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Gavery PCSGA 2010

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  • 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

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