1. DNA Methylation & Epigenetic Regulation in the Pacific Oyster Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences

3. Results: characterization of DNA methylation in Pacific oysters

4. Current directions: method development

6. Background: color disease resistance growth TRAITS pathogens toxins nutrition EPIGENOME (DNA methylation) ENVIRONMENT GENES (DNA)

7. Background: color disease resistance growth TRAITS pathogens toxins nutrition EPIGENOME (DNA methylation) ENVIRONMENT GENES (DNA)

8. Epigenetics Heritable changes in trait or phenotype, caused by a mechanism other than mutation to the DNA sequence Most well understood epigenetic mechanism is DNA methylation occurs in CpG in animals functions regulates gene expression essential for development genome stability Me C G G C

9. Characterization of DNA methylation in Pacific oysters describe distribution of methylation elucidate functional significance

10. Results Methylation Specific PCR Bisulfite sequencing In silicoanalysis

11. Results Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis

12. Results: gene-targeted approach Methylation Sensitive PCR 5 stress related genes were examined Identified CpG methylation in heat shock protein 70 Bisulfite sequencing 136 bp fragment: 1 of 7 cytosinesmethylated (homology to neuromedin-u receptor) 93 bp fragment: 1 of 2 cytosinesmethylated (homology to bromodomain adjacent to zinc finger domain) Gavery & Roberts, 2010

13. Results Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis

14. Results CpG observed CpG o/e CpG expected Methylation Sensitive PCR Bisulfite sequencing In silicoanalysis predicted methylation status of 12,000 C. gigasgenes

15. in silico approach Principle: Methylated cytosines are highly mutable C  T methylated regions of DNA are depleted of CpGdinucleotides over evolutionary time (CpG to TpG) CpG observed CpG o/e CpG expected m high = unmethylated low = methylated

16. Regulation of Gene Expression Gavery & Roberts, 2010

17. Regulation of Gene Expression ‘housekeeping’ ‘highly regulated’ Gavery & Roberts, 2010

18. Summary: oyster DNA is methylated genes with differing regulatory requirements have different levels of DNA methylation

19. Current Directions Method evaluation/development: challenges with non-model species MBD-isolated genome sequencing (MBD-seq)

20. Goal: MBD-seq genome wide methylation analysis evaluate in silicoresults which genes are methylated? which parts of the genome are methylated?

21. Work Flow: MBD-seq genomic DNA

22. Work Flow: MBD-seq 1. fragmentation

23. Work Flow: MBD-seq 2. enrichment MBD Y MBD Y MBD Y

24. Work Flow: MBD-seq MBD Y MBD Y MBD Y 3. library prep & sequencing

25. Work Flow: MBD-seq 4. mapping genomic DNA – reference sequence methylated unmethylated unmethylated methylated

26. Status: MBD-seq MBD isolation: complete library prep and sequencing: in progress methylated 22% unmethylated 78%

27. Summary genes with differing regulatory requirements have different levels of DNA methylation currently evaluating & developing methods and tools to evaluate epigenetic mechanisms in bivalves Implications…

28. Implications: Environment Endocrine disrupting compounds: cause changes in DNA methylation patterns associated with negative phenotypes can be passed on for multiple generations

29. Implications: Selective Breeding Selective breeding can contribute to improved & predictable performance in oysters Understanding geneticand epigenetic influences will increase predictability

30. Implications: Hybrid Vigor Heterosis (hybrid vigor) mechanism not fully understood epigenetic mechanisms have been proposed better understanding will allow for greater control in predicting and manipulating gene expression in oysters X =

31. Conclusion Elucidating the functional significance of DNA methylation in aquatic invertebrates will improve our understanding of the interactions between the environment, gene expression, and organismal responses.

32. Acknowledgements UW, SAFS Dr. Steven Roberts Samuel White Taylor Shellfish Farms Joth Davis National Shellfisheries Association