cerad epigenetics workshop ¯ster s 130819 partner nvh, peter … seminar august 2013... · 2013....
TRANSCRIPT
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CERAD epigenetics workshop Østerås 130819Partner NVH, Peter AleströmCorrelations between gene expression and epigenetic markers as a result of single and multiple stressor exposure induction, studied in zebrafish founder and offspring generations
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zCERAD epigenetics
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International Commission on Radiological Protection) RAPs https://wiki.ceh.ac.uk/display/rpemain/ICRP
Model organisms
https://wiki.ceh.ac.uk/display/rpemain/ICRPhttps://wiki.ceh.ac.uk/display/rpemain/ICRP
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Zebrafish is an excellent lab animal: • a robust small size aquarium fish with short life cycle (4 generations/year) • a transparent embryo which develops in a dish of water. • hatches day 3, starts feeding day 6• embryo development well characterized • the genome fully sequenced and annotated• multigenerational studies• keeping and monitoring a defined and optimized environment• mutants, advanced GM methods and tg models• low cost• 3R-oriented
zebrafish model publications
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gammaUV
multiple stressors
Centre for Environmental Radioactivity
(CERAD) 2013-2022
gammaUV
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MBT
6 M
ONTH
S
PUBE
RTY
MBT
PUBE
RTY
6 M
ONTH
S
F1 F2
zEmbryo96 w
•RNA•ChIP
•RNA•ChIP
•RNA•ChIP
•RNA•ChIP
•DNA damage•oncogenmarkers
pathology•liver•gonads•gut
F0 F1
MBT PU
BERT
Y
zebrafish F0
•DNA damage•oncogenmarkers
pathology•liver•gonads•gut
UVγ-rad
multip
le
stress
ors
early life stagestransgenerational
hESC
•RNA•ChIP
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RNA-seq uncovers the complete transcriptome MeDIP-seq uncovers the DNA methylomeChIP-seq uncovers mechanisms of the epigenome
Early life stage effects- zebrafish model
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ecotox exposureendpoints
IPA gene networks
Nourizadeh-Lillabadi R, Lyche JL, Almaas C, Stavik B, Moe SJ, Aleksandersen M, Berg V, Jakobsen KS, Stenseth NC, Skåre JU, Alestrøm P, Ropstad E. 2009. J Toxicol Environ Health A. 72:112-30.
genes regulated in ovary in response to lifelong exposure to mixture of Lake Mjøsa
Lyche, J. L. et al. Parental exposure to natural mixtures of POPs reduced embryo production and altered gene transcription in zebrafish embryos. Aquatic Toxicology (2012).doi:10.1016/j.aquatox.2012.08.019; Kraugerud, M. et al. Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio). Aquat. Toxicol. 116-117, 16–23 (2012); Lyche, J. L. et al. Natural mixtures of POPs affected body weight gain and induced transcription of genes involved in weight regulation and insulin signaling. Aquat. Toxicol. 102, 197–204 (2011); Berg, V. et al. Accumulation and effects of natural mixtures of persistent organic pollutants (POP) in Zebrafish after two generations of exposure. J. Toxicol. Environ. Health Part A 74, 407–423 (2011); Lyche, J. L. et al. Natural mixtures of persistent organic pollutants (POP) increase weight gain, advance puberty, and induce changes in gene expression associated with steroid hormones and obesity in female zebrafish. J. Toxicol. Environ. Health Part A 73, 1032–1057 (2010); Nourizadeh-Lillabadi, R. et al. Transcriptional regulation in liver and testis associated with developmental and reproductive effects in male zebrafish exposed to natural mixtures of persistent organic pollutants (POP). J. Toxicol. Environ. Health Part A 72, 112–130 (2009).
