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NSA 2011

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  • And NSA for the SEF award.
  • Transcript

    • 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