Exploring the role of DNA methylation as a source of phenotypic variation in Crassostrea gigas<br />Mackenzie Gavery & Ste...
Outline<br /><ul><li>Background: oysters
Results: characterization of DNA methylation in Crassostrea gigas
Discussion: functional role</li></li></ul><li>Oyster: Biology<br />free swimming larvae (2 weeks)<br />egg<br />sperm<br /...
Oysters: Economic value<br />Major Producers of Crassostrea gigas<br />(FAO Fishery Statistics, 2006)<br />
Oysters: Threats<br />
DNA methylation<br />An epigenetic mechanism found in plants and animals<br />In animals: occurs primarily  in a CpG conte...
DNA methylation: invertebrates<br />Only a handful of species have been evaluated<br />Model invertebrates lack DNA methyl...
Characterization of DNA methylation in oysters<br />Describe distribution of methylation<br />Elucidate functional signifi...
Results<br />in silicoanalysis<br />Genome wide methylation analysis<br />
in silico approach<br />Principle:<br />Methylated cytosines are highly mutable<br />C  T<br />Methylated regions of DNA ...
in silico approach<br />Principle:<br />Methylated cytosines are highly mutable<br />C  T<br />Methylated regions of DNA ...
Results: in silico<br />Gavery & Roberts, 2010<br />
Results: in silico<br />=methylated CpG<br />‘inducible’<br />‘housekeeping’<br />Gavery & Roberts, 2010<br />
Summary of Results:<br />Genes with differing regulatory requirements have different levels of DNA methylation<br />‘house...
Results<br />in silicoanalysis<br />Genome wide methylation analysis<br />
MBD-seq<br />Methyl-binding domain isolated - genome sequencing<br />MBD<br />Y<br />MBD<br />Y<br />MBD<br />Y<br />
MBD-seq: Results<br />CpG O/E<br />(modified from Gavery and Roberts 2010)<br />Predicted degree of DNA methylation<br />
MBD-seq: Results<br />Enrichment level in MBD library<br />Measured degree of DNA methylation<br />(unpublished)<br />CpG ...
MBD-seq: Results<br />Enrichment level in MBD library<br />Measured degree of DNA methylation<br />(unpublished)<br />CpG ...
MBD-seq: Results<br />Enrichment level in MBD library<br />Measured degree of DNA methylation<br />(unpublished)<br />CpG ...
Summary of Results:<br />Experimental analysis confirms in silicoresults<br />Genes with differing regulatory requirements...
Discussion:<br />Why?<br />
Discussion: Functional Role<br />‘inducible’<br />‘housekeeping’<br />Gavery & Roberts, 2010<br />
Discussion: Functional Role<br />‘inducible’<br />Conventional transcription of genes<br />required for essential function...
Discussion: Functional Role<br />Increased variation in environmental response genes<br />‘inducible’<br />‘housekeeping’<...
Discussion: Functional Role<br />Increased variation in environmental response genes<br />TF<br />TF<br />a)  alternative ...
Discussion: Functional Role<br />Increased variation in environmental response genes<br />alternative splicing<br />sequen...
Discussion: Functional Role<br />Increased variation in environmental response genes<br />alternative splicing<br />sequen...
Discussion: Functional Role<br />Increased variation in environmental response genes<br />alternative splicing<br />sequen...
Discussion: Functional Role<br />The distribution of DNA methylation may function to promote variation in environmental re...
Conclusions/Future Directions:<br />Oysters have a functioning DNA methylation system <br />Ubiquitously expressed and ind...
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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

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

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

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