Polycomb repression regulates Schwann cell proliferation and axon regeneration after nerve injury - PubMed (original) (raw)

. 2018 Nov;66(11):2487-2502.

doi: 10.1002/glia.23500. Epub 2018 Oct 11.

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Polycomb repression regulates Schwann cell proliferation and axon regeneration after nerve injury

Ki H Ma et al. Glia. 2018 Nov.

Abstract

The transition of differentiated Schwann cells to support of nerve repair after injury is accompanied by remodeling of the Schwann cell epigenome. The EED-containing polycomb repressive complex 2 (PRC2) catalyzes histone H3K27 methylation and represses key nerve repair genes such as Shh, Gdnf, and Bdnf, and their activation is accompanied by loss of H3K27 methylation. Analysis of nerve injury in mice with a Schwann cell-specific loss of EED showed the reversal of polycomb repression is required and a rate limiting step in the increased transcription of Neuregulin 1 (type I), which is required for efficient remyelination. However, mouse nerves with EED-deficient Schwann cells display slow axonal regeneration with significantly low expression of axon guidance genes, including Sema4f and Cntf. Finally, EED loss causes impaired Schwann cell proliferation after injury with significant induction of the Cdkn2a cell cycle inhibitor gene. Interestingly, PRC2 subunits and CDKN2A are commonly co-mutated in the transition from benign neurofibromas to malignant peripheral nerve sheath tumors (MPNST's). RNA-seq analysis of EED-deficient mice identified PRC2-regulated molecular pathways that may contribute to the transition to malignancy in neurofibromatosis.

Keywords: Schwann; axon; chromatin; injury; myelin; nerve; neurofibromatosis.

© 2018 Wiley Periodicals, Inc.

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Figures

Figure 1.

Figure 1.. Schwann cell EED is required for timely axon regeneration.

A, Electron micrographs of the sciatic nerves at the indicated time points after crush and uninjured nerves of Eed cKO mice and littermate controls. The arrows and inset show macrophages engulfing myelin debris. Scale bars of uninjured and injured nerve images are 5 μm and 8 μm, respectively. B, C, Myelinated and amyelinated axons (> 1μm in diameter), myelin debris, macrophages, and engulfed myelin by macrophages were counted in randomly selected fields that accounted for over 40% of an entire sciatic nerve cross section from each animal and normalized per surface area (10,000 μm2). Data: mean ± STDEV; **p < 0.005, *p < 0.05; n=3 per genotype (one-way ANOVA). D, For g-ratio analysis (axon diameter/diameter of myelinated fiber), the diameter of axon and outer diameter of myelinated fiber were measured on over 500 randomly selected fibers per genotype. Data: weighted mean ± pooled STDEV; ***p < 0.0005, **p < 0.005, *p < 0.05; n=3 per genotype and age (one-way ANOVA). E, qRT-PCR analysis was used to identify the expression level of myelin genes from 2 month Eed cKO and control sciatic nerves in uninjured condition or 14 day after crush. Expression levels were normalized with Gapdh. Asterisks indicate p_-_value between genotypes in the respective condition. Data: mean ± SD; **p < 0.005, ***p < 0.0005; n=4 for control and n=3 for Eed cKO (one-way ANOVA).

Figure 2.

Figure 2.. Increased activation of AKT in Eed cKO nerves after injury.

Western blot analysis of lysates from distal stumps of control and Eed cKO sciatic nerves 1 d after cut was performed using the indicated antibodies. n=5 for control and n=3 for Eed cKO nerves. Data: mean ± STDEV; **p < 0.005, *p < 0.05 (one-way ANOVA).

Figure 3.

Figure 3.. Schwann cell Nrg1 type I is regulated by PRC2.

A, ChIP-seq mapping of H3K27me3 was performed in uninjured rat sciatic nerves. The transcription start site (TSS) is on the left. Statistical (Stat) Enrichment indicates regions with more sequencing reads than random chance. B, H3K27me3-ChIP assays were performed with distal stumps of rat sciatic nerves 1 d post-cut or sham surgery, and percent recovery relative to input was calculated by qPCR analysis. Data: mean ± SD; *p < 0.05; n=5 for sham and n=6 for 1 d post-cut (one-way ANOVA). C, qRT-PCR analysis was used to identify the expression level of Nrg1 type I and type III from 2 month Eed cKO and control sciatic nerves of uninjured condition or 1 day after cut. Expression levels were normalized with Gapdh. Data: mean ± SD; Asterisks indicate p_-_value between genotypes in the respective condition. *p < 0.05, ***p < 0.0005; n=5 per genotype and condition (one-way ANOVA). D, Rat sciatic nerve explants were cultured for 1 d in the presence of GSK-J4 at indicated concentrations or DMSO vehicle and subjected to qRT-PCR together with immediately frozen nerve segments after dissection (indicated as uninjured). Uninjured level of Nrg1 is set as 1. Expression levels were normalized to 18S rRNA. Data: mean ± SD; n=5 per condition (one-way ANOVA). E, Nerve lysates were obtained from uninjured control and Eed cKO nerves, and were blotted with an antibody to the extracellular domain of NRG1. The 30 kd band was normalized to α-tubulin, and the bar graph shows the average of the 3 replicates per condition.

