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DNA Methylation Patterns & Epigenetic Regulation in the Pacific Oyster  Mackenzie Gavery & Steven Roberts University of Wa...
Outline <ul><li>Background : epigenetics, DNA methylation  </li></ul><ul><li>Results : characterization of DNA  methylatio...
Background:  GENES (DNA) TRAITS color growth disease resistance toxins ENVIRONMENT nutrition pathogens
GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance toxins Background:  ENVIRONMENT nutrition p...
GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance toxins Background:  ENVIRONMENT nutrition p...
Epigenetics  <ul><li>Heritable changes in trait or phenotype, caused by a mechanism other than mutation to the DNA sequenc...
<ul><li>Effects of disruptions: </li></ul><ul><ul><li>tumor promotion </li></ul></ul><ul><ul><li>alteration of development...
Characterization of DNA methylation in Pacific oysters : <ul><li>describe distribution of methylation </li></ul><ul><li>el...
Results <ul><li>Methylation Specific PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico  analysis </li...
Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico  analysis </l...
Results: gene-targeted approach <ul><li>Methylation Sensitive PCR </li></ul><ul><ul><li>5 stress related genes were examin...
Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico  analysis </l...
Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico  analysis </l...
Regulation of Gene Expression high mid low Predicted DNA Methylation
Regulation of Gene Expression high mid low Predicted DNA Methylation
Implications: <ul><li>evidence suggests DNA methylation plays a regulatory role in Pacific oysters </li></ul><ul><ul><li>i...
Implications: Environment Low   High
<ul><li>Selective breeding can contribute to improved & predictable performance in oysters </li></ul><ul><li>Understanding...
Implications: Hybrid Vigor <ul><ul><li>Heterosis (hybrid vigor) </li></ul></ul><ul><ul><ul><li>mechanism not fully underst...
Implications: Nutrition <ul><li>diet can modify traits by affecting DNA methylation.  </li></ul>Waterland & Jirtle,  Molec...
Future Directions <ul><li>Method evaluation/development: </li></ul><ul><ul><li>challenges associated with non-model specie...
Summary <ul><li>Characterization of DNA methylation in Pacific oyster suggests a role in gene regulation, specifically gen...
Acknowledgements <ul><li>UW, SAFS </li></ul><ul><ul><li>Dr. Steven Roberts </li></ul></ul><ul><ul><li>Samuel White </li></...
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Gavery PCSGA 2010

