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

CH AP 2 Myc/Max CH 3 3 MECP2 mSin3A HDAC CH 3 3 MECP2 DNA Methylation inhibits gene expression by two independent mechanisms

CH 3 devlopment CH 3 Site specific demethylation CH 3 mature cells CH 3 maintenance methylation Model 1: fixed Model 1: DNA methylation patterns are fixed during development maintained faithfully by the maintenance methyltransferase in somatic cells

CAT SV40 CAT pMet An Ectopically Methylated Reporter Gene is Demethylated when it is Directed by an Active Promoter Acetylated Chloramphenicol (dpm) SV40CAT pMetCAT 0 Promoter Constructs

CH 3 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

CH 3 demethylase X TSA HAT binding CH 3 Ac demethylase

TSA Enhances Processive Demethylation of GFP

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 control Serum starved +TSA

Sequences associated with acetylated histones are actively demethylated MetCAT -TSA +TSA CMVGFP - TSA +TSA SV40CAT -TSA +TSA pre +AB -AB NO IP CONTROL Anti H3 IP + TSA NO IP CONTROL Anti H3 IP +TSA -TSA pre +AB -AB pre +AB -AB

CH 3 demethylase X TSA HAT binding CH 3 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 aa Inhibitors of Acetyltransferases (INHAT subunits) Inhibit Acetylation Through Histone Masking CH 3 K INHAT K CH 3 Ac IDAC ?

Set/Taf1 -  inhibits histone acetylation and expression of CMV-GFP Histone acetylation : CMV-GFP expression

The INHATs Set/Taf1-  and pp32 inhibit TSA induced demethylation of GFP sequences control Set/Taf1-   Set/Taf1- 

Dose dependent inhibition of GFP demethylation by Set/Taf-1  but not  Set/Taf1-  Dose µg % demethylation Set/Taf1-   Set/Taf1-  Set/Taf1-   Set/Taf1-  -TSA

DNA bound to INHATs is protected from demethylase, DNA bound to acetylated histones is demethylated - acetyl-

TF HAT TR HDAC TSA INHATs The epigenome is guarded by the interdependence of DNA methylation and histone acetylation demethylase DNMT

a breast colon stomach uterus rectum kidney p< totalaverage normaltumor Set/TAF-1ß Oncogene Message is Significantly Increased in Multiple Tumor Tissues

MBD2/demethylase1 MBD3/demethylase2 MBD Coiled coil domain PLC motif Amino acid sequence of demethylase 1 and 2

Demethylase assay

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) slow Histone 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?

Inhibition of MBD2/demethylase mRNA by an antisense adenoviral vector control GFP dMTase aS 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