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

Thank you for your time!

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