The role of DNA methylation in
mediating the effects of
estrogens in oysters
Mackenzie Gavery & Steven Roberts
School of A...
Outline
17α ethinylestradiol
(EE2)
Outline
— Background
— DNA methylation
— EDCs & bivalves
— Results
— Implications
17α ethinylestradiol
(EE2)
GENES (DNA)
TRAITS
color
growth
disease resistance
ENVIRONMENT
Background
GENES (DNA)
EPIGENOME
(DNA methylation)
TRAITS
color
growth
disease resistance
ENVIRONMENT
Background
GENES (DNA)
EPIGENOME
(DNA methylation)
TRAITS
color
growth
disease resistance
ENVIRONMENT
Background
GENES (DNA)
EPIGENOME
(DNA methylation)
TRAITS
color
growth
disease resistance
ENVIRONMENT
Background
Me
C
G
C
G
Me
C
G
C
G
Gene A
TF X
Me
C
G
C
G
Gene A
TF X
Me
C
G
C
G
Gene A
TF X
Me
C
G
C
G
Gene A
TF X
Reproduction in oysters
—  Pacific oysters are sequential hermaphrodites
—  Sex determination has a genetic component, b...
Reproduction in oysters
Nonylphenol
•  offspring of exposed
larvae had é intersex
(Nice et al. 2003)
17α ethinylestradiol...
Hypotheses
—  EE2 exposure will result in phenotypes such as
skewed sex ratios and increased rate of gonad
development
—...
Hypotheses
—  EE2 exposure will result in phenotypes such as
skewed sex ratios and increased rate of gonad
development
—...
Estrogen Experiment
500 ng/L EE2: 150 oysters (n=50/tank)
Control: 150 oysters (n=50/tank)
Estrogen Experiment
Control: 150 oysters (n=50/tank)
Day 0 Day 7 Day 60
—  Samples: histology, gonad tissue
500 ng/L EE2:...
Estrogen Experiment
Day 0 Day 7 Day 60
500 ng/L EE2: 150 oysters (n=50/tank)
Control: 150 oysters (n=50/tank)
—  Samples:...
Results: Day 60
sex determination
0%
20%
40%
60%
80%
100%
control EE2
PercentofTotalOysters
Hermaphrodite
Unknown
Male
Fem...
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
c...
Results: DNA methylation
control 150 oysters (n=50/tank)
Day 0 Day 7 Day 60
EE2 (500ng/L) 150 oysters (n=50/tank)
Results: DNA methylation
control 150 oysters (n=50/tank)
Day 0 Day 7 Day 60
EE2 (500ng/L) 150 oysters (n=50/tank)
MBD-Chip
NimbleGen
3 x 720k Array
compare
DNA
methylation
control
EE2
exposed
MBD
MBD
Results: DNA methylation
—  Results:
— 45 differentially methylated regions (DMR)
—  DMRs were located in 38 different genes
Results: DNA methyl...
)"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
...
)"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
...
)"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
...
Summary
—  EE2 treatment did not
affect sex ratios, but
exposed females were
larger than controls
—  DMRs were identifie...
Implications
—  DNA methylation may play a role in mediating
responses to EDCs in bivalves
—  Epigenetic marks may provi...
Acknowledgements
—  Roberts Lab:
Steven Roberts
Samuel White
Emma Timmins-Schiffman
Claire Ellis
Brent Vadopalas
Jake Hea...
Next Steps
—  Validation of differentially
methylated regions using
pyrosequencing
—  Gene expression analysis
—  Histo...
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The role of DNA methylation in mediating the effects of estrogens in oysters

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Presentation given at 2014 Salish Sea Conference.

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

  1. 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. 2. Outline 17α ethinylestradiol (EE2)
  3. 3. Outline — Background — DNA methylation — EDCs & bivalves — Results — Implications 17α ethinylestradiol (EE2)
  4. 4. GENES (DNA) TRAITS color growth disease resistance ENVIRONMENT Background
  5. 5. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  6. 6. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  7. 7. GENES (DNA) EPIGENOME (DNA methylation) TRAITS color growth disease resistance ENVIRONMENT Background
  8. 8. Me C G C G
  9. 9. Me C G C G Gene A TF X
  10. 10. Me C G C G Gene A TF X
  11. 11. Me C G C G Gene A TF X
  12. 12. Me C G C G Gene A TF X
  13. 13. Reproduction in oysters —  Pacific oysters are sequential hermaphrodites —  Sex determination has a genetic component, but influenced by environmental factors
  14. 14. Reproduction in oysters Nonylphenol •  offspring of exposed larvae had é intersex (Nice et al. 2003) 17α ethinylestradiol (EE2) •  é 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. 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. 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. 17. Estrogen Experiment 500 ng/L EE2: 150 oysters (n=50/tank) Control: 150 oysters (n=50/tank)
  18. 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. 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. 20. Results: Day 60 sex determination 0% 20% 40% 60% 80% 100% control EE2 PercentofTotalOysters Hermaphrodite Unknown Male Female
  21. 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. 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. 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. 24. MBD-Chip NimbleGen 3 x 720k Array compare DNA methylation control EE2 exposed MBD MBD Results: DNA methylation
  25. 25. —  Results: — 45 differentially methylated regions (DMR) —  DMRs were located in 38 different genes Results: DNA methylation
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   Gene  Ontology  (GO  Slim)   Count      transport   10      cell  organiza:on  and  biogenesis   8      other  metabolic  processes   7      signal  transduc:on   6      protein  metabolism   5      RNA  metabolism   5      developmental  processes   4      cell  cycle  and  prolifera:on   3      death   2      stress  response   2      cell-­‐cell  signaling   1      DNA  metabolism   1  
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  29. 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. 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. 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)
  32. 32. Next Steps —  Validation of differentially methylated regions using pyrosequencing —  Gene expression analysis —  Histological analysis of female gonad samples

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