Plasticity of DNA methylation in a nerve injury model of pain

Abstract

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10(-4)) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10(-15)) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings-obtained in mixed tissue-show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.

Keywords: DRG, dorsal root ganglion; HCP, high-CpG promoter; LCP, low-CpG promoter; RRBS; RRBS, reduced representation bisulfite sequencing; SNL, spinal nerve ligation; dDMCs, dynamically differentially methylated CpGs; dorsal root ganglion; methylation; pain; peripheral nervous system; rat; spinal nerve ligation; tDMCs, tissue-specific differentially methylated CpGs; tIMCs, tissue-invariant methylated CpGs.

Figure 1 (See previous page).

Gene methylation response to nerve injury. (A) Genome-wide quantification of CpG methylation by RRBS. Genomic DNA from the L5 dorsal root ganglion (DRG) of Brown Norway rats was isolated 24 h after spinal nerve ligation (SNL) or a sham procedure (negative control). Genomic DNA (gDNA) was isolated and subjected to a restriction digest with MspI. DNA fragments were ligated to adapters, bisulfite treated converting unmethylated cytosines to uracils, and sequenced. Resulting paired-end reads—1.1 billion in total from 8 independent libraries analyzed in the present study—were aligned to the rat genome. Cytosine methylation levels were called only for CpG sites covered in a given library by ≥10 independent sequence reads. (B) Nerve injury eliciting methylome alterations: Evidence at the whole-data set level. DNA methylation was markedly altered after SNL. Hierarchical clustering—using all methylation levels measured at 917,097 CpG sites within genes—clearly separated control animals (left) from SNL animals (right). (C) Nucleotide-resolution analysis of the methylation profile of HCN2. Top panel: Changes in methylation were noted in clusters of juxtaposed CpGs. Shown as an example is HCN2, an ion channel modulating inflammatory and neuropathic pain ⁠. The x-axis indicates the position of captured CpG sites within a gene. RRBS captured 216 CpGs located in gene body of HCN2, while no CpGs were captured in the promoter region for this gene. The negative log-p value of the significance level computed by a likelihood ratio test using a generalized linear model (GLM) is shown on the y-axis. Higher values indicate stronger significance. Differences with a P > 10⁻³ were considered non-significant (CpG positions shown below the dotted line). Differences at individual CpG sites were highly significant ranging from P < 10⁻³ to P < 10⁻¹⁴. The bars in the colored band above the scatterplot indicate for each CpG whether the mean methylation level was higher (red) or lower (blue) in the SNL group compared with controls. The direction of change is shown regardless of significance at the level of a specific CpG. Regions that are significantly changed (P < 10⁻³) according to a sign test of the direction of juxtaposed CpGs are represented by brown stars. Bottom panel: Random permutation of group assignment and CpG positions confirmed that both statistical testing procedures were robust as indicated by low false-positive rates of 0.004 for single CpG testing of HCN2 (1/216 CpG above significance threshold) and of 0 (no false-positive) for regions. Additional examples are shown in Figure S1. (D) Gene types and regions undergoing hyper- and hypo-methylation. The fraction of CpGs with significantly altered methylation was calculated across different gene regions for the entire dataset. Low CpG content promoter (LCP) genes and high CpG content promoter (HCP) genes differed. LCP genes were altered in the promoter, exon, and intron regions. HCP genes harbored a comparable fraction of altered methylation sites only in exons and introns, while HCP gene promoters were unaffected. Hypermethylation (red) accounted for a greater fraction of changes than hypomethylation (blue) in all regions. (E) Axon guidance pathway genes differentially methylated after SNL. The most significantly enriched molecular mechanism in an unbiased global analysis of genes undergoing differential methylation after SNL was the axon guidance pathway (P < 10⁻¹¹). Depicted are 35 differentially methylated genes with dense connectivity. Variable methylation predominantly occurred in the gene body. Only FES and CDK5 showed methylation alterations in their promoter (black stars). A total of 98 out of 468 axon guidance pathway genes were differentially methylated (complete list provided as Table S1).

Figure 2.

Transcriptome up- and down-regulation can co-exist with CpG hyper- or hypo-methylation in the promoter and gene body. An integrated transcriptome-methylome analysis supported a negative finding, notably the absence of any tight correlation between transcriptome- and methylome alterations following SNL. Shown is the direction of CpG methylation change and gene expression change following nerve injury. Genes were divided into 2 groups: high CpG promoter (HCP; panel A and B) and low CpG promoter (LCP) genes (panel C and D). Promoter- (left, panel A and C) and gene body methylation (right, panel B and D) were analyzed separately. The methylation values of dDMCs (x-axis) were plotted against the expression value of their corresponding gene (y-axis). Each circle represents a differentially methylated CpG (dDMC), thereby allowing multiple pairings of individual dDMCs with the same gene. The analysis was performed on significantly changed CpG sites located in genes that underwent a significant change in expression. The results shown demonstrate that all possible scenarios exist: hyper- and hypo-methylation of CpG associated with increase or decrease in RNA levels from the same gene. Red: hypermethylated dDMC, blue: hypomethylated dDMC.

Figure 3 (See previous page).

Methylome remodeling of gene deserts. (A) Desert dDMCs. Nearly half of the CpGs undergoing statistically significant hyper- or hypo-methylation in response to nerve injury, dDMCs, were discovered in intergenic regions, 44% of dDMCs in the study (left panel). The distribution was even across the intergenic span, as seen from the combined analysis of the location of 5,042 hypermethylated and 1,612 hypomethylated sites (right panel). The likelihood of a CpG to be differentially methylated (after SNL) was independent of its distance from the closest gene, thereby authenticating the classification of all these sites as "desert dDMCs." (B) Uniformity of desert dDMC clusters. Neighboring dDMCs in gene deserts underwent unidirectional methylation change forming clusters. All clusters consisting of ≥5 dDMC members were examined. The largest clusters (unselected) are shown. Non-randomness was significant with P < 0.05 to P < 2 × 10⁻⁶ for sizes ranging from 7 to 21. Red bar: hypermethylated dDMC. Blue: hypomethylated dDMC. A complete depiction of clusters is provided in Figure S3. (C) DRG-specific partitioning of the gene desert methylome. Cytosine methylation sites were dichotomized into tissue-differentially methylated CpGs (tDMCs) and tissue-invariant methylated CpGs (tIMCs), demarcating CpGs that are DRG specific, tDMCs, and others that were equally methylated in all organs, tIMCs. Rat control DRG (new replicates), spleen, muscle, and liver were compared. Sample regions with tDMCs and tIMCs are shown (left). The absolute methylation level is represented on the y-axis. A principal components analysis (PCA) confirmed organ-specific methylation of gene deserts (right). (D) Desert remodeling after SNL (dDMCs) occurs at CpG with a DRG-specific methylation level (tDMCs). CpG undergoing methylation changes in response to SNL, dDMCs, were a subset of tDMCs. The enrichment of dDMCs in the tDMC vs. tIMC subsets of CpG sites was highly significant (P < 10⁻¹⁵). Gene desert methylation can be divided according to a dual model: The majority of CpG methyl-marks remain stable organism-wide regardless of tissue type or pathology; a substantial minority of CpG methyl-marks, tDMCs, are DRG-specific and encompass all sites capable of responding to changes in the environmental such as at the CpG sites identified in the present study as dDMCs through their response to nerve injury.