This study aims to find the difference of genomewide DNA methylation in Schwann cells (SCs) before and after peripheral nerve system (PNS) injury by Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) and seek meaningful differentially methylated genes related to repairment of injured PNS. SCs harvested from sciatic nerve were named as activated Schwann cells (ASCs), and the ones harvested from brachial plexus were named as normal Schwann cells (NSCs). Genomic DNA of ASCs and NSCs were isolated and MeDIP-Seq was conducted. Differentially methylated genes and regions were discovered and analyzed by bioinformatic methods. MeDIP-Seq analysis showed methylation differences were identified between ASCs and NSCs. The distribution of differentially methylated regions (DMRs) peaks in different components of genome was mainly located in distal intergenic regions. GO and KEGG analysis of these methylated genes were also conducted. The expression patterns of hypermethylated genes (Dgcr8, Zeb2, Dixdc1, Sox2, and Shh) and hypomethylated genes (Gpr126, Birc2) detected by qRT-PCR were opposite to the MeDIP analysis data with significance (
Schwann cells (SCs) play an important role in the peripheral nervous system (PNS) and have been shown to have a variety of functions especially in participating in the formation of myelin, maintaining the morphology and function of myelin sheath as well as involving in the regeneration of nerve injury [
Recently, a large number of studies have been focusing on epigenetics research of SCs related to PNS injury, and some studies [
Adult Wistar rats (
After 7 days, sciatic nerves and brachial plexus were acquired from 6 Wistar rats. After washing away the blood in phosphate-buffered saline (PBS) and removing the epineurium and connective tissue under the dissecting microscope, the tissues were cut into 1 × 1 mm explants with scissors [
The purified SCs were cultured to the third generation and were planted on poly-D-lysine-coated coverslips in 24-well plates. After 72 hours, SCs were briefly washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. Then these cells were washed 3 times with PBS and permeabilized with 0.5% Triton X-100 15 min. After blocking with 0.5% (v/v) goat serum in PBS for 1 hour at room temperature, these cells were incubated with S100ß antibody (mouse anti-rat antibody, 1 : 500 volume dilution) overnight at 4°C. After washing 3 times in PBS, SCs were incubated with conjugated secondary antibodies (FITC) goat anti-mouse IgG for 1 hour (diluted 1 : 1000 in PBS). Lastly, the nuclei were stained with DAPI and images were captured using a laser scanning confocal microscope.
Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used in cell proliferation assays. These cells were seeded into 96-well plates, and 3 duplicate wells were set. At the same time 3 control wells were set correspondingly. The density of cells was 1000 per well with 100 uL growth medium. Then cells were treated with the CCK8 (10 uL, 2 hours per well) and measuring the numbers of cells per well was conducted by the absorbance (450 nm) of reduced water-soluble tetrazolium salt (WST) for 7 days continuously.
The genomic DNA of ASCs and NSCs was extracted and purified, respectively. Then sonication method was used to fragment these DNA into 100–500 bp fragments. DNA fragment 3′ end repair was carried out by adding Adenine (A) and sequencing adapters were simultaneously added to their ends by Paired-End DNA Sample Prep Kit (Illumina, USA). Double stranded DNA was denatured as single stranded DNA. Subsequently, sequencing adapters-ligated DNA fragments and 5-methylcytosine (5-mC) antibody beads (Diagenode, USA) were immunoprecipitated. These methylated DNA fragments were enriched. Then immunoprecipitated products were amplified and validated by quantitative real-time polymerase chain reaction (qRT-PCR), and those of 200–300 bp were excised from the gel and purified. DNA library quality control and sequencing run were processed by Illumina HiSeq 2000 platform (Illumina, USA). Finally, we got the Illumina sequencing raw data of DNA. Concise experimental procedure for DNA methylation sequencing was shown in Figure
Concise experimental procedure for DNA methylation sequencing.
Data analysis was carried out according to the following steps. First, the previous sequencing data of DNA was used and the high quality data of them were screened out according to the screening criteria as follows:
Seven genes were verified by qRT-PCR with the Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, USA). Total RNA was isolated from the ASCs and NSCs by using TRIZOL (Invitrogen Corp, Carlsbad, CA) and was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNA following the manufacturer’s directions. Real-time PCR was performed using SYBR green dye in a thermal cycler with the following parameters: an initial denaturation step at 95°C for 30 min; 40 cycles at 95°C for 5 seconds and 60°C for 30 seconds. Complete experimental process was performed for each sample in triplicate. All primers were synthesized by Shanghai Shenggong Inc, and mRNA-specific primers were listed in Table S2, in Supplementary Material available online at
All data were analyzed using SPSS statistical software (version 11.5 for Windows). Statistical analysis was performed using two-tailed Student’s
The results of observation under optical microscope showed no morphological differences between ASCs and NSCs (Figures
(a) The shape of activated Schwann cells (ASCs) under optical microscope. (b) The shape of normal Schwann cells (NSCs) under optical microscope. Both of these SCs, long spindle cells, were arranged in fish shape and nucleus was ovoid or oblong. Scale bar: 200 um. (c) SCs were marked with S-100ß by immunofluorescence. (d) The nucleus of SCs was marked with DAPI by immunofluorescence. (e) SCs and nucleus of SCs were merged together by immunofluorescence. Both of the two kinds of SCs had positive results under fluorescence microscope. Scale bar: 100 um. (f) The growth rate of SCs was showed by optical density (OD) value of 450 nm from 1 day to 7 days through cells proliferation assay. The growth rate of ASCs was obviously higher than that of NSCs starting from 2 days.
