DNA Methylation & Epigenetic Regulation in the Pacific Oyster <br />Mackenzie Gavery & Steven Roberts<br />University of W...
Outline<br /><ul><li>Background: epigenetics, DNA methylation
Results: characterization of DNA methylation in Pacific oysters
Current directions: method development
Implications</li></li></ul><li>Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br /...
Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br />toxins<br />nutrition<br />EPI...
Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br />toxins<br />nutrition<br />EPI...
Epigenetics <br />Heritable changes in trait or phenotype, caused by a mechanism other than mutation to the DNA sequence<b...
Characterization of DNA methylation in Pacific oysters<br />describe distribution of methylation<br />elucidate functional...
Results<br />Methylation Specific PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
Results<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
Results: gene-targeted approach<br />Methylation Sensitive PCR<br />5 stress related genes were examined<br />Identified C...
Results<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
Results<br />CpG observed<br />CpG o/e<br />CpG expected<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In ...
in silico approach<br />Principle:<br />Methylated cytosines are highly mutable<br />C  T<br />methylated regions of DNA ...
Regulation of Gene Expression<br />Gavery & Roberts, 2010<br />
Regulation of Gene Expression<br />‘housekeeping’<br />‘highly regulated’<br />Gavery & Roberts, 2010<br />
Summary:<br />oyster DNA is methylated<br />genes with differing regulatory requirements have different levels of DNA meth...
Current Directions<br />Method evaluation/development:<br />challenges with non-model species<br />MBD-isolated genome seq...
Goal: MBD-seq<br />genome wide methylation analysis<br />evaluate in silicoresults<br />which genes are methylated?<br />w...
Work Flow: MBD-seq<br />genomic DNA<br />
Work Flow: MBD-seq<br />1. fragmentation<br />
Work Flow: MBD-seq<br />2. enrichment<br />MBD<br />Y<br />MBD<br />Y<br />MBD<br />Y<br />
Work Flow: MBD-seq<br />MBD<br />Y<br />MBD<br />Y<br />MBD<br />Y<br />3. library prep & sequencing<br />
Work Flow: MBD-seq<br />4. mapping<br />genomic DNA – reference sequence<br />methylated<br />unmethylated<br />unmethylat...
Status: MBD-seq<br />MBD isolation: complete<br />library prep and sequencing: in progress<br />methylated <br />22%<br />...
Summary<br />genes with differing regulatory requirements have different levels of DNA methylation<br />currently evaluati...
Implications: Environment<br />Endocrine disrupting compounds:<br />cause changes in DNA methylation patterns <br />associ...
Implications: Selective Breeding<br />Selective breeding can contribute to improved & predictable performance in oysters<b...
Implications: Hybrid Vigor<br />Heterosis (hybrid vigor)<br />mechanism not fully understood<br />epigenetic mechanisms ha...
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  • And NSA for the SEF award.
  • NSA 2011

    1. 1. DNA Methylation & Epigenetic Regulation in the Pacific Oyster <br />Mackenzie Gavery & Steven Roberts<br />University of Washington<br />School of Aquatic and Fishery Sciences<br />
    2. 2. Outline<br /><ul><li>Background: epigenetics, DNA methylation
    3. 3. Results: characterization of DNA methylation in Pacific oysters
    4. 4. Current directions: method development
    5. 5. Implications</li></li></ul><li>Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br />toxins<br />nutrition<br />ENVIRONMENT<br />GENES (DNA)<br />
    6. 6. Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br />toxins<br />nutrition<br />EPIGENOME<br />(DNA methylation)<br />ENVIRONMENT<br />GENES (DNA)<br />
    7. 7. Background: <br />color<br />disease resistance<br />growth<br />TRAITS<br />pathogens<br />toxins<br />nutrition<br />EPIGENOME<br />(DNA methylation)<br />ENVIRONMENT<br />GENES (DNA)<br />
