chromatin dna methylation1

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

chromatin dna methylation1

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