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