Presentation given at 2014 Salish Sea Conference.

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 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. 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. )"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
D,?"*%$0-$8,+6082*0&%,-",8,1+*"-'?>?%&+-$012$5E
<%$12$',51"*(*+&%#-8*(10,7-'?>?%&+-9-
<"",'+&%-/*($&%58*%+$&%&%#-1"*+,&%-G
I$0(*/?0&%-
I*"+&8*+"*1&%5",0,$'&%#-@$8+*"-",8,1+*"-K-
I$"%*'&%,-'6%+2$',-9-
LG-?>&M?&+&%51"*+,&%-0&#$',-.=JGN-
+&&- .6%,&%-#$(($-82$&%O-@0$#,00$"-*?+,"-$"(
L0*%#$+*"-8*(10,7-1"*+,&%-K-
C6%8*0&%59
A0?+$(&%,-'6%+2,+$',-
A0?+$",/*7&%-G
A"$%?0&%'-
="$%'0$+&*%-@$8+*"-A?@9O-(&+*82*%/"&$0-
&'-3&"?'- <1*1+*'&'-&%2&>&+*"-P<)
P%+,"@,"*%5&%/?8,/-1"*+,&%-QQ-
R,08250&S,-1"*+,&%-KQ-
N&1"&%5>,+$59-
N*T5/,%'&+6-0&1*1"*+,&%-",8,1+*"5",0$+,/-1"*+,&%-E-
U%8*%3,%+&*%$0-(6*'&%5V>
D<.;-/,26/"*#,%$',-W?>&M?&%*%,X-@0$3*1"*+,&%-9O-(&+*82*%/"&$0-
D*',-",'&'+$%+-+*-@0?*7,+&%,-1"*+,&%-E-
),1+&/$',-FKY-/*($&%58*%+$&%&%#-1"*+,&%-K-
EY-S.$-ZZ5<[C*-"&>*%?80,*1"*+,&%-
Z*0?+,-8$""&,"-@$(&06-K-(,(>,"-G-
Z*0?+,-8$""&,"-@$(&06-Q4-(,(>,"-G-
)"*+,&%-+"$%'1*"+-1"*+,&%-Z,89E<-
Z($00-&%+,#"$0-(,(>"$%,-1"*+,&%-9Q
Z"8-S&%$',5$''*8&$+,/-12*'12*1"*+,&%-K5:
.D<-+*1*&'*(,"$',-9-
+CD<-1',?/*?"&/&%,-'6%+2$',-<O-(&+*82*%/"&$0-
V$'*"&%-
V$8?*0$"-1"*+,&%-'*"+&%#5$''*8&$+,/-1"*+,&%-9GI
]<Z;-8*(10,7-'?>?%&+-^

27. )"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
D,?"*%$0-$8,+6082*0&%,-",8,1+*"-'?>?%&+-$012$5E
<%$12$',51"*(*+&%#-8*(10,7-'?>?%&+-9-
<"",'+&%-/*($&%58*%+$&%&%#-1"*+,&%-G
I$0(*/?0&%-
I*"+&8*+"*1&%5",0,$'&%#-@$8+*"-",8,1+*"-K-
I$"%*'&%,-'6%+2$',-9-
LG-?>&M?&+&%51"*+,&%-0&#$',-.=JGN-
+&&- .6%,&%-#$(($-82$&%O-@0$#,00$"-*?+,"-$"(
L0*%#$+*"-8*(10,7-1"*+,&%-K-
C6%8*0&%59
A0?+$(&%,-'6%+2,+$',-
A0?+$",/*7&%-G
A"$%?0&%'-
="$%'0$+&*%-@$8+*"-A?@9O-(&+*82*%/"&$0-
&'-3&"?'- <1*1+*'&'-&%2&>&+*"-P<)
P%+,"@,"*%5&%/?8,/-1"*+,&%-QQ-
R,08250&S,-1"*+,&%-KQ-
N&1"&%5>,+$59-
N*T5/,%'&+6-0&1*1"*+,&%-",8,1+*"5",0$+,/-1"*+,&%-E-
U%8*%3,%+&*%$0-(6*'&%5V>
D<.;-/,26/"*#,%$',-W?>&M?&%*%,X-@0$3*1"*+,&%-9O-(&+*82*%/"&$0-
D*',-",'&'+$%+-+*-@0?*7,+&%,-1"*+,&%-E-
),1+&/$',-FKY-/*($&%58*%+$&%&%#-1"*+,&%-K-
EY-S.$-ZZ5<[C*-"&>*%?80,*1"*+,&%-
Z*0?+,-8$""&,"-@$(&06-K-(,(>,"-G-
Z*0?+,-8$""&,"-@$(&06-Q4-(,(>,"-G-
)"*+,&%-+"$%'1*"+-1"*+,&%-Z,89E<-
Z($00-&%+,#"$0-(,(>"$%,-1"*+,&%-9Q
Z"8-S&%$',5$''*8&$+,/-12*'12*1"*+,&%-K5:
.D<-+*1*&'*(,"$',-9-
+CD<-1',?/*?"&/&%,-'6%+2$',-<O-(&+*82*%/"&$0-
V$'*"&%-
V$8?*0$"-1"*+,&%-'*"+&%#5$''*8&$+,/-1"*+,&%-9GI
]<Z;-8*(10,7-'?>?%&+-^
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

