Effects of low dose rates gamma irradiation on histone modifications during zebrafish embryogenesis Leif Lindeman Norwegian University of Life Sciences 60 Co exposure facility Figaro Centre for Environmental Radioactivity (CERAD CoE) will provide new scientific knowledge and tools for better protection of people and the environment from harmful effects of radiation (cerad.nmbu.no). Vetbio
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Effects of low dose rates gamma irradiation on histone modifications during zebrafish embryogenesis
Leif Lindeman Norwegian University of Life Sciences
60Co exposure facility Figaro
Centre for Environmental Radioactivity (CERAD CoE) will provide new
scientific knowledge and tools for better protection of people and the
environment from harmful effects of radiation (cerad.nmbu.no).
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OBJECTIVE: to assess epigenetic changes in zebrafish embryos after exposure to low dose rates of external 60Co gamma radiation.
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Why: To learn more about environmental chronic low dose radiation How: Zebrafish embryos as a model for study epigenetic effects
Epigenetics – Changes in gene expression and chromatin organization that are propagated through cell divisions and not result in alterations in DNA sequence.
Main components:
- post translational histone modifications
-DNA methylation
-ncRNAs
Cells with identical genomes can have different epigenomes
Herceg et al., 2013
Same genotype
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Phenotype 1
Phenotype 2
The complex of histones and DNA is referred to as chromatin
Klug and Cummings, 1997
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Histone modifications have a regulatory role on gene expression
chromatin compaction H3K9me3 H3K27me3 open chromatin H3K4me3 H3K9ac H3K36me3 H4ac
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Modifications associated with:
Scharf and Imhof 2011
Chromatin immunoprecipitation
(ChIP) is a method for mapping
the genomic position of DNA
binding proteins
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Dahl & Collas, Front Biosci. 2008
sequencing
Philippe Collas (ed.), Chromatin
Immunoprecipitation Assays,
Methods in Molecular Biology 567,
Humana Press, a part of Springer
Science+Business Media, LLC 2009
Experimental design gamma exposure studies
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Hurem et al. in progress
Changes in gene expression is correlated with radiation dose
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Upregulated
Downregulated
Total number of genes
Direct exposure of 0.03 Gy gamma radiation during embryogenesis causes enrichment of histone modifications
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hnf4a
3h 10 mGy/h
2.3 hpf embryos
5.5 hpf embryos ChIP-PCR
TSS - 4 kb +0.5 kb +2 kb
Control Exposed Control Exposed Control Exposed Control Exposed
Exposure of 6,7 Gy gamma radiation during gametogenesis causes enrichment of histone modifications in F1 offspring
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TSS - 4 kb +0.5 kb +2 kb
Control Exposed Control Exposed Control Exposed Control Exposed
F0 5 mpf zebrafish
6 mpf zebrafish
F1 5.5 hpf embryos ChIP
28d 10 mGy/h
A dose response appear at K4 and K9 and histone H3 on b-actin2 as a response to increasing amount of gamma- radiation in 5.5 hpf embryos
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3h
2.3 hpf embryos
5.5 hpf embryos ChIP-PCR
TSS
Bactin2 (non-DEG) bactin2 (non DEG)
0 mGy
1.5 mGy
3 mGy
30 mGy
120 mGy
Dose responses appear at H3K4me3, H3K9m3 and H3K27me3 after gamma radiation during embryogenesis
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Gene Differentially expressed
Histone PTM dose response
bactin2 Non DEG H3K4me3, H3K9ac
Cebpa H3K27me2
hnf4a H3K4me3,K27me3, K9me3, K9ac
vegfab H3K4me3, H3K9ac
gmnn H3K4me3, H3K9ac
il15 Not expressed No enrichment
Conclusions:
Gamma radiation affects epigenetic signatures after exposure
• at radiation doses at 1.5 mGy
• through gametogenesis (28d) and/or embryogenesis (3h).
• Histone PTMs dose responsis appear in exposed embryos
Zebrafish appears as a sensitive model for low dose exposure
experiments and ChIP as a sensitive method for detection of epigenetic
changes at histone PTMs
More loci are needed to get a more comprehensive picture. ChIP-seq
gives genome wide profiling.
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NMBU Campus Oslo Peter Aleström Jan L Lyche Jorke Kamstra Leonardo Martin Selma Hurem NMBU Campus Ås Brit Salbu Deborah Oughton