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7/28/2019 Chromatin DNA Methylation1
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The bilateral interrelationship
between chromatin and DNA
methylation and its impact on cancer
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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.
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DNA methylation aberrations in
cancer cells Certain few genes are regionally
hypermethylated
The genome is globally hypomethylated
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CH3
CH3
CH3
CH3
CH3
CH3
AP 2 Myc/MaxCH
3
CH3 MECP2
mSin3A
HDACCH3
CH3
MECP2
DNA Methylation inhibits gene expression
by two independent mechanisms
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CH3
CH3
CH3
CH3
devlopment
CH3
CH3
Site specific demethylation
CH3
CH3
mature
cells
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
maintenance methylation
Model 1: DNA methylation patterns are fixed during developmentmaintained faithfully by the maintenance methyltransferase in somatic cells
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CA T
SV40
CA T
pMet
An Ectopically Methylated Reporter Gene is Demethylatedwhen it is Directed by an Active Promoter
Acetylated
Chloramphenico
l(dpm)
140000
120000
10000
80000
60000
40000
20000
0
SV40CAT
pMetCAT0
Promoter Constructs
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CH3
CH3
CH3
CH3
CH3
CH3
methylase
deme
thylase
active
inactiveactive
inactive
Model 2: The steady state methylation pattern is a dynamicequilibrium between methylase and demethylaseactivities
The direction of the arrow is determined by interactingfactors that determine the state of activity of the gene
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CH3
CH3
CH3
X
TSA
HAT binding
CH3
CH3CH3
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EGFP
Xba I
pCMV
Dpn I Hpa IIDpnI HpaII
+TSA
A T C G A T C G
-TSA +TSA
TSA Enhances Processive Demethylation of GFP
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CMV-GFP does not replicate in HEK293 cellstherefore demethylation must be active
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TSA induces demethylation of a promotererless
GFP DNAtherefore demethylase does not require specific promoter binding
sites
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Time and TSA dose dependence of active
demethylation
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TSA induced demethylation is not a consequence of alteration
incell cycle kinetics
EGFP
pCMV
control
Serum
starved
+TSA
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Sequences associatedwith acetylated histones are actively demethylated
MetCAT
-TSA
+TSA
CMVGFP
-TSA
+TSASV40CAT
-TSA
+TSA
NO IPCONTROL
Anti H3 IP
+ TSA
NO IPCONTROL
Anti H3 IP+TSA
Anti H3 IP-TSA
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CH3
CH3
CH3
X
TSA
HAT binding
CH3
CH3CH3
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Why do certain housekeeping genes
become hypermethylated in cancer? Why doesnt 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.
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TAF-1TAF-1
Inhibitors of Acetyltransferases (INHAT subunits)Inhibit Acetylation Through Histone Masking
CH3CH3
CH3
K
INHAT
K
CH3CH3
?
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Set/Taf1-b inhibits histone acetylation and
expression of CMV-GFP
Set/TaF1-b
H4
H3
H2AH2B phosphorimage
120-225
Set/TaF1-b
Set/TaF1-bcoomasie
Amido black
Set/TaF1-b 120-225
Set/TaF1-b
GFP-Westen blot
Histone acetylation:
CMV-GFP expression
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The INHATs Set/Taf1-b and pp32 inhibit TSA
induced demethylation of GFP sequences
100
50
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Dose dependent inhibition of GFP demethylation by
Set/Taf-1b but not DSet/Taf1-b
Dose g
0.5 1 1.5 2
%d
emethylation
100
50
Set/Taf1-b
DSet/Taf1-b
Set/Taf1-b DSet/Taf1-b
-TSA
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DNA bound to INHATs is protected
from demethylase, DNA bound to acetylated histones is
demethylated
Input
IP
Histone
Set/TAF-1
-acetyl-
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TF
HAT
TR
HDAC
TSAINHATs
The epigenome is guarded by the interdependence of
DNA methylation and histone acetylation
demethylase DNMT
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breast
00.40.81.21.6
colon
3210
4
stomach321
0
4
uterus
00.20.40.6
rectum3210
kidney
0
0.40.8
1.2
p
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MBD2/demethylase1
MBD3/demethylase2
MBD
Coiled coil
domain
PLC
motifAmino acid sequence of demethylase 1 and 2
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Demethylase assay
demethylase activity
CpGpCpGpCpGpCpGpCpG
CH 3
GpC pGpCp GpCp GpC
CH 3
* * *
Cp*
CH 3
Cp* Cp*
Cp*
CH 3
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Ectopic expression of Mbd2bhis-dMTase induces
demethylation of GFP reporter sequences
CMV-GFP
Promoterless-GFP
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MBD2/demethylase activates specific promoters but
not others in a time dependent manner
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Dose dependent activation by
MBD2/demethylase
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Ectopic expression of MBD2/demethylase
increases global demethylase activity in HEK
cells
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Expression of MBD2/demethylase increases
demethylation at the SV40 promoter
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Mechanisms of protection of the epigenome:
DNA replication DNA methylation
slow
Histone acetylation demethylation (stable)
slow
Histone deacetylation methylation (stable)
transient and fast
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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
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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?
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Inhibition of MBD2/demethylase mRNA by an antisense
adenoviral vector
dMTase
18 rRNA
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control
GFP
dMTase anti
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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 oftumor invasion promoting genes.
MBD2/demethylase is not required for normal cell growth.
MBD2/demethylase is a promising anticancer drug target.
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Nancy Detich
Steffan Hamm
Nadia Cervoni
Johanne Theberge
Paul Campbell
Veronica Bovenzi
Orval Mamer
George Just
Debu Chakravarti
Sang-beom Seo
Shafaat Rabbani
Pouya PakneshanYongjing Guo