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  • And NSA for the SEF award.

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  • 1. DNA Methylation & 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
    • 3. Results: characterization of DNA methylation in Pacific oysters
    • 4. Current directions: method development
    • 5. Implications
  • Background:
    color
    disease resistance
    growth
    TRAITS
    pathogens
    toxins
    nutrition
    ENVIRONMENT
    GENES (DNA)
  • 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