Figure 4.

Figure 4.. H3K27me3-occupied silent genes induced in the Eed cKO.

A, Box-and-Whisker plot shows RPKM distribution of genes in uninjured wildtype nerves, grouped by H3K27me3 peak score, which was determined by ChIP-seq analysis (n=2) using Hypergeometric optimization of motif enrichment (HOMER) (Heinz et al., 2010). B, Distribution of genes by RPKM values of indicated genotypes. Genes with the H3K27me3 score greater than 10 were indicated by red dots. RPKM, Reads Per Kilobase of transcript per Million mapped reads. RPKM values are averaged across three samples per genotypes (p < 0.05).

Figure 5.

Figure 5.. Polycomb activity regulates early transcriptional response after nerve injury.

A, RNA-seq analysis identified a number of genes dysregulated by Eed cKO in uninjured or 1 d after injury conditions. Gene expression was determined by RNA-seq analysis of 3 samples per genotype and condition (_p-_value, < 0.05). The brackets indicate the number of genes associated with H3K27me3 around the transcription start site (±7 Kb). **B, C,** RNA-seq analysis was used to identify the expression level of early and late injury genes in _Eed_ cKO nerves and control nerves at 1 d post-cut relative to uninjured control nerves. Note that the y-axis is on a log scale. Data: mean of n=3 with _p_-value, < 0.05 per genotype. The late injury genes were identified from microarray analysis of peripheral nerves 3, 5 or 7 d after injury (> 2 fold) (GEO accession: GSE22291, GSE38693, GSE33454) (Barrette et al., 2010; Arthur-Farraj et al., 2012; Kim et al., 2012)(Kim et al. 2012). See Supporting Information Table 3 and 4 for the complete list of genes analyzed in Figure 5.

Figure 6.

Figure 6.. The expression of critical axonal growth genes semaphorin 4F and ciliary neurotrophic factor is dependent on EED-mediated transcriptional regulation during nerve repair.

A, RNA-seq analysis identified a number of genes dysregulated by Eed cKO among genes differentially expressed during nerve regeneration at 14 d post-crush compared to uninjured nerves. See Supporting Information Table 2 and 5 for the list of dysregulated genes by Eed cKO in overall transcriptome and in the injury responsive transcriptome 14 d post-crush, respectively (n=3 per condition for control and n=2 for Eed cKO 14 d post-crush). B, Immunohistochemistry on transverse sections of distal stumps displays the JUN expression in nerves of indicated genotypes 5 d after cut. Scale bars, 40 μm. C, D, qRT-PCR analysis was used to identify the expression level of injury-responsive genes from 2 month Eed cKO and control sciatic nerves in uninjured condition or 14 day after crush. Expression levels were normalized with Gapdh. Data: mean ± SD; Asterisks indicate p-value between genotypes in the respective condition. *p < 0.05, **p < 0.005, ***p < 0.0005; n=4 for control and n=3 for Eed cKO (one-way ANOVA).

Figure 7.

Figure 7.. Eed cKO exhibits impaired proliferation in injured nerves.

A, mRNA expression relative to uninjured control level set as 1 (not shown) at indicated time points was assessed by qRT-PCR with primer sequences specific to p16/Ink4a and p19/Arf transcripts of Cdkn2a. Expression levels were normalized with Gapdh. Data: mean ± SD; ***p < 0.0005; n=5 and n=4 per genotype and condition at 1 d post-cut and 7 d post-crush, respectively, and n=4 for control and n=3 for _Eed_ cKO at 14 d-post crush (one-way ANOVA). **B, C,** The expression of a proliferation marker Ki-67 and p19/ARF among SOX10-positive nuclei at 5 d after denervation was assessed by immunohistochemistry on transverse sections of indicated genotypes. Scale bars, 20 μm. n=3 per genotype. Data: mean ± STDEV; **_p_ < 0.005 (one-way ANOVA). **D,** Representative genes that were commonly upregulated in PRC2-deficient MPNSTs relative to non-deficient MPNSTs (> 3 fold, RNA-seq) (Lee et al., 2014) and Eed cKO nerves relative to wildtype nerves of uninjured or 1d, 14d post-injury conditions (> 2 fold, RNA-seq) are listed (FC, fold change). Gray shading indicates H3K27me3 occupancy of genes in peripheral nerves of uninjured or post-injury conditions. See Supporting Information Table 6 for expression fold changes by Eed cKO among PRC2-deficient MPNST genes and H3K27me3 peak score.

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