    8. 8. Epigenetics <br />Heritable changes in trait or phenotype, caused by a mechanism other than mutation to the DNA sequence<br />Most well understood epigenetic mechanism is DNA methylation<br />occurs in CpG in animals<br />functions<br />regulates gene expression<br />essential for development<br />genome stability<br />Me<br />C<br />G<br />G<br />C<br />
    9. 9. Characterization of DNA methylation in Pacific oysters<br />describe distribution of methylation<br />elucidate functional significance<br />
    10. 10. Results<br />Methylation Specific PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
    11. 11. Results<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
    12. 12. Results: gene-targeted approach<br />Methylation Sensitive PCR<br />5 stress related genes were examined<br />Identified CpG methylation in heat shock protein 70<br />Bisulfite sequencing<br />136 bp fragment: 1 of 7 cytosinesmethylated (homology to neuromedin-u receptor)<br />93 bp fragment: 1 of 2 cytosinesmethylated (homology to bromodomain adjacent to zinc finger domain)<br />Gavery & Roberts, 2010<br />
    13. 13. Results<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />
    14. 14. Results<br />CpG observed<br />CpG o/e<br />CpG expected<br />Methylation Sensitive PCR<br />Bisulfite sequencing<br />In silicoanalysis<br />predicted methylation status of 12,000 C. gigasgenes<br />
    15. 15. in silico approach<br />Principle:<br />Methylated cytosines are highly mutable<br />C  T<br />methylated regions of DNA are depleted of CpGdinucleotides over evolutionary time (CpG to TpG)<br />CpG observed<br />CpG o/e<br />CpG expected<br />m<br />high = unmethylated<br />low = methylated<br />
    16. 16. Regulation of Gene Expression<br />Gavery & Roberts, 2010<br />
    17. 17. Regulation of Gene Expression<br />‘housekeeping’<br />‘highly regulated’<br />Gavery & Roberts, 2010<br />
    18. 18. Summary:<br />oyster DNA is methylated<br />genes with differing regulatory requirements have different levels of DNA methylation<br />
    19. 19. Current Directions<br />Method evaluation/development:<br />challenges with non-model species<br />MBD-isolated genome sequencing (MBD-seq)<br />
    20. 20. Goal: MBD-seq<br />genome wide methylation analysis<br />evaluate in silicoresults<br />which genes are methylated?<br />which parts of the genome are methylated?<br />
    21. 21. Work Flow: MBD-seq<br />genomic DNA<br />
    22. 22. Work Flow: MBD-seq<br />1. fragmentation<br />
    23. 23. Work Flow: MBD-seq<br />2. enrichment<br />MBD<br />Y<br />MBD<br />Y<br />MBD<br />Y<br />
    24. 24. Work Flow: MBD-seq<br />MBD<br />Y<br />MBD<br />Y<br />MBD<br />Y<br />3. library prep & sequencing<br />
    25. 25. Work Flow: MBD-seq<br />4. mapping<br />genomic DNA – reference sequence<br />methylated<br />unmethylated<br />unmethylated<br />methylated<br />
    26. 26. Status: MBD-seq<br />MBD isolation: complete<br />library prep and sequencing: in progress<br />methylated <br />22%<br />unmethylated<br />78%<br />
    27. 27. Summary<br />genes with differing regulatory requirements have different levels of DNA methylation<br />currently evaluating & developing methods and tools to evaluate epigenetic mechanisms in bivalves<br />Implications…<br />
    28. 28. Implications: Environment<br />Endocrine disrupting compounds:<br />cause changes in DNA methylation patterns <br />associated with negative phenotypes<br />can be passed on for multiple generations<br />
    29. 29. Implications: Selective Breeding<br />Selective breeding can contribute to improved & predictable performance in oysters<br />Understanding geneticand epigenetic influences will increase predictability<br />
    30. 30. Implications: Hybrid Vigor<br />Heterosis (hybrid vigor)<br />mechanism not fully understood<br />epigenetic mechanisms have been proposed<br />better understanding will allow for greater control in predicting and manipulating gene expression in oysters <br />X<br />=<br />
    31. 31. Conclusion<br />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.<br />
    32. 32. Acknowledgements<br />UW, SAFS<br />Dr. Steven Roberts<br />Samuel White<br />Taylor Shellfish Farms<br />Joth Davis<br />National Shellfisheries Association<br />

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