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doi: 10.1038/srep25668.

Etiology matters - Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy

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Etiology matters - Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy

Konrad J Dębski et al. Sci Rep. .

Abstract

This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation, and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns.

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Figures

Figure 1
Figure 1. Pattern of neurodegeneration in three models of acquired epilepsy.
Nissl-stained sections presenting dentate gyrus of animals from amygdala stimulation (a2), pilocarpine (b2) and TBI (c2) model and respective controls (a1,b1,c1). Arrows indicate neuronal loss.
Figure 2
Figure 2. Differentially methylated regions and their distribution in three models of epilepsy.
(a–c) - Unsupervised hierarchical cluster analysis of differential methylation in (a) amygdala stimulation, (b) TBI, and (c) pilocarpine models. Hypermethylation – yellow, hypomethylation – blue, green – control, magenta - SE/TBI. (d) - Heatmap summarizing overlapping regions with consistent change in DNA methylation in at least 2 of 3 models of epilepsy (p < 0.01). Hypermethylation – yellow, hypomethylation – blue, green – control, magenta - SE/TBI. (e) - Rat genome ideogram summarizing the probability of increased (yellow) or decreased (blue) DNA methylation in injured vs. control animals in three animal epilepsy models. Outer ring represents chromosomes. Inner rings indicate location of methylation events in each model (amygdala stimulation - light blue, TBI - light green, pilocarpine - light red, hypermethylation – yellow, hypomethylation – blue). Distance from black line in the middle of each ring represents increasing probability of methylation change (presented as -log10 of p-value). Arrowheads indicate areas in chromosomes 6 and 15 that lacked increased methylation in the amygdala stimulation model. (f) - Rat genome ideogram summarizing DNA methylation changes common for pairs of models (inner gray rings). Outer ring represents chromosomes. Red bars - common increased methylation events, blue bars - common decreased methylation events. (g) - Venn diagram presenting differentially methylated DNA regions detected in each model of epilepsy and regions overlapping between models with change in methylation to the same direction. (h–j) - Genomic distribution of DNA methylation changes in (h) amygdala stimulation, (i) TBI, and (j) pilocarpine models. Frequency of observed methylation changes compared with non-differentially methylated regions (p < 0.01), with upper and lower 95% confidence intervals for different genomic features. Magenta - increased methylation, green - decreased methylation, O/E - observed/expected ratio, CpG - CpG islands, SNP - single nucleotide polymorphism, TSS - transcriptional start site, UTR - untranslated region. (k) - Ratio of increased to decreased methylation events across genomic features for each epilepsy model with upper and lower 95% confidence intervals. Blue, green and light red bars represent the focal amygdala stimulation, TBI, and systemic pilocarpine models, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided Fisher’s exact test).
Figure 3
Figure 3. Differentially expressed genes over three models of TLE.
(a–c) - Unsupervised hierarchical clustering of samples and transcripts according to differential expression profiles (upregulation - red, downregulation - blue, cut-off p < 0.001) for the amygdala stimulation (a), TBI (b) and pilocarpine model (c). Green bars on the top of the heatmaps refer to control and magenta bars to SE/TBI animals. Data from pilocarpine model were previously published by Kobow et al. and reanalyzed here as described in Methods section. (d) - Heatmap presenting genes with altered expression levels in at least two animal models (cut-off p < 0.001). Blue color indicates downregulation and red upregulation of gene expression in a given model of epilepsy. The blue bar on the top of the heatmap refers to control and the magenta bar to SE/TBI animals. Black shadings on the left side of the heatmap represent the significant differences. (e–f) - Venn diagrams presenting upregulated (e) or downregulated (f) genes common between models. (g) - Direct and indirect experimentally observed interaction between genes or gene products according to IPA. Note that interactions were found between Msn (moesin) and Cd44 (Cd44 antigen precursor), and that feedback interaction was observed for S100a6, Msn and Cd44. (h) - Word clouds presenting enrichment analysis of terms from the GO database from the Biological Process branch and terms from biological pathways databases (KEGG and Reactome) with gProfiler for each model. The length of blue and red bars over each word cloud are proportional to the number of down- and upregulated genes, respectively. The numbers on the right side of the bars (G1 and G2) represent the number of down- and upregulated genes, respectively. Word clouds under the blue bars describe enriched terms for downregulated genes whereas words under the red bars represent upregulated genes. Only significantly enriched terms (enrichment p < 0.01) are presented. The color of the font indicates the enrichment p-value. Darker colors indicate more significant enrichment. The font size is related to the significance of enrichment of a given term but only within a given cloud.
Figure 4
Figure 4. Correlation between methylation and gene expression.
(a) - Examples of GSEA enrichment profiles of association between gene expression and alterations in promoter methylation, A strong association (FDR < 0.25 was considered significant) was observed between increased promoter methylation and gene activation in the TBI model, between decreased promoter methylation and gene repression in the focal amygdala stimulation model, and between decreased promoter methylation and gene activation in the systemic pilocarpine model. ES - enrichment score, PRL - pre-ranked list, FDR - false discovery rate. (b) - Gene set enrichment analysis (GSEA) presenting associations between gene expression and methylation changes in different genomic features in three epilepsy models. The results are presented as false discovery rates of nominal p-values of gene sets of increased or decreased methylation events at promotors (5 kb upstream of transcription start), CpG islands in promoters (CGI in promoter), TSSs, 3′UTRs, exons, introns and 5′UTRs against the rank of mRNA-Seq data from the same samples. Cell background color representations are as follows: gray – non-significant association, green - significant association with decreased gene expression, magenta - significant association with increased gene expression (FDR < 0.25 was considered as significant). NA - analysis not available due to missing differentially methylated regions in gene sets. (c) - Venn diagrams showing overlaps between genes and differentially methylated DNA regions used in GSEA. (d) - List of differentially expressed genes (cut-off p < 0.001), in which methylation changes occurred in gene body or promoter regions in each model. Magenta and green arrows indicate increased and decreased levels of mRNA, respectively. Orange and blue arrows indicate increased and decreased methylation events, respectively, which occurred in promoter regions, TSSs, exons, introns, 3′UTRs, 5′UTRs and CGIs (CpG islands).

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