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The role of DNA methylation in mediating the effects of estrogens in oysters

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Slides from Salish Sea Conference, May 2014

Slides from Salish Sea Conference, May 2014

Published in: Science, Technology
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  • 1. The role of DNA methylation in mediating the effects of estrogens in oysters Mackenzie Gavery & Steven Roberts School of Aquatic and Fishery Sciences University of Washington
  • 2. Outline 17α ethinylestradiol (EE2)
  • 3. Outline  Background  DNA methylation  EDCs & bivalves  Results  Implications 17α ethinylestradiol (EE2)
  • 4. GENES (DNA) TRAITS color growth disease resistance ENVIRONMENT Background
  • 5. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  • 6. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  • 7. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  • 8. Me C G C G
  • 9. Me C G C G Gene A TF X
  • 10. Me C G C G Gene A TF X
  • 11. Me C G C G Gene A TF X
  • 12. Me C G C G Gene A TF X
  • 13. Reproduction in oysters  Pacific oysters are sequential hermaphrodites  Sex determination has a genetic component, but influenced by environmental factors
  • 14. Reproduction in oysters Nonylphenol • offspring of exposed larvae had  intersex (Nice et al. 2003) 17α ethinylestradiol (EE2) •  rate of oocyte development (Andrew 2010) Estradiol • induces sex reversal (Mori 1969)  Pacific oysters are sequential hermaphrodites  Sex determination has a genetic component, but influenced by environmental factors
  • 15. Hypotheses  EE2 exposure will result in phenotypes such as skewed sex ratios and increased rate of gonad development  DNA methylation patterns will be altered in oysters exposed to EE2
  • 16. Hypotheses  EE2 exposure will result in phenotypes such as skewed sex ratios and increased rate of gonad development  DNA methylation patterns will be altered in oysters in upon exposure to EE2
  • 17. Estrogen Experiment 500 ng/L EE2: 150 oysters (n=50/tank) Control: 150 oysters (n=50/tank)
  • 18. Estrogen Experiment Control: 150 oysters (n=50/tank) Day 0 Day 7 Day 60  Samples: histology, gonad tissue 500 ng/L EE2: 150 oysters (n=50/tank)
  • 19. Estrogen Experiment Day 0 Day 7 Day 60 500 ng/L EE2: 150 oysters (n=50/tank) Control: 150 oysters (n=50/tank)  Samples: histology, gonad tissue
  • 20. Results: Day 60 sex determination 0% 20% 40% 60% 80% 100% control EE2 PercentofTotalOysters Hermaphrodite Unknown Male Female
  • 21. Results: Day 60 size of females 30 35 40 45 50 55 60 length width mm shell size (mm) control EE2 17 18 19 20 21 22 23 24 control EE2 grams total weight (g)
  • 22. Results: DNA methylation control 150 oysters (n=50/tank) Day 0 Day 7 Day 60 EE2 (500ng/L) 150 oysters (n=50/tank)
  • 23. Results: DNA methylation control 150 oysters (n=50/tank) Day 0 Day 7 Day 60 EE2 (500ng/L) 150 oysters (n=50/tank)
  • 24. MBD-Chip NimbleGen 3 x 720k Array compare DNA methylation control EE2 exposed MBD MBD Results: DNA methylation
  • 25.  Results:  45 differentially methylated regions (DMR)  DMRs were located in 38 different genes Results: DNA methylation
  • 26. Protein names 5-hydroxytryptamine receptor 1B ATP-binding cassette sub-family G member 1 Angiotensin-converting enzyme Neuronal acetylcholine receptor subunit alpha-6 Anaphase-promoting complex subunit 1 Arrestin domain-containing protein 3 Calmodulin Corticotropin-releasing factor receptor 2 Carnosine synthase 1 E3 ubiquitin-protein ligase DTX3L Dynein gamma chain, flagellar outer arm Elongator complex protein 2 Ryncolin-1 Glutamine synthetase Glutaredoxin 3 Granulins Translation factor Guf1, mitochondrial Apoptosis inhibitor IAP Interferon-induced protein 44 Kelch-like protein 24 Liprin-beta-1 Low-density lipoprotein receptor-related protein 6 Unconventional myosin-Vb NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial Nose resistant to fluoxetine protein 6 Peptidase M20 domain-containing protein 2 60 kDa SS-A/Ro ribonucleoprotein Solute carrier family 28 member 3 Solute carrier family 45 member 3 Protein transport protein Sec16A Small integral membrane protein 14 Src kinase-associated phosphoprotein 2-B DNA topoisomerase 1 tRNA pseudouridine synthase A, mitochondrial Vasorin Vacuolar protein sorting-associated protein 13C WASH complex subunit 7
