase is essential for tumorigenesis.
•
MBD2/demethylase controls genes required for invasion.
DNA methylation aberrations in
cancer cells
• Certain few genes are regionally
hypermethylated
• The genome is globally hypomethylated
DNA Methylation inhibits gene expression
by two independent mechanisms
CH3
CH3
CH3
CH3
AP 2
CH3
CH3
Myc/Max
CH3
CH3
CH3
CH3
MECP2
MECP2
mSin3A
HDAC
Model 1: DNA methylation patterns are fixed during development
maintained faithfully by the maintenance methyltransferase in somatic cells
CH3
CH3
devlopment
CH3
CH3
CH3
Site specific demethylation
CH3
CH3
CH3
mature cells
CH3
CH3
CH3
CH3
maintenance methylation
CH3
CH3
CH3
CH3
An Ectopically Methylated Reporter Gene is Demethylated
when it is Directed by an Active Promoter
CA T
Acetylated
Chloramphenicol (dpm)
SV40
140000
120000
100000
80000
60000
40000
SV40CAT
pMetCAT
20000
0
CA T
Promoter Constructs
pMet
Model 2: The steady state methylation pattern is a dynamic
equilibrium between methylase and demethylase activities
CH3
active
CH3
CH3
inactive
inactive
methylase
demethylase
CH3
CH3
active
CH3
The direction of the arrow is determined by interacting
factors that determine the state of activity of the gene
TSA
CH3
CH3
CH3
CH3
HAT binding
X
CH3
CH3
TSA Enhances Processive Demethylation of GFP
DpnI
HpaII
Dpn I
EGFP
+TSA
pCMV
Hpa II Xba I
-TSA
+TSA
A TC G
ATC G
CMV-GFP does not replicate in HEK293 cells
therefore demethylation must be active
TSA induces demethylation of a promotererless
GFP DNA
therefore demethylase does not require specific promoter binding
sites
Time and TSA dose dependence of active
demethylation
TSA induced demethylation is not a consequence of alteration
in cell cycle kinetics
EGFP
pCMV
control
Serum
starved
+TSA
Sequences associated
with acetylated histones are actively demethylated
+TSA
CMVGFP
SV40CAT
+TSA
- TSA
-TSA
NO IP
CONTROL
NO IP
CONTROL
Anti H3 IP
+TSA
Anti H3 IP
+ TSA
Anti H3 IP
-TSA
+TSA
MetCAT
-TSA
TSA
CH3
CH3
CH3
CH3
HAT binding
X
CH3
CH3
Why do certain housekeeping genes
become hypermethylated in cancer?
• Why doesn’t TSA induce demethylation of all genes?
– A number of methylated tumor suppressors were shown not to be induced by
TSA.
Hypothesis: certain proteins bind to specific promoters and
inhibit histone acetylation and demethylation.
Inhibitors of Acetyltransferases (INHAT subunits)
Inhibit Acetylation Through Histone Masking
?
K
CH3
CH3
TAF-1∂
TAF-1ß
INHAT
K
CH3
CH3
CH3
Set/Taf1-b inhibits histone acetylation and
expression of CMV-GFP
Histone acetylation:
Set/TaF1-b
Set/TaF1-b
120-225
Set/TaF1-b
H3
H2B
H2A
H4
CMV-GFP expression
Set/TaF1-b
coomasie
phosphorimage
Set/TaF1-b
120-225
GFP-Westen blot
Amido black
The INHATs Set/Taf1-b and pp32 inhibit TSA
induced demethylation of GFP sequences
100
50
Dose dependent inhibition of GFP demethylation by
Set/Taf-1b but not DSet/Taf1-b
-TSA
DSet/Taf1-b
100
% demethylation
Set/Taf1-b
DSet/Taf1-b
50
Set/Taf1-b
0.5
1 1.5
Dose µg
2
DNA bound to INHATs is protected
from demethylase, DNA bound to acetylated histones is
demethylated
Input
IP
-acetylHistone
Set/TAF-1ß
The epigenome is guarded by the interdependence of
DNA methylation and histone acetylation
TF
TR
HAT
HDAC
demethylase
INHATs
DNMT
TSA
Set/TAF-1ß Oncogene Message is Significantly Increased
in Multiple Tumor Tissues
1.6
1.2
0.8
0.4
0
4
3
2
1
0
4
3
2
1
0
breast
0.6
0.4
0.2
0
stomach
colon
3
2
1
0
rectum
1.2
0.8
0.4
0
kidney
total
1.0 average
0.8
0.6
0.4
0.2
0
normal
p<0.00039
uterus
tumor
MBD
MBD2/demethylase1
MBD3/demethylase2
PLC Coiled coil
motif domain
Amino acid sequence of demethylase 1 and 2
Demethylase assay
demethylase activity
CH 3
CH 3
*
*
*
CpGpCpGpCpGpCpGpCpG
GpC pGpCp GpCp GpC
CH 3
CH 3
Cp*
Cp*
Cp*
Cp*
Ectopic expression of Mbd2bhis-dMTase induces
demethylation of GFP reporter sequences
CMV-GFP
Promoterless-GFP
MBD2/demethylase activates specific promoters but
not others in a time dependent manner
Dose dependent activation by
MBD2/demethylase
Ectopic expression of MBD2/demethylase
increases global demethylase activity in HEK
cells
Expression of MBD2/demethylase increases
demethylation at the SV40 promoter
Mechanisms of protection of the epigenome:
DNA replication
DNA methylation
slow
Histone acetylation
demethylation (stable)
transient and fast
slow
Histone deacetylation
methylation (stable)
Regional hypermethylation in
cancer
•
Increasing association of chromatin modifying proteins (such as INHAT) to
promoters of growth suppressing genes.
•
Selective advantage
•
Recruitment of DNMTs- inaccessibility to demethylase
•
Regional hypermethylation
Global hypomethylation is a
hallmark of cancer
•
Repetitive, satellite, centromeric and pericentromeric sequences are
hypomethylated in cancer.
•
Agents that inhibit DNA methyltransferase such as 5-aza-CdR stimulate tumor
invasion and metastasis.
•
Agents that stimulate DNA methylation such as SAM protect from
tumorigenesis.
•
Is there a role for MBD2/demethylase in cancer and metastasis?
Inhibition of MBD2/demethylase mRNA by an antisense
adenoviral vector
dMTase
18 rRNA
control
GFP
dMTase anti
DNA methylation is a reversible reaction,
chromatin structure defines the direction of
the reaction
•
Chromatin modifying proteins cause regional hypermethylation preventing
access to demethylase
•
Increased MBD2/demethylase is responsible for global hypomethylation and
maintaining tumor invasion genes hypomethylated and active
•
Inhibition of MBD2/demethylase causes hypermethylation and silencing of
tumor invasion promoting genes.
•
MBD2/demethylase is not required for normal cell growth.
•
MBD2/demethylase is a promising anticancer drug target.
Nancy Detich
Steffan Hamm
Nadia Cervoni
Johanne Theberge
Paul Campbell
Veronica Bovenzi
Orval Mamer
George Just
Debu Chakravarti
Sang-beom Seo
Shafaat Rabbani
Pouya Pakneshan
Yongjing Guo