2. The dynamic epigenome; an interface between nurture and nature Moshe Szyf Department of pharmacology and Therapeutics McGill University, Montreal Canada Moshe Szyf Department of pharmacology and Therapeutics McGill University, Montreal Canada OHAO SPRING SYMPOSIUM Wednesday, March 26, 2008 Toronto ON

3. The programming of the genome is controlled by the epigenome The epigenome is composed of two components: 1) the chromatin which is associated with the DNA and 2) DNA methylation which is part of the covalent structure of the genome and is therefore a stable long- term signal DNA methylation is an interface between the dynamic environment and the static genome The dynamic epigenome and its implications

4. CH 3 Maintenance DNA methyltransferase DNA replication CH 3 De novo methyltransferase Demethylase CH 3 CH (DNMT3a, 3b and DNMT1?) (DNMT1) DNA methylation reactions

5. Anders H. Lund et al. Genes Dev. 2004; 18: 2315-2335 Covalent modifications of the N-terminal tail of the canonical core histones

6. X CH 3 Transcription factor Transcription factor XAc AcAc CH 3 Sin3A MECP2 SUV39 HDACAc CH 3 HP1 X Fig. 3 DNA methylation silences gene expression by two mechanisms

7. N CH C N OHNHCH3 C DNA C N CH CHCHCHCH NHNHO DNA HOH CH3OH dMTase DNMT AdoMet AdoHcy The reversible methylation reaction The environment is dynamic, the chromatin is dynamic, would DNA methylation remain static throughout life?

8. environment CH 3 methylase demethylase active chromatin inactive chromatin dynamic Hypothesis: The steady state methylation pattern is a dynamic equilibrium between methylase and demethylase activities nutrientstoxinssocialbehavioralpathologicalphysiological

9. Methylated DNA is actively demethylated in a replication independent manner upon induction of histone acetylation by TSA CH 3 Human HEK cells GFP CH 3 X TSA demethylase Human HEK cells GFP No origin of replication-no replication-no passive demethylation

10. HDAC inhibitors induce replication-independent active demethylation Cervoni et al., J. Biol. Chem 276, 40788 (2001) Cervoni et al., J. Biol. Chem. 277, 25026 (2002) Detich et al.,J Biol. Chem. 278, 27586 (2003).

11. N CH C N O HNH CH3 C DNA C N CH CHCHCHCH N HNH O DNA dMTaseDNMT Is there in vivo evidence for a dynamic methylation pattern in postmitotic tissue?

12. LOW LGHIGH LG Glucocorticoid receptor gene GR GENE GR RECEPTORS GR GENE GR RECEPTORS Environmental programming of gene activity STRESS RESPONSE PUP (Day 1-6) ADULT (Day 90)

13. MECHANISM How are the long-term effects of maternal care on gene expression in the offspring maintained into adulthood? How are these differences transmitted across generations?

14. HIPPOCAMPAL GR(1 7 ) REGION 16 (5’ NGFI-A RE) METHYLATION TIMELINE LOW HIGH Mean C-Methylation 0 0.2 0.4 0.6 0.8 1.0 1.2 GR (1 7 ) PROMOTER NGFI-A EMBRYO DAY 20 BIRTH DAY 1 ADULT DAY 90 WEANING DAY 21 PUP DAY 6 Age * * *

15. In the adult (day 90) rat hippocampal GR gene expression of Low LG-ABN offspring is reversed by TSA DNMT NGFI-A X High LGLow LG NGFI-A CH 3 Ac Demethylase HDAC TSA

16. demethylase DNMT SAM X NGFIA binding X Detich et al. J Biol. Chem. 278, 20812-20820 (2003). SAM inhibits replication independent demethylation NGFI-A CH 3 Ac Is it possible to reverse the effects of maternal care on DNA methylation and behavior in the adult rat?

17. (a)(b) * ** Methionine treatment reverses Open Field Behavior of Adult (Day-90) Male Offspring of High LG-ABN maternal care

18. PKA Behavioural gene programming 5-HT cAMP NGFI-AHAT Demethylase MBD2? DNMT

19. DNA methylation and inter-individual phenotypic variance Working hypothesis: environment epigenetic changes inter-individual epigenetic variation gene expression programming Phenotypic variation.

20. Suicide study and control group

21. Methylation of the GR locus and its chromosomal neighborhood in suicide completers

22. Genome wide organization of differentially methylated promoters

23. What are the implications of a life-long dynamic epigenome? chemicalsocial Signaling pathways Epigenome phenotype Non-genotoxic agents might have a profound effect on our genome and our health icluding obesity, diabetes, cnacer autoimmune disease

24. Acknowledgements Suchin Tendulkar, Steven Andrews Jing-Ni Ou Nancy Detich Nadia Cervoni Nada BORGHOL Steffan Hamm George Just MICHAEL MEANEY & IAN WEAVER SERGIY DYMOV, Patrick MacGowen Gustavo Turecki SHAFAAT RABBANI, Bushra Ateq, Nicholai Shukeir, Pouya Pakenshan Michael Hallet, Matt Suderman THIS RESEARCH IS SUPPORTED BY GRANTS FROM NCIC, CIHR, HSFP & THE NICHD