The document summarizes research on DNA methylation patterns and epigenetic regulation in the Pacific oyster. Results showed DNA methylation occurs in stress-related genes and may play a regulatory role in immune and stress responses. Understanding genetic and epigenetic influences could increase predictability in selective breeding of oysters. Further evaluating epigenetic mechanisms in bivalves using new methods like whole genome bisulfite sequencing may provide insights with implications for selective breeding, hybrid vigor, and how nutrition affects gene expression through DNA methylation.

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

2. Outline Background : epigenetics, DNA methylation Results : characterization of DNA methylation in Pacific oysters Implications Future directions

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

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

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

6. 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 at CpG sites in animals regulates gene expression influenced by the environment Me C G C G

7. Effects of disruptions: tumor promotion alteration of development inhibition of reproduction Compounds that impact normal epigenetic functions: Endocrine disruptors: estrogen, BPA, pesticides DNA methylation

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

9. Results Methylation Specific PCR Bisulfite sequencing In silico analysis

10. Results Methylation Sensitive PCR Bisulfite sequencing In silico analysis

11. 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 CpG methylated (homology to neuromedin-u receptor ) 93 bp fragment: 1 of 2 CpG methylated (homology to bromodomain adjacent to zinc finger domain )

12. Results Methylation Sensitive PCR Bisulfite sequencing In silico analysis

13. Results Methylation Sensitive PCR Bisulfite sequencing In silico analysis predicted methylation status of 12,000 C. gigas sequences from GigasBase sequences were grouped by Gene Ontology term an average predicted methylation status was determined

14. Regulation of Gene Expression high mid low Predicted DNA Methylation

15. Regulation of Gene Expression high mid low Predicted DNA Methylation

16. Implications: evidence suggests DNA methylation plays a regulatory role in Pacific oysters implications for immune/stress responses

17. Implications: Environment Low High

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

19. 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 =

20. Implications: Nutrition diet can modify traits by affecting DNA methylation. Waterland & Jirtle, Molecular and Cellular Biology , 2003

21. Future Directions Method evaluation/development: challenges associated with non-model species new approaches: Whole genome bisulfite sequencing (BS-seq) Methylated DNA immunoprecipitation (MeDIP) MeDIP-seq MeDIP-chip

22. Summary Characterization of DNA methylation in Pacific oyster suggests a role in gene regulation, specifically genes with inducible expression DNA methylation could be an important mechanism contributing to phenotypic variation in oysters Important evaluate & develop methods and tools to evaluate epigenetic mechanisms in bivalves

23. Acknowledgements UW, SAFS Dr. Steven Roberts Samuel White Lisa Crosson Emma Timmins-Schiffman Taylor Shellfish Farms Joth Davis NSA-PCS NOAA Aquaculture Program