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The bilateral interrelationship between chromatin and DNA methylation and its impact on cancer

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.

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The bilateral interrelationship between chromatin and DNA methylation and its impact on cancer

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  1. The bilateral interrelationship between chromatin and DNA methylation and its impact on cancer

  2. 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.

  3. DNA methylation aberrations in cancer cells • Certain few genes are regionally hypermethylated • The genome is globally hypomethylated

  4. Ac Ac Ac Ac M M M M X

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

  6. CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 Model 1: DNA methylation patterns are fixed during development maintained faithfully by the maintenance methyltransferase in somatic cells Site specific demethylation devlopment maintenance methylation maturecells

  7. SV40CAT 1 4 0 0 00 Acetylated Chloramphenicol (dpm) 1 2 0 0 00 pMetCAT 0 1 0 0 0 0 8 0 0 0 0 6 0 0 0 0 4 0 0 0 0 2 0 0 0 0 0 Promoter Constructs An Ectopically Methylated Reporter Gene is Demethylated when it is Directed by an Active Promoter CA T SV40 CA T pMet

  8. methylase demethylase CH3 CH3 CH3 CH3 CH3 CH3 Model 2: The steady state methylation pattern is a dynamic equilibrium between methylase and demethylaseactivities active inactive inactive active The direction of the arrow is determined by interacting factors that determine the state of activity of the gene

  9. CH3 CH3 CH3 Ac Ac Ac Ac Ac TSA demethylase CH3 HAT binding X CH3 CH3 Ac demethylase Ac

  10. DpnI HpaII D p n I H p a I I X b a I -TSA +TSA EGFP A T C G A T C G pCMV -TSA +TSA TSA Enhances Processive Demethylation of GFP

  11. CMV-GFP does not replicate in HEK293 cellstherefore demethylation must be active

  12. TSA induces demethylation of a promotererless GFP DNAtherefore demethylase does not require specific promoter binding sites

  13. Time and TSA dose dependence of active demethylation

  14. TSA induced demethylation is not a consequence of alteration incell cycle kinetics control Serum starved +TSA

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

  16. CH3 CH3 CH3 Ac Ac Ac Ac Ac TSA demethylase CH3 HAT binding X CH3 CH3 Ac demethylase Ac

  17. 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.

  18. a a INHAT K K Inhibitors of Acetyltransferases (INHAT subunits) Inhibit Acetylation Through Histone Masking IDAC ? Ac Ac Ac Ac Ac CH3 CH3 Ac Ac TAF-1∂ TAF-1ß pp32 CH3 CH3 CH3

  19. Set/Taf1-b inhibits histone acetylation and expression of CMV-GFP Histone acetylation: CMV-GFP expression

  20. The INHATs Set/Taf1-b and pp32 inhibit TSA induced demethylation of GFP sequences 100 50 control Set/Taf1-b DSet/Taf1-b

  21. Dose dependent inhibition of GFP demethylation by Set/Taf-1b but not DSet/Taf1-b DSet/Taf1-b Set/Taf1-b 100 -TSA DSet/Taf1-b % demethylation 50 Set/Taf1-b 0.5 1 1.5 2 Dose µg

  22. -acetyl- DNA bound to INHATs is protectedfrom demethylase, DNA bound to acetylated histones is demethylated

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

  24. a 1.2 0.8 0.4 0 total average 1.0 0.8 0.6 0.4 0.2 0 normal tumor p<0.00039 Set/TAF-1ß Oncogene Message is Significantly Increased in Multiple Tumor Tissues breast uterus 1.6 0.6 1.2 0.4 0.8 0.2 0.4 0 0 stomach rectum 3 4 3 2 2 1 1 0 0 colon kidney 4 3 2 1 0

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

  26. Demethylase assay

  27. Ectopic expression of Mbd2bhis-dMTase induces demethylation of GFP reporter sequences CMV-GFP Promoterless-GFP

  28. MBD2/demethylase activates specific promoters but not others in a time dependent manner

  29. Dose dependent activation by MBD2/demethylase

  30. Ectopic expression of MBD2/demethylase increases global demethylase activity in HEK cells

  31. Expression of MBD2/demethylase increases demethylation at the SV40 promoter

  32. Mechanisms of protection of the epigenome: DNA replication DNA methylation slow Histone acetylation demethylation (stable) slow Histone deacetylation methylation (stable) transient and fast

  33. 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

  34. 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?

  35. Inhibition of MBD2/demethylase mRNA by an antisense adenoviral vector dMTase aS control GFP dMTase 18 rRNA

  36. control GFP dMTase anti

  37. 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.

  38. 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

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