After MeDIP-Seq, we generated a total of 6,592,337,700 bp data from ASCs groups and 7,136,742,100 bp data from NSCs groups. 17474976 reads were obtained in NSCs groups and 16816796 reads in ASCs groups were obtained, respectively. To our satisfaction, 16415908 reads of NSCs group and 15729752 reads of ASCs group were uniquely mapped to the latest rat genome, which showed that more than 93% of MeDIP-Seq reads were aligned (mapped) on latest rat genome in each group. In addition, more than 80% of MeDIP-Seq reads were high quality (HQ) mapped reads in Table
Reads genome alignment statistics.
Class | ASCs | NSCs | ||
---|---|---|---|---|
Number | % | Number | % | |
Total reads | 16816796 | 17474976 | ||
Total mapped |
15729752 | 93.54 | 16415908 | 93.94 |
Total HQ mapped reads | 13005381 | 82.68 | 13137926 | 80.03 |
Promoter (≤1 kb) | 90241 | 0.69 | 84170 | 0.64 |
Promoter (1-2 kb) | 81955 | 0.63 | 79542 | 0.61 |
1st exon | 20000 | 0.15 | 17909 | 0.14 |
Other exons | 458938 | 3.53 | 430799 | 3.28 |
1st intron | 625989 | 4.82 | 610668 | 4.65 |
Other introns | 2953913 | 22.72 | 2887216 | 21.99 |
3′ UTR | 91283 | 0.70 | 87248 | 0.66 |
5′ UTR | 9569 | 0.07 | 9244 | 0.07 |
Distal intergenic regions | 8463689 | 65.10 | 8722453 | 66.43 |
Downstream (≤3 kb) | 204583 | 1.57 | 200206 | 1.52 |
In |
97569 | 0.62 | 79283 | 0.48 |
ASCs: activated Schwann cells; NSCs: normal Schwann cells; HQ: high quality.
A total of 13005381 HQ mapped reads in ASCs group and 13137926 HQ mapped reads in NSCs group were distributed in the chromosomes of the DMRs. Of them, HQ mapped reads mainly were found in chr1 and detailed results were showed in Table
Chromosome distribution of total HQ mapped reads.
Class | ASCs | NSCs | ||
---|---|---|---|---|
Number | % | Number | % | |
Total HQ mapped reads | 13005381 | 13137926 | ||
chr1 | 1454481 | 11.1836862 | 1543628 | 11.7494 |
chr2 | 1074744 | 8.26384094 | 1098163 | 8.35872 |
chr3 | 816906 | 6.28129234 | 808555 | 6.15436 |
chr4 | 814638 | 6.2638534 | 810404 | 6.16843 |
chr5 | 754336 | 5.80018379 | 755245 | 5.74859 |
chr6 | 662551 | 5.09443745 | 661399 | 5.03427 |
chr7 | 742593 | 5.70989039 | 748669 | 5.69853 |
chr8 | 633675 | 4.87240628 | 627670 | 4.77754 |
chr9 | 604079 | 4.64483893 | 601617 | 4.57924 |
chr10 | 615858 | 4.73540914 | 597108 | 4.54492 |
chr11 | 406076 | 3.12236912 | 403700 | 3.07278 |
chr12 | 386267 | 2.97005524 | 381848 | 2.90646 |
chr13 | 645303 | 4.96181542 | 707230 | 5.38312 |
chr14 | 588247 | 4.52310471 | 584761 | 4.45094 |
chr15 | 503096 | 3.86836802 | 509972 | 3.88168 |
chr16 | 431447 | 3.31744991 | 434373 | 3.30625 |
chr17 | 488717 | 3.7578061 | 480979 | 3.661 |
chr18 | 431679 | 3.31923378 | 428225 | 3.25946 |
chr19 | 363485 | 2.7948816 | 353620 | 2.6916 |
chr20 | 372963 | 2.86775912 | 361241 | 2.7496 |
chrX | 209019 | 1.60717322 | 231048 | 1.75863 |
chrM | 5221 | 0.04014492 | 8471 | 0.06448 |
chr: chromosome; ASCs: activated Schwann cells; NSCs: normal Schwann cells; HQ: high quality.
