Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas
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Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas

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Epigenetics in Context: from Ecology to Evolution (ESF-EMBO). San Feliu de Guixols, Spain. Sept 18 - 23, 2011

Epigenetics in Context: from Ecology to Evolution (ESF-EMBO). San Feliu de Guixols, Spain. Sept 18 - 23, 2011

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Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas Presentation Transcript

  • Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas
    Mackenzie Gavery & Steven Roberts
    University of Washington
    School of Aquatic and Fishery Sciences
    Seattle, WA USA
  • Outline
    • Background: oysters
    • Results: characterization of DNA methylation in Crassostrea gigas
    • Discussion: functional role
  • Oyster: Biology
    free swimming larvae (2 weeks)
    egg
    sperm
    2-3 years
    juvenile
  • Oysters: Economic value
    Major Producers of Crassostrea gigas
    (FAO Fishery Statistics, 2006)
  • Oysters: Threats
  • DNA methylation
    An epigenetic mechanism found in plants and animals
    In animals: occurs primarily in a CpG context
    Function: gene regulation
    Can be affected by environmental factors
    Me
    C
    G
    G
    C
  • DNA methylation: invertebrates
    Only a handful of species have been evaluated
    Model invertebrates lack DNA methylation
    Most: 30 – 60 % methylation
    Primarily in exonic regions
    Important regulatory functions – honey bee
    (e.g. Kucharski et al., 2008; Elango et al., 2009; Lyko et al., 2010)
  • Characterization of DNA methylation in oysters
    Describe distribution of methylation
    Elucidate functional significance
  • Results
    in silicoanalysis
    Genome wide methylation analysis
  • in silico approach
    Principle:
    Methylated cytosines are highly mutable
    C  T
    Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG)
    CpG observed
    CpG O/E
    CpG expected
    m
    low = methylated
  • in silico approach
    Principle:
    Methylated cytosines are highly mutable
    C  T
    Methylated regions of DNA are depleted of CpG dinucleotides over evolutionary time (CpG to TpG)
    CpG observed
    CpG O/E
    CpG expected
    m
    high = unmethylated
  • Results: in silico
    Gavery & Roberts, 2010
  • Results: in silico
    =methylated CpG
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Summary of Results:
    Genes with differing regulatory requirements have different levels of DNA methylation
    ‘housekeeping’, ubiquitously expressed = methylated
    ‘inducible genes’ = unmethylated
  • Results
    in silicoanalysis
    Genome wide methylation analysis
  • MBD-seq
    Methyl-binding domain isolated - genome sequencing
    MBD
    Y
    MBD
    Y
    MBD
    Y
  • MBD-seq: Results
    CpG O/E
    (modified from Gavery and Roberts 2010)
    Predicted degree of DNA methylation
  • MBD-seq: Results
    Enrichment level in MBD library
    Measured degree of DNA methylation
    (unpublished)
    CpG O/E
    (modified from Gavery and Roberts 2010)
    Predicted degree of DNA methylation
  • MBD-seq: Results
    Enrichment level in MBD library
    Measured degree of DNA methylation
    (unpublished)
    CpG O/E
    (modified from Gavery and Roberts 2010)
    Predicted degree of DNA methylation
  • MBD-seq: Results
    Enrichment level in MBD library
    Measured degree of DNA methylation
    (unpublished)
    CpG O/E
    (modified from Gavery and Roberts 2010)
    Predicted degree of DNA methylation
  • Summary of Results:
    Experimental analysis confirms in silicoresults
    Genes with differing regulatory requirements have different levels of DNA methylation
    ‘housekeeping’ ubiquitously expressed = methylated
    ‘inducible genes’ = unmethylated
  • Discussion:
    Why?
  • Discussion: Functional Role
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    ‘inducible’
    Conventional transcription of genes
    required for essential functioning
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    Increased variation in environmental response genes
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    Increased variation in environmental response genes
    TF
    TF
    a) alternative splicing
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    Increased variation in environmental response genes
    alternative splicing
    sequence variation
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    Increased variation in environmental response genes
    alternative splicing
    sequence variation
    transient methylation
    ‘inducible’
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    Increased variation in environmental response genes
    alternative splicing
    sequence variation
    transient methylation
    ‘inducible’
    Conventional transcription of genes
    required for essential functioning
    ‘housekeeping’
    Gavery & Roberts, 2010
  • Discussion: Functional Role
    The distribution of DNA methylation may function to promote variation in environmental response genes
    Planktonic larvae
    Sessile
    Variable environments
  • Conclusions/Future Directions:
    Oysters have a functioning DNA methylation system
    Ubiquitously expressed and inducible genes have different levels of methylation – indicating a functional role
    Future work:
    Test the hypothesis that the DNA methylation system functions to enhance random variation in aquatic invertebrates
    Investigate epigenetic effects of synthetic estrogens in oysters
  • Acknowledgements
    Samuel White (UW, SAFS)
    Joth Davis (Taylor Shellfish Farms)
    US Environmental Protection Agency
    Graduate School, UW
    email: mgavery@uw.edu
    website: students.washington.edu/mgavery