28. )"*+,&%-%$(,'
4526/"*76+"61+$(&%,-",8,1+*"-9:-
<=)5>&%/&%#-8$'',++,-'?>5@$(&06-A-(,(>,"-9-
<%#&*+,%'&%58*%3,"+&%#-,%B6(,-
D,?"*%$0-$8,+6082*0&%,-",8,1+*"-'?>?%&+-$012$5E
<%$12$',51"*(*+&%#-8*(10,7-'?>?%&+-9-
<"",'+&%-/*($&%58*%+$&%&%#-1"*+,&%-G
I$0(*/?0&%-
I*"+&8*+"*1&%5",0,$'&%#-@$8+*"-",8,1+*"-K-
I$"%*'&%,-'6%+2$',-9-
LG-?>&M?&+&%51"*+,&%-0&#$',-.=JGN-
+&&- .6%,&%-#$(($-82$&%O-@0$#,00$"-*?+,"-$"(
L0*%#$+*"-8*(10,7-1"*+,&%-K-
C6%8*0&%59
A0?+$(&%,-'6%+2,+$',-
A0?+$",/*7&%-G
A"$%?0&%'-
="$%'0$+&*%-@$8+*"-A?@9O-(&+*82*%/"&$0-
&'-3&"?'- <1*1+*'&'-&%2&>&+*"-P<)
P%+,"@,"*%5&%/?8,/-1"*+,&%-QQ-
R,08250&S,-1"*+,&%-KQ-
N&1"&%5>,+$59-
N*T5/,%'&+6-0&1*1"*+,&%-",8,1+*"5",0$+,/-1"*+,&%-E-
U%8*%3,%+&*%$0-(6*'&%5V>
D<.;-/,26/"*#,%$',-W?>&M?&%*%,X-@0$3*1"*+,&%-9O-(&+*82*%/"&$0-
D*',-",'&'+$%+-+*-@0?*7,+&%,-1"*+,&%-E-
),1+&/$',-FKY-/*($&%58*%+$&%&%#-1"*+,&%-K-
EY-S.$-ZZ5<[C*-"&>*%?80,*1"*+,&%-
Z*0?+,-8$""&,"-@$(&06-K-(,(>,"-G-
Z*0?+,-8$""&,"-@$(&06-Q4-(,(>,"-G-
)"*+,&%-+"$%'1*"+-1"*+,&%-Z,89E<-
Z($00-&%+,#"$0-(,(>"$%,-1"*+,&%-9Q
Z"8-S&%$',5$''*8&$+,/-12*'12*1"*+,&%-K5:
.D<-+*1*&'*(,"$',-9-
+CD<-1',?/*?"&/&%,-'6%+2$',-<O-(&+*82*%/"&$0-
V$'*"&%-
V$8?*0$"-1"*+,&%-'*"+&%#5$''*8&$+,/-1"*+,&%-9GI
]<Z;-8*(10,7-'?>?%&+-^
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
— 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)

32. Next Steps
— Validation of differentially
methylated regions using
pyrosequencing
— Gene expression analysis
— Histological analysis of
female gonad samples