Differential methylation of Schwann cells statistics.
Class | Number | % |
---|---|---|
Genome regions | 11610434 | 100 |
DMR | 176610 | 1.52113177 |
Hypermethylated | 77799 | 44.0512995 |
Hypomethylated | 98811 | 55.9487005 |
DMR: differentially methylated region.
Calculating CpG enrichment of provided short reads compared to the reference genome.
Class | CpGs number | CpGs rel. frequency (%) | CpGs obs/exp | Enrichment rel. frequency (%) | Enrichment obs/exp |
---|---|---|---|---|---|
BSgenome.Rnorvegicus.UCSC | 24713205 | 0.851414595 | 0.247437644 | — | — |
ASCs_1 | 73092752 | 2.01814666 | 0.393297098 | 2.37034539 | 1.589479639 |
ASCs_2 | 66573452 | 1.986667228 | 0.38941539 | 2.333372296 | 1.57379202 |
ASCs_3 | 56781764 | 2.026584608 | 0.394905638 | 2.380255893 | 1.595980428 |
NSCs_1 | 68221902 | 1.916242372 | 0.381639103 | 2.25065718 | 1.542364761 |
NSCs_2 | 62275625 | 1.922886765 | 0.382886143 | 2.258461126 | 1.547404574 |
NSCs_3 | 67670669 | 1.982610575 | 0.391849804 | 2.328607692 | 1.583630515 |
rel: relative; obs: observed; exp: expected; ASCs: activated Schwann cells; NSCs: normal Schwann cells.
All statistics of calculations and plot are using MEDIPS, R package for MeDIP-Seq. BSgenome, Rnorvegicus, and UCSC were all genome databases.
(a) The distribution of differentially methylated regions (DMRs) peaks in different components of genome. (b) The distribution of DMRs peaks in activated Schwann cells (ASCs). (c) The distribution of DMRs peaks in normal Schwann cells (NSCs).
A total of 176612 methylated genes between ASCs and NSCs were detected by high-throughput MeDIP sequencing analysis (
Hypermethylated and hypomethylated genes were significantly enriched (
GO-enrichment analysis of biological processes (a), cellular components (b), and molecular functions (c).
Signaling pathway analysis of hypermethylated and hypomethylated genes was conducted. A total of 279 signaling pathways were identified. Of them, 9 signaling pathways including cAMP signaling pathway, Wnt signaling pathway, ERK signaling pathway, PI3-Akt signaling pathway, Hippo/YAP signaling pathway, MAPK signaling pathway, Rap1 signaling pathway, cGMP-PKG signaling pathway, and calcium signaling pathway were considered to be significant (
Significant KEGG signaling pathways of methylated genes.
Pathway | Gene number |
|
Partial related methylated genes |
---|---|---|---|
Calcium signaling pathway | 136 | 0.01 | Phka2, Adcy7, Hrh2, Drd1, Grm1, Vdac1, Grm5, Pde1b, Tnnc2, Nos1, Camk4 |
cAMP signaling pathway | 143 | 0.02 | Vav3, Drd2, Adcy10, Rhoa, Sox9, Mc2r, Gli3, Cngb3, Rras, Adcy1, Rac1 |
cGMP-PKG signaling pathway | 126 | 0.02 | Adcy7, Irs1, Npr2, Mylk, Rhoa, Adrb1, Ins2, Adcy5, Gna12, Itpr3, Prkg1 |
Wnt signaling pathway | 105 | 0.03 | Fzd5, Nlk, Smad4, Chd8, Plcb1, Sfrp4, Sost, Mapk8, Gsk3b, Lrp5, Rac1 |
Rap1 signaling pathway | 150 | 0.03 | Kit, Magi1, Krit1, Plce1, Pgf, Csf1, Tln1, Cdh1, Skap1, Fgf8, Rac1, Actg1 |
ERK signaling pathway | 62 | 0.04 | Mtor, Nck1, Erbb3, Egf, Araf, Pak7, Shc3, Plcg2, Ptk2, Tgfa, Nrg1, Kras |
PI3-Akt signaling pathway | 209 | 0.04 | Itga4, Ccne1, Gng7, Cdc37, Tlr2, Ccnd3, Gngt1, Hgf, Syk, Fgf1, Tnr, Tnc |
Hippo/YAP signaling pathway | 104 | 0.04 | Fzd5, Mpp5, Crb2, Gsk3b, Tead1, Ccnd1, Actb, Smad3, Dlg1, Fzd4, Bmp7 |
MAPK signaling pathway | 172 | 0.04 | Atf2, Cd14, Rras2, Ntf3, Rap1b, Grb2, Dusp1, Stk4, Mapk3, Jun, Nf1 |
KEGG: Kyoto Encyclopedia of Genes and Genomes.
