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.
N
DNA (CG)N
N N
NNH 2
O
HOHO
CH 2SCH 2CH 2CH(NH 2
CH3
S-Adenosylmethionine (Adomet or SAM)
+COOH
O
HOHO
N
N N
NNH2
S-Adenosylhomocysteine (AdoHcy or SAH)
CH2SCH2CH2CH(NH 2
COOH
DNA (CmG)
N
CH3
The DNA methylation reaction
DNA MeTase M
MMM
Mature somatic cells
MM
DNA deMeTase
MM M
Early Development
DNA methylation aberrations in cancer cells
• Certain few genes are regionally hypermethylated
• The genome is globally hypomethylated
CH3
CH3
CH3
CH3CH3
CH3
AP 2 Myc/MaxCH3
CH3 MECP2
mSin3A
HDACCH3
CH3 MECP2
DNA Methylation inhibits gene expressionby two independent mechanisms
CH3
CH3
CH3
CH3
devl
opm
ent
CH3
CH3
Site specific demethylation
CH3
CH3m
atur
e ce
lls
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
maintenance methylation
Model 1: Model 1: DNA methylation patterns are fixedfixed during development maintained faithfully by the maintenance methyltransferase in somatic cells
What is a deMeTase activity?
MM
DNA deMeTase
M
M+
CA T
SV40
CA T
pMet
An Ectopically Methylated Reporter Gene is Demethylated An Ectopically Methylated Reporter Gene is Demethylated when it is Directed by an Active Promoter when it is Directed by an Active Promoter
A
cety
late
d C
hlor
amph
enic
ol (d
pm)
1400001200001000080000600004000020000
0
SV40CATpMetCAT
0
Promoter Constructs
CH3
CH3
CH3
CH3
CH3
CH3
methylase
demethylase
active
inactiveactive
inactive
Model 2: The steady state methylation pattern is a dynamic equilibrium between methylase and demethylase activities
The direction of the arrow is determined by interacting factors that determine the state of activity of the gene
CH3
CH3
CH3
demethylase
X
TSA
HAT bindingCH3
CH3
CH3
Ac
Ac
AcAc
Ac
Ac
Ac
demethylase
EGFP
XXbbaa II
pCMV
DDppnn II HHppaa IIIIDpnIDpnI HpaII
-TSA
-TSA
+TSA
+TSA
A T C G A T C G-TSA +TSA
TSA Enhances Processive Demethylation of GFPTSA Enhances Processive Demethylation of GFP
CMV-GFP does not replicate in HEK293 cellstherefore 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
aaa
0
1020
30
40
50
60 SG0/G1
G2
aa
EGFPpCMV
control
Serum starved
+TSA
Sequences associated Sequences associated with acetylated histones are actively demethylatedwith acetylated histones are actively demethylated
MetCAT-TSA
+TSA
CMVGFP
- TSA
+TSASV40CAT-TSA
+TSAprepre +A
B+A
B-A
B-AB
NO IPNO IPCONTROLCONTROL
Anti H3 IP+ TSA
NO IPNO IPCONTROLCONTROL
Anti H3 IPAnti H3 IP+TSA+TSA
Anti H3 IPAnti H3 IP-TSA-TSA
prepre +AB
+AB
-AB-AB
prepre +AB
+AB
-AB-AB
CH3
CH3
CH3
demethylase
X
TSA
HAT bindingCH3
CH3
CH3
Ac
Ac
AcAc
Ac
Ac
Ac
demethylase
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.
pp32
TAF-1∂TAF-1ß
Inhibitors of Acetyltransferases (INHAT subunits) Inhibitors of Acetyltransferases (INHAT subunits) Inhibit Acetylation Through Histone MaskingInhibit Acetylation Through Histone Masking
CH3CH3CH3
K
INHAT
K
CH3CH3
AcAc
Ac
AcAc
Ac
Ac
IDAC?
Set/Taf1-inhibits histone acetylation and expression of CMV-GFP
Set/TaF1-
H4
H3H2AH2B phosphorimage
120-225Set/TaF1-b
Set/TaF1-b coomasie
Amido black
Set/TaF1- 120-225Set/TaF1-b
GFP-Westen blot
Histone acetylation:
CMV-GFP expression
The INHATs Set/Taf1- and pp32 inhibit TSA induced demethylation of GFP sequences
100
50
control
Set/Taf1-
Set/Taf1-
Dose dependent inhibition of GFP demethylation by
Set/Taf-1 but not Set/Taf1-
Dose µg0.5 1 1.5 2
% d
emet
hyla
tion100
50Set/Taf1-
Set/Taf1-
Set/Taf1- Set/Taf1-
-TSA
DNA bound to INHATs is protectedfrom demethylase, DNA bound to acetylated histones is
demethylated
Input
IPHistone
Set/TAF-1ß
-acetyl-
TF
HAT
TR
HDAC
TSAINHATs
The epigenome is guarded by the interdependence of DNA methylation and histone acetylation
demethylase DNMT
breastbreast
00.40.81.21.6
coloncolon3210
4
stomachstomach3210
4
uterusuterus
00.20.40.6
rectumrectum3210
kidneykidney
00.40.81.2
p<0.00039p<0.00039
totaltotalaverageaverage
normalnormal tumortumor00.20.40.60.81.0
Set/TAF-1ß Oncogene Message is Significantly Increased Set/TAF-1ß Oncogene Message is Significantly Increased in Multiple Tumor Tissuesin Multiple Tumor Tissues
MBD2/demethylase1
MBD3/demethylase2
MBD
Coiled coil domain
PLC motif
Amino acid sequence of demethylase 1 and 2
Demethylase assay
demethylase activity
CpGpCpGpCpGpCpGpCpGCH 3
GpCpGpCp GpCpGpC
CH 3 * * *
Cp*CH 3
Cp* Cp*
Cp*CH 3
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
slowHistone acetylation demethylation (stable)
slowHistone deacetylation methylation (stable)
transient and fast
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?
Antisense to dMTase inhibits tumorigenesis ex vivo
0
20
40
60
80
PCMV T7 ATG (His)6
Xpress EK MCS BGHpA
pcDNA-His-dMTase
Not 1Not 1
1.36 kbcoiled-coil
MDBDcoiled-coil
MDBDSense
Antisense
Inhibition of MBD2/demethylase mRNA by an antisense
adenoviral vector
controlGFP
dMTase aSdMTase
18 rRNA
dMTase antisense treatment of human A549 cancer cells in vivo
100
200
300
400
500
600
700
800
900
days post infection
anti dM Tase
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 DetichSteffan HammNadia CervoniJohanne ThebergePaul CampbellVeronica Bovenzi
Orval MamerGeorge Just
Debu ChakravartiSang-beom Seo
Shafaat RabbaniPouya PakneshanYongjing Guo