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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 ,[object Object]
Results: characterization of DNA methylation in Crassostrea gigas
Discussion: functional role,[object Object]
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MBD-seq: Results Enrichment level in MBD library Measured degree of DNA methylation (unpublished) CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation
MBD-seq: Results Enrichment level in MBD library Measured degree of DNA methylation (unpublished) CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation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

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

  • 1. 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
  • 2.
  • 3. Results: characterization of DNA methylation in Crassostrea gigas
  • 4.
  • 5. Oysters: Economic value Major Producers of Crassostrea gigas (FAO Fishery Statistics, 2006)
  • 7. 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
  • 8. 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)
  • 9. Characterization of DNA methylation in oysters Describe distribution of methylation Elucidate functional significance
  • 10. Results in silicoanalysis Genome wide methylation analysis
  • 11. 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
  • 12. 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
  • 13. Results: in silico Gavery & Roberts, 2010
  • 14. Results: in silico =methylated CpG ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 15. Summary of Results: Genes with differing regulatory requirements have different levels of DNA methylation ‘housekeeping’, ubiquitously expressed = methylated ‘inducible genes’ = unmethylated
  • 16. Results in silicoanalysis Genome wide methylation analysis
  • 17. MBD-seq Methyl-binding domain isolated - genome sequencing MBD Y MBD Y MBD Y
  • 18. MBD-seq: Results CpG O/E (modified from Gavery and Roberts 2010) Predicted degree of DNA methylation
  • 19. 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
  • 20. 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
  • 21. 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
  • 22. 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
  • 24. Discussion: Functional Role ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 25. Discussion: Functional Role ‘inducible’ Conventional transcription of genes required for essential functioning ‘housekeeping’ Gavery & Roberts, 2010
  • 26. Discussion: Functional Role Increased variation in environmental response genes ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 27. Discussion: Functional Role Increased variation in environmental response genes TF TF a) alternative splicing ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 28. Discussion: Functional Role Increased variation in environmental response genes alternative splicing sequence variation ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 29. Discussion: Functional Role Increased variation in environmental response genes alternative splicing sequence variation transient methylation ‘inducible’ ‘housekeeping’ Gavery & Roberts, 2010
  • 30. 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
  • 31. Discussion: Functional Role The distribution of DNA methylation may function to promote variation in environmental response genes Planktonic larvae Sessile Variable environments
  • 32. 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
  • 33. 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