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

Outline17α 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

GC

G

Me

C

GC

G

Gene A

TF X

Me

C

GC

G

Gene A

TF X

Me

C

GC

G

Gene A

TF X

Me

C

GC

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 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

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

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)

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

Results: Day 60

sex determination

0%

20%

40%

60%

80%

100%

control EE2

Pe

rce

nt o

f To

tal O

yste

rs

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

gra

ms

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)

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 methylation

Organism Proteinnames

Didelphisvirginiana 5-hydroxytryptaminereceptor1B

Homosapiens ATP-bindingcassettesub-familyGmember1

Gallusgallus Angiotensin-convertingenzyme

Rattusnorvegicus Neuronalacetylcholinereceptorsubunitalpha-6

Homosapiens Anaphase-promotingcomplexsubunit1

Musmusculus Arrestindomain-containingprotein3

Patinopectensp. Calmodulin

Xenopuslaevis Corticotropin-releasingfactorreceptor2

Homosapiens Carnosinesynthase1

Homosapiens E3ubiquitin-proteinligaseDTX3L

Chlamydomonasreinhardtii Dyneingammachain,flagellarouterarmDaniorerio Elongatorcomplexprotein2

Cerberusrynchops Ryncolin-1

Acomyscahirinus Glutaminesynthetase

Daniorerio Glutaredoxin3

Homosapiens Granulins

Musmusculus TranslationfactorGuf1,mitochondrial

Cydiapomonellagranulosisvirus ApoptosisinhibitorIAP

Musmusculus Interferon-inducedprotein44

Rattusnorvegicus Kelch-likeprotein24

Musmusculus Liprin-beta-1

Musmusculus Low-densitylipoproteinreceptor-relatedprotein6

Homosapiens Unconventionalmyosin-Vb

Pongopygmaeus NADHdehydrogenase[ubiquinone]flavoprotein1,mitochondrialCaenorhabditiselegans Noseresistanttofluoxetineprotein6

Xenopustropicalis PeptidaseM20domain-containingprotein2

Homosapiens 60kDaSS-A/Roribonucleoprotein

Musmusculus Solutecarrierfamily28member3

Macacafascicularis Solutecarrierfamily45member3

Homosapiens ProteintransportproteinSec16ABostaurus Smallintegralmembraneprotein14Xenopuslaevis Srckinase-associatedphosphoprotein2-B

Xenopuslaevis DNAtopoisomerase1Musmusculus tRNApseudouridinesynthaseA,mitochondrialMusmusculus VasorinHomosapiens Vacuolarproteinsorting-associatedprotein13CHomosapiens WASHcomplexsubunit7

Organism Proteinnames

Didelphisvirginiana 5-hydroxytryptaminereceptor1B

Homosapiens ATP-bindingcassettesub-familyGmember1

Gallusgallus Angiotensin-convertingenzyme

Rattusnorvegicus Neuronalacetylcholinereceptorsubunitalpha-6

Homosapiens Anaphase-promotingcomplexsubunit1

Musmusculus Arrestindomain-containingprotein3

Patinopectensp. Calmodulin

Xenopuslaevis Corticotropin-releasingfactorreceptor2

Homosapiens Carnosinesynthase1

Homosapiens E3ubiquitin-proteinligaseDTX3L

Chlamydomonasreinhardtii Dyneingammachain,flagellarouterarmDaniorerio Elongatorcomplexprotein2

Cerberusrynchops Ryncolin-1

Acomyscahirinus Glutaminesynthetase

Daniorerio Glutaredoxin3

Homosapiens Granulins

Musmusculus TranslationfactorGuf1,mitochondrial

Cydiapomonellagranulosisvirus ApoptosisinhibitorIAP

Musmusculus Interferon-inducedprotein44

Rattusnorvegicus Kelch-likeprotein24

Musmusculus Liprin-beta-1

Musmusculus Low-densitylipoproteinreceptor-relatedprotein6

Homosapiens Unconventionalmyosin-Vb

Pongopygmaeus NADHdehydrogenase[ubiquinone]flavoprotein1,mitochondrialCaenorhabditiselegans Noseresistanttofluoxetineprotein6

Xenopustropicalis PeptidaseM20domain-containingprotein2

Homosapiens 60kDaSS-A/Roribonucleoprotein

Musmusculus Solutecarrierfamily28member3

Macacafascicularis Solutecarrierfamily45member3

Homosapiens ProteintransportproteinSec16ABostaurus Smallintegralmembraneprotein14Xenopuslaevis Srckinase-associatedphosphoprotein2-B

Xenopuslaevis DNAtopoisomerase1Musmusculus tRNApseudouridinesynthaseA,mitochondrialMusmusculus VasorinHomosapiens Vacuolarproteinsorting-associatedprotein13CHomosapiens WASHcomplexsubunit7

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

Organism Proteinnames

Didelphisvirginiana 5-hydroxytryptaminereceptor1B

Homosapiens ATP-bindingcassettesub-familyGmember1

Gallusgallus Angiotensin-convertingenzyme

Rattusnorvegicus Neuronalacetylcholinereceptorsubunitalpha-6

Homosapiens Anaphase-promotingcomplexsubunit1

Musmusculus Arrestindomain-containingprotein3

Patinopectensp. Calmodulin

Xenopuslaevis Corticotropin-releasingfactorreceptor2

Homosapiens Carnosinesynthase1

Homosapiens E3ubiquitin-proteinligaseDTX3L

Chlamydomonasreinhardtii Dyneingammachain,flagellarouterarmDaniorerio Elongatorcomplexprotein2

Cerberusrynchops Ryncolin-1

Acomyscahirinus Glutaminesynthetase

Daniorerio Glutaredoxin3

Homosapiens Granulins

Musmusculus TranslationfactorGuf1,mitochondrial

Cydiapomonellagranulosisvirus ApoptosisinhibitorIAP

Musmusculus Interferon-inducedprotein44

Rattusnorvegicus Kelch-likeprotein24

Musmusculus Liprin-beta-1

Musmusculus Low-densitylipoproteinreceptor-relatedprotein6

Homosapiens Unconventionalmyosin-Vb

Pongopygmaeus NADHdehydrogenase[ubiquinone]flavoprotein1,mitochondrialCaenorhabditiselegans Noseresistanttofluoxetineprotein6

Xenopustropicalis PeptidaseM20domain-containingprotein2

Homosapiens 60kDaSS-A/Roribonucleoprotein

Musmusculus Solutecarrierfamily28member3

Macacafascicularis Solutecarrierfamily45member3

Homosapiens ProteintransportproteinSec16ABostaurus Smallintegralmembraneprotein14Xenopuslaevis Srckinase-associatedphosphoprotein2-B

Xenopuslaevis DNAtopoisomerase1Musmusculus tRNApseudouridinesynthaseA,mitochondrialMusmusculus VasorinHomosapiens Vacuolarproteinsorting-associatedprotein13CHomosapiens WASHcomplexsubunit7

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

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 RobertsSamuel White Emma Timmins-SchiffmanClaire EllisBrent VadopalasJake Heare

Taylor Shellfish:

Joth DavisMolly Jackson

Irv Shultz (Battelle PNNL)