Differentially methylated genes related to repairment of peripheral nerve injury including adhesion, secretion, proliferation, neuronal regeneration, and axonal regeneration were uncovered. For adhesion, 580 methylated genes were identified, such as Sox-2, Shh, Lphn1, and Cdh9. 553 differentially methylated genes related to secretion including Lphn1, Pim3, and Plcd1 were found. We also confirmed 787 methylated genes associated with proliferation such as Dixdc1, Lrp6, Edn3, and Ephb1. For neuronal regeneration, 473 methylated genes were identified, like Bhlhb9, Cckar, Klhl1, Dlg2, and others. In addition, 215 differentially methylated genes related to axonal regeneration including Ifrd1, Unc5c, and Ntrk2 were found. Detailed results have been summarized in Table
Five aspects of genes related to repairment of peripheral nerve injury.
Biological function | Gene number | Partial related methylated genes |
---|---|---|
Adhesion | 580 | Sox-2, Shh, Itpkb, Pkd1, Reln, Ptk2b, Nck2, Ass1 |
Secretion | 553 | Lphn1, Pim3, Fst, Pim3, Pcsk6, Lax1, Sct, Sytl3 |
Proliferation | 787 | Dixdc1, Lrp6, Edn3, Nkx2, Cyr61, Src, Sox8, Stk4 |
Neuronal regeneration | 473 | Bhlhb9, Cckar, Fzd2, Thy1, Pbx3, Otx2, Lhx8, Btg2 |
Axonal regeneration | 215 | Ifrd1, c-Jun, Bcl2, Tnn, Mbp, Slit3, Ist1, Drgx, Thy1 |
In addition to validating the MeDIP analysis results, qRT-PCR was used to quantify parts of mRNAs of corresponding methylated genes in ASCs compared with NSCs, such as 5 differentially hypermethylated genes (Dgcr8, Zeb2, Dixdc1, Sox2, and Shh) and 2 differentially hypomethylated genes (Gpr126, Birc2), resulting in the fact that they were closely related with repairment of peripheral nerve injury after searching PubMed. As shown in Figure
Validation of the differential expression of 7 mRNAs of corresponding methylated genes identified in the MeDIP in ASCs compared with NSCs by qRT-PCR. Data indicate relative expression following normalization. Values are means ± SE (
Epigenetics is the branch of traditional genetics, whose meaning is that this action changes expression and function of genes and generates heritable phenotypes under the condition of unchanged DNA sequences [
ASCs serve a critical role in nerve remyelination after PNS injury, which are specialised glial cells and myelin-forming cells in PNS [
DNA methylation peak number shows the popularity of methylation in genome. More DNA methylation peaks mean more loci in genome are methylated. Our data indicated that the distribution of DMRs peaks in different components of genome showed that uniquely mapped reads in distal intergenic regions had a relatively higher methylation level but only 567 DMRs peaks in CGls account for only 0.32 percent. It should also be noted that DNA methylation does not occur exclusively at CGls. In fact, recently tissue-specific DNA methylation has been found at CpG island shores, outlying areas close to CGls (up to 2 kb distance), and strongly related to gene expression inactivation [
In order to disclose the underlying molecular mechanisms of SCs in repairing injured PNS, we characterized the possible GO functional terms and signaling pathways of differentially methylated genes using the GO function and KEGG analysis. Considering the results of GO function analysis, we linked the differentially methylated genes with biological regulation, cellular process, regulation of biological process, metabolic process, single-organism process, and other biological processes which are probably very important for the injured PNS repair process of SCs. As previous articles reported, our KEGG pathway analysis showed that cAMP signaling pathway, Wnt signaling pathway, ERK signaling pathway, PI3-Akt signaling pathway, Hippo/YAP signaling pathway, MAPK signaling pathway, and others were among the most relevant pathways for injured PNS repair process of SCs. Calcitonin gene-related peptides could activate the cAMP-PKA-ERK signaling cascade, which may play a direct role in initiating inflammatory processes in the PNS [
Also of note is that there were numerous evidences for our differentially methylated genes, which have proven to play important roles during PNS injury repairment. Glenn and Talbot [
The authors declare that they have no conflicts of interest.
Xian-Hu Zhou, Wei Lin, and Yi-Ming Ren contributed equally to this work.
The authors are grateful for the support by the State Program of National Natural Science Foundation of China (81371957), the State Key Program of the National Natural Science Foundation of China (81330042), the Special Program for Sino-Russian Joint Research Sponsored by the Ministry of Science and Technology, China (2014DFR31210), the International Cooperation Program of National Natural Science Foundation of China (81620108018), and the Key Program Sponsored by the Tianjin Science and Technology Committee, China (13RCGFSY19000, 14ZCZDSY00044).