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Gavery PCSGA 2010

  1. 1. DNA Methylation Patterns & Epigenetic Regulation in the Pacific Oyster Mackenzie Gavery & Steven Roberts University of Washington School of Aquatic and Fishery Sciences
  2. 2. Outline <ul><li>Background : epigenetics, DNA methylation </li></ul><ul><li>Results : characterization of DNA methylation in Pacific oysters </li></ul><ul><li>Implications </li></ul><ul><li>Future directions </li></ul>
  3. 3. Background: GENES (DNA) TRAITS color growth disease resistance toxins ENVIRONMENT nutrition pathogens
  4. 4. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance toxins Background: ENVIRONMENT nutrition pathogens
  5. 5. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance toxins Background: ENVIRONMENT nutrition pathogens
  6. 6. Epigenetics <ul><li>Heritable changes in trait or phenotype, caused by a mechanism other than mutation to the DNA sequence </li></ul><ul><li>Most well understood epigenetic mechanism is DNA methylation: </li></ul><ul><ul><li>occurs at CpG sites in animals </li></ul></ul><ul><ul><li>regulates gene expression </li></ul></ul><ul><ul><li>influenced by the environment </li></ul></ul>Me C G C G
  7. 7. <ul><li>Effects of disruptions: </li></ul><ul><ul><li>tumor promotion </li></ul></ul><ul><ul><li>alteration of development </li></ul></ul><ul><ul><li>inhibition of reproduction </li></ul></ul><ul><li>Compounds that impact normal epigenetic functions: </li></ul><ul><ul><li>Endocrine disruptors: estrogen, BPA, pesticides </li></ul></ul>DNA methylation
  8. 8. Characterization of DNA methylation in Pacific oysters : <ul><li>describe distribution of methylation </li></ul><ul><li>elucidate functional significance </li></ul>
  9. 9. Results <ul><li>Methylation Specific PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico analysis </li></ul>
  10. 10. Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico analysis </li></ul>
  11. 11. Results: gene-targeted approach <ul><li>Methylation Sensitive PCR </li></ul><ul><ul><li>5 stress related genes were examined </li></ul></ul><ul><ul><li>Identified CpG methylation in heat shock protein 70 </li></ul></ul><ul><li>Bisulfite sequencing </li></ul><ul><ul><li>136 bp fragment: 1 of 7 CpG methylated (homology to neuromedin-u receptor ) </li></ul></ul><ul><ul><li>93 bp fragment: 1 of 2 CpG methylated (homology to bromodomain adjacent to zinc finger domain ) </li></ul></ul>
  12. 12. Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico analysis </li></ul>
  13. 13. Results <ul><li>Methylation Sensitive PCR </li></ul><ul><li>Bisulfite sequencing </li></ul><ul><li>In silico analysis </li></ul><ul><ul><li>predicted methylation status of 12,000 C. gigas sequences from GigasBase </li></ul></ul><ul><ul><li>sequences were grouped by Gene Ontology term </li></ul></ul><ul><ul><li>an average predicted methylation status was determined </li></ul></ul>
  14. 14. Regulation of Gene Expression high mid low Predicted DNA Methylation
  15. 15. Regulation of Gene Expression high mid low Predicted DNA Methylation
  16. 16. Implications: <ul><li>evidence suggests DNA methylation plays a regulatory role in Pacific oysters </li></ul><ul><ul><li>implications for immune/stress responses </li></ul></ul>
  17. 17. Implications: Environment Low High
  18. 18. <ul><li>Selective breeding can contribute to improved & predictable performance in oysters </li></ul><ul><li>Understanding genetic and epigenetic influences will increase predictability </li></ul>Implications: Selective Breeding
  19. 19. Implications: Hybrid Vigor <ul><ul><li>Heterosis (hybrid vigor) </li></ul></ul><ul><ul><ul><li>mechanism not fully understood </li></ul></ul></ul><ul><ul><ul><li>epigenetic mechanisms have been proposed </li></ul></ul></ul><ul><ul><ul><li>better understanding will allow for greater control in predicting and manipulating gene expression in oysters </li></ul></ul></ul>X =
  20. 20. Implications: Nutrition <ul><li>diet can modify traits by affecting DNA methylation. </li></ul>Waterland & Jirtle, Molecular and Cellular Biology , 2003
  21. 21. Future Directions <ul><li>Method evaluation/development: </li></ul><ul><ul><li>challenges associated with non-model species </li></ul></ul><ul><ul><li>new approaches: </li></ul></ul><ul><ul><ul><li>Whole genome bisulfite sequencing (BS-seq) </li></ul></ul></ul><ul><ul><ul><li>Methylated DNA immunoprecipitation (MeDIP) </li></ul></ul></ul><ul><ul><ul><ul><li>MeDIP-seq </li></ul></ul></ul></ul><ul><ul><ul><ul><li>MeDIP-chip </li></ul></ul></ul></ul>
  22. 22. Summary <ul><li>Characterization of DNA methylation in Pacific oyster suggests a role in gene regulation, specifically genes with inducible expression </li></ul><ul><li>DNA methylation could be an important mechanism contributing to phenotypic variation in oysters </li></ul><ul><li>Important evaluate & develop methods and tools to evaluate epigenetic mechanisms in bivalves </li></ul>
  23. 23. Acknowledgements <ul><li>UW, SAFS </li></ul><ul><ul><li>Dr. Steven Roberts </li></ul></ul><ul><ul><li>Samuel White </li></ul></ul><ul><ul><li>Lisa Crosson </li></ul></ul><ul><ul><li>Emma Timmins-Schiffman </li></ul></ul><ul><li>Taylor Shellfish Farms </li></ul><ul><ul><li>Joth Davis </li></ul></ul><ul><li>NSA-PCS </li></ul><ul><li>NOAA Aquaculture Program </li></ul>

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