Epigenetics in Context: from Ecology to Evolution (ESF-EMBO). San Feliu de Guixols, Spain. Sept 18 - 23, 2011

1. Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences Seattle, WA USA

3. Results: characterization of DNA methylation in Crassostrea gigas

5. Oysters: Economic value Major Producers of Crassostrea gigas (FAO Fishery Statistics, 2006)

6. Oysters: Threats

7. DNA methylation An epigenetic mechanism found in plants and animals In animals: occurs primarily in a CpG context Function: gene regulation Can be affected by environmental factors Me C G G C

8. DNA methylation: invertebrates Only a handful of species have been evaluated Model invertebrates lack DNA methylation Most: 30 – 60 % methylation Primarily in exonic regions Important regulatory functions – honey bee (e.g. Kucharski et al., 2008; Elango et al., 2009; Lyko et al., 2010)

9. Characterization of DNA methylation in oysters Describe distribution of methylation Elucidate functional significance

10. Results in silicoanalysis Genome wide methylation analysis

11. in silico approach Principle: Methylated cytosines are highly mutable C  T Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG) CpG observed CpG O/E CpG expected m low = methylated

12. in silico approach Principle: Methylated cytosines are highly mutable C  T Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG) CpG observed CpG O/E CpG expected m high = unmethylated

13. Results: in silico Gavery & Roberts, 2010

14. Results: in silico =methylated CpG ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

15. Summary of Results: Genes with differing regulatory requirements have different levels of DNA methylation ‘housekeeping’, ubiquitously expressed = methylated ‘inducible genes’ = unmethylated

16. Results in silicoanalysis Genome wide methylation analysis

17. MBD-seq Methyl-binding domain isolated - genome sequencing MBD Y MBD Y MBD Y

18. MBD-seq: Results CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation

19. MBD-seq: Results Enrichment level in MBD library Measured degree of DNA methylation (unpublished) CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation

20. MBD-seq: Results Enrichment level in MBD library Measured degree of DNA methylation (unpublished) CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation

21. MBD-seq: Results Enrichment level in MBD library Measured degree of DNA methylation (unpublished) CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation

22. Summary of Results: Experimental analysis confirms in silicoresults Genes with differing regulatory requirements have different levels of DNA methylation ‘housekeeping’ ubiquitously expressed = methylated ‘inducible genes’ = unmethylated

23. Discussion: Why?

24. Discussion: Functional Role ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

25. Discussion: Functional Role ‘inducible’ Conventional transcription of genes required for essential functioning ‘housekeeping’ Gavery & Roberts, 2010

26. Discussion: Functional Role Increased variation in environmental response genes ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

27. Discussion: Functional Role Increased variation in environmental response genes TF TF a) alternative splicing ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

28. Discussion: Functional Role Increased variation in environmental response genes alternative splicing sequence variation ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

29. Discussion: Functional Role Increased variation in environmental response genes alternative splicing sequence variation transient methylation ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010

30. Discussion: Functional Role Increased variation in environmental response genes alternative splicing sequence variation transient methylation ‘inducible’ Conventional transcription of genes required for essential functioning ‘housekeeping’ Gavery & Roberts, 2010

31. Discussion: Functional Role The distribution of DNA methylation may function to promote variation in environmental response genes Planktonic larvae Sessile Variable environments

32. Conclusions/Future Directions: Oysters have a functioning DNA methylation system Ubiquitously expressed and inducible genes have different levels of methylation – indicating a functional role Future work: Test the hypothesis that the DNA methylation system functions to enhance random variation in aquatic invertebrates Investigate epigenetic effects of synthetic estrogens in oysters

33. Acknowledgements Samuel White (UW, SAFS) Joth Davis (Taylor Shellfish Farms) US Environmental Protection Agency Graduate School, UW email: mgavery@uw.edu website: students.washington.edu/mgavery