Epigeneticsin aquaticplant, invertebrateand vertebrate models seminar august 2013/Tollefse… ·...

1Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13

Epigenetics in aquatic plant, invertebrate and vertebrate

models

NIVA - experiences and plans

Eivind Farmen (NIVA/MDIR) and Knut Erik Tollefsen (NIVA/UMB)

Contact: [email protected]


Contents

• Radioactivity and multiple stressors

• NIVA – research approach in CERAD

• Epigenetics

• Epigenetic research at NIVA

• CERAD - Plans

• Conclusions


Radioactivity & Multiple stressors

Key issues

• No such thing as an exposure to a single stressor!

• Organisms are constantly in contact with a large number of different anthropogenic & natural stressors

• Often exposed to sub-effect concentrationsof single stressors

• Risk assessment is often performed onsingle stressor effect

• Species & life-stage specific differences in sensitivity

• Uncertainty large as the effect ofcombination of stressors are unknown

Are we adequately adressing the risk of multiple stressors?

Radioactivity

UV radiation

Metals

Organics

Climate change


NIVA - Effect assessment

Adverse effectExposure Response PertubationInteraction

Targets GenomicsTranscriptomicsMetabolomics

ProteomicsEpigenetics

ViabilityReproduction

GrowthDevelopment

Stressor


Risk assessment

Extrapolation between species & organisational levels

� Combined toxicity modelling� Predictive risk assessment� Improved impact & risk assessment� Cell to organism extrapolation� Organism to population extrapolation� Life-stage sensitivity� Species sensitivity determination

-Exposure

-Effect

-JMO -TOH


Epigenetic mechanisms


DNA methylation in ZFL cells

Why in vitro?• Limited knowledge on how contaminants may influence DNA

methylation in aquatic organisms

• In vitro systems offer high-throughput capabilities

• Multiple endpoints analysed simoultanously

Analyse the potential for DNA methylation change in genes

important to adverse disease

Finne, E.F., Hultman, M.T., 1, Anglès d'Auriac, M., Tollefsen, K.E. (Submitted). Development of an aquatic in vitro screening system for determination of potential DNA methylation alterations relevant to ecotoxicology. JTEH.


MethodsCytotoxicity-Metabolic activity

Single gene methylationscreening -HRM: High resolution melt-Direct bisulfite sequencing

Global methylomeanalysis-zebrafish ChIP-chip-(Pyrosequencing)

Expose cells48-96 h

ZF-L zebrafish hepatomacell line


Selection of contaminants

• 5’-Azacytidine (AZA)

• Sodium arsenate (NAS)

• 2,3,7,8-Tetrachlorodibenzodioxin

(TCDD)

• 17α-ethynylestradiol (EE2)

• Diethylstilbestrol (DES)


Cytotoxicity

Concentration (mol/L)

Met

abo

lic in

hib

itio

n (

%)

10- 1 4 10- 1 3 10- 1 2 10- 1 1 10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 40

20

40

60

80

100

120

TCDD

NAS

EE2

DES

AZA


High resolution melt (HRM)

• 0% methylated and 100 % methylatedZF-DNA wassynthesized and mixed to obtainstandard curve

• Shown here: igfbp


Validation of the HRM

Direct bilsulphite sequencing


Screening: selection of genes

• jun proto-oncogene (c-jun)

• cyclin-dependent kinase inhibitor 2B (cdkn2a)

• SHC transforming protein 1 (shc)

• Angiopoetin-like 3 (angpl3)

• glutathione S-transferase P1 (gstp1)

• insulin-like growth factor binding protein 1b

(igfbp1b)


5’-Azacytidine (AZA)AZA

% m

eth

yla

ted

cyto

sin

e

0.1%

DM

SO

0.5

µM A

ZA

0.1%

DM

SO

0.5

µM A

ZA

0.1%

DM

SO

0.5

µM A

ZA

0.1%

DM

SO

0.5

µM A

ZA

0.1%

DM

SO

0.5

µM A

ZA

0.1%

DM

SO

0.5

µM A

ZA

0

20

40

60

80

100igfbp

anglp3

gstp1

cdkn2a

c-jun

shc

* *

* **


Sodium arsenate (NAS)

NAS%

meth

yla

ted

cyto

sin

e

0.1%

DM

SO

10 µ

M N

AS

0.1%

DM

SO

10 µ

M N

AS

0.1%

DM

SO

10 µ

M N

AS

0.1%

DM

SO

10 µ

M N

AS

0.1%

DM

SO

10 µ

M N

AS

0.1%

DM

SO

10 µ

M N

AS

0

20

40

60

80

100igfbp

anglp3

gstp1

cdkn2a

c-jun

shc

** *


What about other genes?

ChIP-chip: Zebrafish tiling array (Mirbahai et al. 2011)

1) 1WANOVA; P<0.05, No-BFR.

2) FC2 applied, resulting in 1252 hypermethylated features and 2208

hypomethylated features (AZA)

3) Filtering on unique genes led to 789 hyper- and 1021

hypomethylated features

4) Roughly 50% were mapped against human orthologs and subjected

to IPA analysis to identify enriched canonical pathways


AZA: pathwaysH

ypo

met

hyl

ated


AZA: pathways

Mol

ecul

arM

echa

nism

sof

Can

cer

Hyp

om

eth

ylat

ed


AZA: pathwaysH

yper

met

hyl

ated


AZA: pathwaysG

-Pro

tein

Cou

pled

Rec

epto

rS

igna

ling

Hyp

erm

eth

ylat

ed


Hyp

om

eth

ylat

edSediment extract: pathways


Sediment extract: pathways

-22

Wnt

/β-c

aten

inS

igna

ling

Hyp

om

eth

ylat

ed


Sediment extract: pathwaysH

yper

met

hyl

ated


Sediment extract: pathwayscA

MP

-med

iate

d si

gnal

ing

Hyp

erm

eth

ylat

ed


Conclusions• Cell line exposure in combination with HRM provides cost efficient

high throughput screening

• Exposure of ZF-L cells to AZA lead to hypomethylation of genes with both low and

high basal methylation (gstp1, igfbp1b, angpl3),

• ChIP-chip (MeDIP) technique enabled functional analysis:

• AZA: Global hypomethylation, BUT hypermethylation also observed

• Sediment extract: Distorted methylomics incl. pathways related to cancers

• In vitro cell system appears to be sensitive to analyse potential

DNA methylation abberations

• Verification by gene expression profiling pending

• Future: development of epigenetics in (L. minor), C. reinhardtii, D.

magna, D. rerio and salmonids using a suite of methods


Future work

L. minor C. reinhardtii D. magna D. rerio Salmonids

Biology √ √ √ √ √Testing √ √ √ √ √Clonalreproduction

√ √ √

Population √ √ √ (√)Genomics √ √ √ √Transcriptomics √ √ √ √Proteomics (√) (√) √ √Metabolomics (√) (√) (√) √ √Epigenetics √ √ √ √


Acknowledgements

CoworkersMaria T. Hultman (NIVA/UMB)Marc Anglès d'Auriac (NIVA)Karina Petersen (NIVA) Harald Hasle Heiaas (NIVA)You Song (UMB/NIVA)Ailbhe Macken (NIVA/UMB)

Financial contribution• NFR-project 196318 alterREACH• Ministry of Environment

(Institutional funding)

Infrastructure funding• The Research Council of Norway

(NFR-AViT 183929)CollaboratorsLeda Mirbahai (UoB)Timothy Williams (UoB)Kevin Chipman (UoB)

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