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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]
2Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Contents
• Radioactivity and multiple stressors
• NIVA – research approach in CERAD
• Epigenetics
• Epigenetic research at NIVA
• CERAD - Plans
• Conclusions
3Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
4Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
NIVA - Effect assessment
Adverse effectExposure Response PertubationInteraction
Targets GenomicsTranscriptomicsMetabolomics
ProteomicsEpigenetics
ViabilityReproduction
GrowthDevelopment
Stressor
5Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
6Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Epigenetic mechanisms
7Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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.
8Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
9Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Selection of contaminants
• 5’-Azacytidine (AZA)
• Sodium arsenate (NAS)
• 2,3,7,8-Tetrachlorodibenzodioxin
(TCDD)
• 17α-ethynylestradiol (EE2)
• Diethylstilbestrol (DES)
10Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
11Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
High resolution melt (HRM)
• 0% methylated and 100 % methylatedZF-DNA wassynthesized and mixed to obtainstandard curve
• Shown here: igfbp
12Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Validation of the HRM
Direct bilsulphite sequencing
13Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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)
14Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
* *
* **
15Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
** *
16Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
17Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
AZA: pathwaysH
ypo
met
hyl
ated
18Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
AZA: pathways
Mol
ecul
arM
echa
nism
sof
Can
cer
Hyp
om
eth
ylat
ed
19Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
AZA: pathwaysH
yper
met
hyl
ated
20Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
AZA: pathwaysG
-Pro
tein
Cou
pled
Rec
epto
rS
igna
ling
Hyp
erm
eth
ylat
ed
21Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Hyp
om
eth
ylat
edSediment extract: pathways
22Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Sediment extract: pathways
-22
Wnt
/β-c
aten
inS
igna
ling
Hyp
om
eth
ylat
ed
23Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Sediment extract: pathwaysH
yper
met
hyl
ated
24Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Sediment extract: pathwayscA
MP
-med
iate
d si
gnal
ing
Hyp
erm
eth
ylat
ed
25Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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
26Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
Future work
L. minor C. reinhardtii D. magna D. rerio Salmonids
Biology √ √ √ √ √Testing √ √ √ √ √Clonalreproduction
√ √ √
Population √ √ √ (√)Genomics √ √ √ √Transcriptomics √ √ √ √Proteomics (√) (√) √ √Metabolomics (√) (√) (√) √ √Epigenetics √ √ √ √
27Knut Erik Tollefsen CERAD Epigenetics WS, 19/8-13
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)