Deep sequencing reveals increased DNA methylation in chronic rat epilepsy

Abstract

Epilepsy is a frequent neurological disorder, although onset and progression of seizures remain difficult to predict in affected patients, irrespective of their epileptogenic condition. Previous studies in animal models as well as human epileptic brain tissue revealed a remarkably diverse pattern of gene expression implicating epigenetic changes to contribute to disease progression. Here we mapped for the first time global DNA methylation patterns in chronic epileptic rats and controls. Using methyl-CpG capture associated with massive parallel sequencing (Methyl-Seq) we report the genomic methylation signature of the chronic epileptic state. We observed a predominant increase, rather than loss of DNA methylation in chronic rat epilepsy. Aberrant methylation patterns were inversely correlated with gene expression changes using mRNA sequencing from same animals and tissue specimens. Administration of a ketogenic, high-fat, low-carbohydrate diet attenuated seizure progression and ameliorated DNA methylation mediated changes in gene expression. This is the first report of unsupervised clustering of an epigenetic mark being used in epilepsy research to separate epileptic from non-epileptic animals as well as from animals receiving anti-convulsive dietary treatment. We further discuss the potential impact of epigenetic changes as a pathogenic mechanism of epileptogenesis.

Figures

Fig. 1

Deep sequencing (Methyl-Seq) revealed increased genomic DNA methylation in chronic rat epilepsy. a Study design, b heat map displaying hierarchical clustering of samples and genomic regions according to differential methylation profiles (yellow methylation up, red methylation down). A specific DNA methylation signature characterized chronic rat epilepsy. CTRL sham injected, healthy controls; PILO pilocarpine injected, chronic epileptic animals. Clustering was performed by taking the trimmed mean normalized values for differential regions as defined by edgeR analysis with a p value <0.01. These values were normalized to the standard normal distribution before performing Euclidean distance based hierarchical clustering on both regions and samples using the heatmap.2 function in the R package gplots

Fig. 2

Genomic distribution of DNA methylation changes (cut-off p < 0.01) in chronic rat epilepsy. a Rat genome ideogram summarizing hypermethylation (red) and hypomethylation events (green) in PILO versus CTRL. DNA methylation targeted the entire genome with almost complete sparing of the X-chromosome (ChrX). b Frequency of observed methylation changes compared to non-differentially methylated regions [−log10 (p value) <0.25], with upper and lower 95 % confidence intervals for different genomic features. Hypermethylation relative to controls is shown in the left panel (red bars), whereas, hypomethylation relative to controls is shown in the right panel (green bars). DNA methylation events were mainly confined to CGIs, but did not frequently target promoters. CGI CpG island, TSS transcriptional start site, O/E observed/expected ratio, 5mC 5-methyl-cytosin. Asterisks indicate significance (p < 0.05 using Fisher’s Exact test)

Fig. 3

mRNA-Seq identified highly distinct gene expression signatures in chronic rat epilepsy and controls. Heat map displaying hierarchical clustering of samples and genes according to differential expression profiles normalized to the standard normal distribution (yellow expression up, red expression down). Treatment groups can be clearly differentiated by their expression profiles. CTRL sham injected, healthy controls; PILO chronic epileptic animals. Clustering was performed by taking the trimmed mean normalized values for genes as defined by edgeR analysis with a p value <0.01. These values were normalized to the standard normal distribution before performing Euclidean distance based hierarchical clustering on both regions and samples using the heatmap.2 function in the R package gplots

Fig. 4

Gene set enrichment analysis (GSEA) of methylated promoters, TSS and gene bodies were performed against the rank of our mRNA-Seq data from same samples. GSEA was separately performed for gene sets showing increased or decreased methylation in chronic rat epilepsy. A strong correlation (FDR FDR false discovery rate, T thousand, CTRL sham injected, healthy controls, PILO pilocarpine injected, chronic epileptic animals. Supplement Table 1 contains comprehensive GSEA statistics

Fig. 5

Left panel showing bisulfite sequencing results (Bis-Seq, n = 3). White dots represent unmethylated and black dots methylated CpGs. Middle panel summarizing schematic gene structure with TSS (green arrow), chromosomal region and region covered in Bis-Seq. Right panel presenting gene expression data from RT-PCR (n = 5). C, CTRL control (white bar); P, PILO chronic epileptic animals (red bar). Asterisks indicate significance (unpaired two-tailed t test, p < 0.05). a Camkk2 showed hypermethylation and concomitant gene repression in PILO versus CTRL. b Hypomethylation of the Il10rb locus and increased gene expression could be confirmed in PILO versus CTRL

Fig. 6

Comparison of differential DNA methylation and gene expression patterns in pilocarpine injected, epileptic animals receiving anti-convulsive ketogenic diet (PILO + KD, blue) or no treatment (PILO, red). The KD treatment partially ameliorated molecular changes associated with chronic rat epilepsy. Effects were more pronounced on a genomic scale than at certain loci of selected candidate genes. a Venn diagram displaying overlap in differential DNA methylation between PILO and PILO + KD compared to CTRL. KD-treated animals showed a distinct genomic methylation profile compared to untreated chronic rat epilepsy. Administration of the KD seemed to have rescued a majority of affected loci. b Venn diagram displaying overlap in differential gene expression between PILO and PILO + KD compared to CTRL. KD-treated animals showed a distinct gene expression pattern compared to untreated chronic rat epilepsy. A majority of differentially expressed genes in PILO were rescued upon KD treatment. Genes exclusively expressed in PILO + KD may have contributed to adverse side effects. c Bisulfite sequencing results. Camkk2 hypermethylation in PILO animals was significantly reduced by KD treatment (Mann–Whitney U test, p < 0.05). Further, hypomethylation of the Il10rb locus in PILO was reversed in KD + PILO. White dots represent unmethylated and black dots methylated CpGs. d Gene expression of Camkk2 and Il10rb in PILO and PILO + KD. KD treatment partially rescued Camkk2 gene expression, but had no significant effect on Il10rb. C, CTRL control (white bar); P, PILO chronic epileptic animals (red bar); KD, PILO + KD pilocarpine injected animals receiving anti-convulsive ketogenic dietary treatment (blue bar). Asterisks indicate significance (univariate one-way ANOVA followed by Bonferroni post hoc test, p < 0.05)