Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury

Abstract

Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair.

Keywords: DNA methylation; Schwann cell; c-Jun; epigenetics; long non-coding RNA; microRNA; nerve injury; nerve regeneration; repair Schwann cell.

Graphical abstract

Figure 1

RNA-Seq Analysis Identifies Enrichment of EMT Genes after Nerve Injury (A) A heatmap of the top 30 significantly downregulated genes between uncut and 7-day cut nerves (n = 3, adjusted p value [p-adj] < 0.05). (B) A heatmap of the top 30 significantly upregulated genes between uncut and 7-day cut nerves (n = 3, p-adj < 0.05). (C and D) Enriched KEGG pathways, GO terms, and protein families (PFAM) for (C) downregulated genes and (D) upregulated genes from RNA-seq analysis 7 days after nerve cut compared to the uncut nerve (n = 3). ^∗p-adj < 0.05; ^∗∗p-adj < 0.01; ^∗∗∗p-adj < 0.001. (E) Enrichment analysis of EMT genes from the RNA-seq study showing the 10 most downregulated and upregulated mRNAs (p-adj < 0.05).

Figure 2

Expression Patterns for Putative Schwann Cell Repair Program Genes and AP-1 TFs after Nerve Injury (A) Time course of putative repair program gene expression after sciatic nerve cut by qPCR (n = 5). (B) Time course of expression of members of the AP-1 TF family after sciatic nerve cut by qPCR (n = 5). Fold change in (A) and (B) is relative to the uncut nerve. (C) Table showing fold change values of repair program genes and all AP-1 TFs 24 hr post-cut relative to uncut nerve. p-adj values are displayed. (D) Relative cell type expression of repair program genes and AP-1 TFs in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow) displayed as percentage of the sum of qPCR copy-number values from 1ug of RNA from each of the three cell types (n = 3). (E) Time course of expression of Egr2 and Mpz by qPCR after sciatic nerve cut. Fold change is relative to the uncut nerve (n = 5). (F) Table showing fold change values of myelin program genes, Egr2 and Mpz, 24 hr post-cut relative to uncut nerve, with p-adj displayed. Error bars in all graphs represent SEM.

Figure 3

lncRNA Expression after Nerve Injury (A) A heatmap of the 52 annotated DE lncRNAs between uncut and 7-day cut sciatic nerve from RNA-seq experiments (p-adj < 0.05). (B) A heatmap of the 17 predicted DE lncRNAs between uncut and 7-day cut sciatic nerve from RNA-seq experiments (p-adj < 0.05). (C) Table of selected known (i) and predicted (ii) lncRNAs, with fold change (FC) and p-adj values from the RNA-seq study along with their closest genes, whether they are also differentially expressed (DE?) in the RNA-seq study, and the corresponding fold change. (D) Time course of expression of the lncRNAs, Meg3 (t1 = transcript 1), Rian, H19, and Sox2ot after sciatic nerve cut by qPCR (n = 4). Fold change is relative to the uncut nerve. Error bars represent SEM. (E) Relative cell type expression of Meg3 (transcript 1), Rian, H19, and Sox2ot in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow) displayed as percentage of the sum of qPCR copy-number values from 1ug of RNA from each of the three cell types (n = 3).

Figure 4

miRNA Expression after Nerve Injury (A) Venn diagram showing the overlap of DE miRNAs between uncut, 3-day cut, and 7-day cut sciatic nerve. (B–D) Heatmap of the top 40 DE miRNAs between (B) uncut and 3-day cut nerves, (C) uncut and 7-day cut nerves, and (D) 3-day cut and 7-day cut nerves (n = 5, p-adj < 0.05). (E) Enrichment analysis of EMT associated miRNAs from small RNA-seq study showing the most upregulated and downregulated miRNAs (FC, Fold change; p-adj < 0.05). (F) Expression of miRNAs in c-Jun flox/flox (control; red) and c-Jun null (P0Cre c-Jun flox/flox; blue) 7-day cut nerves. ^∗p < 0.05, n = 3. Fold change is relative to the uncut control nerve (c-Jun flox/flox). Error bars represent SEM.

Figure 5

Overview of the Methylome in the Injured Nerve (A) Validation of WGSB-seq results by Sanger sequencing of bisulfite-treated DNA. Strong correlation of total methylation percentage of 20 individual CpGs within six DMRs in WGSB-seq compared with Sanger sequencing. DMRs related to genes Cln8, Mob3b, Arl4C, and Nr1h4 and two DMRs in Nedd4l were used. (B) Percentage of DM CpGs (i) or DMRs(ii) in coding/regulatory regions (dotted line) or other genomic loci in 7-day cut nerve. (C) Enrichment of DM CpGs across genomic loci with odds ratio and confidence intervals (x axis), genomic loci (y axis), and significant p-adj values shown. Error bars represent SEM. (D) Distance of DM CpGs to the transcription start site (TSS), binned by distance and gene orientation, with total number of DM CpGs shown above each bin. (E–G) Percentage total methylation of 20 individual CpGs within six DMRs by Sanger sequencing in 7-day cut nerves compared with cultured (E) mouse Schwann cells, (F) nerve fibroblasts, and (G) activated macrophages (n = 3, ^∗p-adj < 0.05). (H) Table demonstrating lack of DM around myelin genes after nerve injury. For each topological associated domain (TAD) locus containing a known myelin gene, the percentage of DM CpGs is shown. (I) Enrichment of significant transcription-factor-binding motifs in close proximity to DM CpGs.

Figure 6

Enhancer-Specific Differential Methylation in the Injured Nerve (A) Pathway enrichment of genes associated with DM enhancer loci. (B) Differential CpG methylation after 7-day nerve cut compared to uncut nerve along the Nedd4l gene. The two DMRs, both of which were significantly demethylated after nerve cut, are marked in red, and mouse (mm10) encode enhancers and active rat (rn5) nerve enhancers, identified by Hung et al., (2015), are shown. DMR2 is located in close proximity to an active rat injured nerve enhancer. Mean RNA-seq data for uncut (blue) and 7-day cut (red) nerves are shown in addition to the location of all introns (arrowed blue lines) and exons (vertical blue lines) within the Nedd4l gene. (C) Genomic location and mean DM between uncut and 7-day cut nerves of individual CpGs within both DMRs of the Nedd4l gene measured by WGSB-seq and by bisulfite Sanger sequencing. (D) Mean methylation percentage for the two DMRs within Nedd4l enhancers in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow). ^∗p-adj < 0.05, n = 3. (E) Relative cell type expression of Nedd4l mRNA in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow) displayed as percentage of the sum of qPCR copy-number values from 1 μg RNA from each of the three cell types (n = 3). Sox2 and Cq1b are included as positive controls for Schwann cells and macrophages, respectively. (F) Time course of Nedd4l mRNA expression in the distal segment after sciatic nerve cut (n = 4). (G) Mean methylation percentage for DMRs near Arl4c, Cln8, and Nr1h4 genes in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow). ^∗p-adj < 0.05. (H) Relative cell-type expression of Arl4c, Cln8, and Nr1h4 mRNA in cultured mouse Schwann cells (blue), nerve fibroblasts (red), and activated macrophages (yellow) displayed as percentage of the sum of qPCR copy-number values from 1 μg RNA from each of the three cell types (n = 3). Sox2 and Cq1b are included as positive controls for Schwann cells and macrophages, respectively. Error bars in all graphs represent SEM.