Proton irradiation induces persistent and tissue-specific DNA methylation changes in the left ventricle and hippocampus

Abstract

Background: Proton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression.

Results: The DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled.

Conclusions: The results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.

Keywords: DNA methylation; Epigenetic; Hippocampus; Left ventricle; Proton irradiation; RNAseq; Ventricle.

Fig. 1

5hmC is enriched in the gene bodies of active genes while 5mC is depleted at the 5’ regions of active genes. (a and b). Pie chart depicts accumulation of 5hmC or 5mC DIP-Seq signal in the indicated genomic regions. Data that matched multiple categories was matched to the closest annotation. (c and d). Heatmaps depict DIP-Seq sequence density (500 bp bins) at RefSeq genes rank-ordered by levels of gene expression (RNA-Seq data from other studies). The color ramp shows min-max DIP/ChIP signal density normalized to the 80^th quantile. The black bar illustrates the transcribed region with the 5’ end on the left

Fig. 2

a-d Pie chart depicts accumulation of 5hmC (a, b) and 5mC (c, d) DIP-Seq signal in the indicated genomic repetitive sequence annotation (DIP-Seq counts per million repeat sequences). Data that matched multiple categories were matched exclusively to the closest annotation

Fig. 3

a-d Strip plots indicate the accumulation of normalized 5hmC and 5mC DiP-Seq sequences (counts per million repeat sequences). The decreased accumulation of 5mC in ventricle tRNA and rRNA repeats, as well as the increased accumulation of 5hmC in ventricle tRNA repeats, was significant (p < 0.05, n = 4)

Fig. 4

a and b Venn diagrams depict directional overlap between differentially hydroxymethylated and methylated regions (FDR-adjusted p < 0.01) in a 25 kb window. The three Venn diagrams with the highest level of significance for the overlap are highlighted by the black boxes they are placed in

Fig. 5

a Bar graph depicts gene ontology categories significantly enriched in the indicated radiation-regulated differentially methylated regions (FDR-adjusted p < 0.01) identified from left ventricle and hippocampus. Note the gene categories-associated with cardiovascular function (red) and neuronal function (green) are restricted to the indicated tissues. (b and c). Diagrams depict selected genes that regulate vascular development or neurogenesis that were significantly associated with decreased methylation in the indicated tissue. (d and e). UCSC genome browser diagrams depict mC-DIP-Seq signal upstream the Activin receptor 1c gene in ventricle and upstream the Synaptopodin gene in hippocampus that showed significant differences following radiation exposure. The wiggle tracks depict median-scaled tag count density above background at an FDR of 5 %. The highlighted region was statistically significant (FDR-adjusted p < 0.001)