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transcriptome
and
epigenome
Correlation between the
=> gene expressionpredictions
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Already before zygotic genome activation (ZGA) >1000 developmentally-regulated promoters are marked with the activation mark H3K4me3
Lindeman et al. 2011 Dev Cell
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Lindeman et al. 2011 Dev Cell
non-exposedgenome wide transcriptome
multi-stressor exposed genome widetranscriptome
compare
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zebrafish miRNAs:
miRNAs (22nt): 740 in the Ensembl annotation
piRNAs (25-30nt): 105 (Wei 2012)
other ncRNAs: 225 tRNA (73-93nt) 3‘ or 5‘end derived
RNAs of 18-28nt (see 30-34nt mse-tsRNA in sperm and
oocytes doi:10.1038/cr.2012.181)
addition of non-template nt’s affects transcript
stability: 3’A (t1/2 up) and U (t1/2 down)
70% expressed at low levels
Wei et al. 2012. RNA 18, 915–929,
Aanes, Collas, Alestrom, Review subm. to Briefings in Functional Genomics
ncRNAsthousands of small and long non-coding RNAs during zebrafish embryogenesis are largely unknown ...................... Fish-miR project 2013-2015 aims at RNA-seq characterization with functional studies (UiN, UiT, NVH, Oregon Univ)
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Multi-Ztress Submitted Aug 14:2 year postdoc (Cecilia Winata, GIS)
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Effect of UV exposure on farmed fish
•In Europe most aquaculture is based on intensive sea cage production (salmon, Atlantic cod, Atlantic halibut, seabass, seabream)•Such intensively raised fish are exposed to unnaturally high levels of UV exposure.•It has been demonstrated that increased exposure to UV-‐B radiation has negative effects on growth, condition and immune function in juvenile Atlantic salmon.•Excessive UV exposure has also been reported to result in ¨summer lesion syndrome¨ in Atlantic salmon, with affected fish showing lesions in the skin behind the pectoral fins, rapidly deepening to involve the muscle layers.•Also, it has been suggested that increased UV exposure is one of the causal factors for the increased levels of fin rot and cataracts reported in farmed Atlantic salmon.•These abnormalities are both a major production and welfare concern to the aquaculture industry.•The objective of this CERAD RA5 study is to investigate whether high UV exposure can increase the incidence of a number of common production abnormalities in farmed salmon, including skin lesions, fin rot and cataracts.•Further the effect of high levels of UV exposure on growth rates and immune function (plasma IgM concentration and lysozyme activity) will be investigated.
CERAD RA5
Ian Mayer 2013
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Marie Curie Action application Multi-Ztress
Provisional work plan
Assessment of morphological effects of UV exposure on early embryos
1st first generaSon zebrafish embryos (F0) exposed to low dose of UV radiaSon
second generaSon zebrafish embryos (F1) indirectly exposed to UV radiaSon
Perform transcriptome profiling with RNA-‐seq and epigenome profiling with ChIP-‐seq
Analysis of next generaSon sequencing data
ValidaSon experiments and funcSonal analysis of several idenSfied target genes
Arrangement of future acSviSes, including grant applicaSons aiming at sub-‐goal 3 (Use the results to predict and test effects on juvenile/adult zebrafish and AtlanSc salmon)
At least two publicaSons in an internaSonal peer-‐reviewed journals
PresentaSon of findings in at least 1 internaSonal conference (EZM 2015)
month
3
6
12
15
18
22
24
48
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ongoing exposure experimentsgamma at Figaro
UV at NVH by NRPA
Gamma: 0, 0.4, 1, 4, 16 og 40 mGy/h for 24, 72, 120 h. Jan, Vidar, Hans-Christian, Ole-Christian, Terje, Thomas, Leonardo, Peter, Ana
UV: UVB 1, 3, 10 min. Terje, Thomas, Ellen
RNA-seq and ChIP-seq of ZGA stage zebrafish embryos with 0.4, 1, 4, 16 og 40 mGy/h gamma and defined doses UV
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ViewPoint ZebraBox
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IN Cell Investigator Zebrafish Analysis GE Healthcare Life Sciences
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early life stage mechanisms markers
mechanism specific tg reporter fish
Long term goal ..........
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NVHPeter AleströmLiqun Jenny Zhang (iPSC)Håvard Aanes (Bioinform.)Leonardo Martin (ncRNA)Jan L Lyche (toxicogenomics)Elisabeth LIe (Envir. tox)Jan E Paulsen(Food tox)Ian Mayer (Reprod. physiol)
Zebrafish facilityErik Ropstad (Head)Jan R T Sørby (Facility man.)Ana C. S. Tavara(Vet-technician)
CERAD-NVHNVH in kind PhD studentPhD quote student
Univ. OsloPhilippe CollasOlga ØstrupAndrew H ReinerLeif LindemanIngrid S Andersen
Singapore (GIS)S. MathavanCecilia Winata
Singapore NUSZhiyuan Gong
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NVHIn-kind: One senior researcher yearIn-kind: NVH CERAD PhD stip 1 (2014-2016) - bioinformaticsIn-kind: NVH-MatInf kvotestip (Sept 2013-Aug 2015) - zebrafish HTP screening
SøknaderIn-kind: Marie Curie postdoc (submitted Aug 14) - transgenerational gene expression and epigeneticsIn-kind: NVH CERAD PhD stip 2 (deadline 2. Sept) - transgenic reporter models
CERADLeif Lindeman: zebrafish and other species, epigenetics and small ncRNAs