  • 27. Protein names 5-hydroxytryptamine receptor 1B ATP-binding cassette sub-family G member 1 Angiotensin-converting enzyme Neuronal acetylcholine receptor subunit alpha-6 Anaphase-promoting complex subunit 1 Arrestin domain-containing protein 3 Calmodulin Corticotropin-releasing factor receptor 2 Carnosine synthase 1 E3 ubiquitin-protein ligase DTX3L Dynein gamma chain, flagellar outer arm Elongator complex protein 2 Ryncolin-1 Glutamine synthetase Glutaredoxin 3 Granulins Translation factor Guf1, mitochondrial Apoptosis inhibitor IAP Interferon-induced protein 44 Kelch-like protein 24 Liprin-beta-1 Low-density lipoprotein receptor-related protein 6 Unconventional myosin-Vb NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial Nose resistant to fluoxetine protein 6 Peptidase M20 domain-containing protein 2 60 kDa SS-A/Ro ribonucleoprotein Solute carrier family 28 member 3 Solute carrier family 45 member 3 Protein transport protein Sec16A Small integral membrane protein 14 Src kinase-associated phosphoprotein 2-B DNA topoisomerase 1 tRNA pseudouridine synthase A, mitochondrial Vasorin Vacuolar protein sorting-associated protein 13C WASH complex subunit 7 Gene Ontology (GO Slim) Count transport 10 cell organization and biogenesis 8 other metabolic processes 7 signal transduction 6 protein metabolism 5 RNA metabolism 5 developmental processes 4 cell cycle and proliferation 3 death 2 stress response 2 cell-cell signaling 1 DNA metabolism 1
  • 28. Protein names 5-hydroxytryptamine receptor 1B ATP-binding cassette sub-family G member 1 Angiotensin-converting enzyme Neuronal acetylcholine receptor subunit alpha-6 Anaphase-promoting complex subunit 1 Arrestin domain-containing protein 3 Calmodulin Corticotropin-releasing factor receptor 2 Carnosine synthase 1 E3 ubiquitin-protein ligase DTX3L Dynein gamma chain, flagellar outer arm Elongator complex protein 2 Ryncolin-1 Glutamine synthetase Glutaredoxin 3 Granulins Translation factor Guf1, mitochondrial Apoptosis inhibitor IAP Interferon-induced protein 44 Kelch-like protein 24 Liprin-beta-1 Low-density lipoprotein receptor-related protein 6 Unconventional myosin-Vb NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial Nose resistant to fluoxetine protein 6 Peptidase M20 domain-containing protein 2 60 kDa SS-A/Ro ribonucleoprotein Solute carrier family 28 member 3 Solute carrier family 45 member 3 Protein transport protein Sec16A Small integral membrane protein 14 Src kinase-associated phosphoprotein 2-B DNA topoisomerase 1 tRNA pseudouridine synthase A, mitochondrial Vasorin Vacuolar protein sorting-associated protein 13C WASH complex subunit 7 Gene Ontology (GO Slim) Count transport 10 cell organization and biogenesis 8 other metabolic processes 7 signal transduction 6 protein metabolism 5 RNA metabolism 5 developmental processes 4 cell cycle and proliferation 3 death 2 stress response 2 cell-cell signaling 1 DNA metabolism 1  ATP-binding cassette protein  Serotonin receptor  Low density lipoprotein receptor  Granulin
  • 29. Summary  EE2 treatment did not affect sex ratios, but exposed females were larger than controls  DMRs were identified within 1 week of EE2 exposure  Genes with DMRs are functionally diverse (e.g. growth, immune, reproduction)
  • 30. Implications  DNA methylation may play a role in mediating responses to EDCs in bivalves  Epigenetic marks may provide early indicators of EDC exposure in aquatic species
  • 31. Acknowledgements  Roberts Lab: Steven Roberts Samuel White Emma Timmins-Schiffman Claire Ellis Brent Vadopalas Jake Heare  Taylor Shellfish: Joth Davis Molly Jackson  Irv Shultz (Battelle PNNL)

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