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

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

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

Me

C

G C

G

Me

C

G C

G Gene A

TF X

Reproduction in oysters �  Pacific oysters are sequential hermaphrodites

�  Sex determination has a genetic component, but influenced by environmental factors

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

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

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

Estrogen Experiment

500 ng/L EE2: 150 oysters (n=50/tank)

Control: 150 oysters (n=50/tank)

Estrogen Experiment


Day 0 Day 7 Day 60

�  Samples: histology, gonad tissue


Estrogen Experiment

Day 0 Day 7 Day 60



�  Samples: histology, gonad tissue

Results: Day 60 sex determination

0%

20%

40%

60%

80%

100%

control EE2

Per

cent

of T

otal

Oys

ters

Hermaphrodite

Unknown

Male

Female

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

gram

s

total weight (g)

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:

� 45 differentially methylated regions (DMR)

�  DMRs were located in 38 different genes


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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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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

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)

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

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)

Next Steps

�  Validation of differentially methylated regions using pyrosequencing

�  Gene expression analysis

�  Histological analysis of

female gonad samples

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

Science

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