Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation–dependent and –independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.


Supplemental Figure 10. Ezh2 overexpression does not alter the epigenetic aging clock of RGCs.
(A) DNA methylation aging signature of RGCs is increased by optic nerve injury, but not affected by Ezh2 or Ezh2-Y726D overexpression (one-way ANOVA followed by Tukey's multiple comparisons; P < 0.0001; n = 3 reduced representation bisulfite sequencing libraries for all).
(B) Heatmaps of mRNA levels of 5mC DNA methyltransferases and demethylases showing that most of them are not significantly changed by Ezh2 or Ezh2-Y726D overexpression in RGCs.Note that Aicda mRNA was not detected in the control vs. Ezh2-Y726D overexpression RNA-seq.
For replate experiments, electroporated cells were plated on pre-coated dishes and cultured for 3 days.Cells were then forced to detach from dishes by pipetting, replated on pre-coated coverslips, and cultured for 24 hours.
For culture of conditioning lesioned DRG neurons, L4/5 DRGs were dissected from Ezh2 f/f and Advillin-Cre; Ezh2 f/f mice 3 days after sciatic nerve transection (see Sciatic nerve crush or transection in Methods).After enzymatic digestion and dissociation, filtered cells were immediately plated on pre-coated coverslips and cultured for 24 hours.

Analysis of in vitro DRG neuron axon growth.
Fluorescent images of cultured DRG neurons were obtained with a Zeiss inverted fluorescence microscope controlled by the AxioVision software using a 5× objective.The longest axon of each neuron was manually traced and measured with the built-in "measure/curve spline" function of the AxioVision software.Only neurons with axons longer than twice the diameter of their somas were included.In most experiments, at least 60 neurons were analyzed in each condition.Measurement was done by experimenters blinded to experimental conditions.Tissue clearing of optic nerves.Two days after intravitreal CTB injection, mice were anesthetized and transcardially perfused with PBS followed by 4% PFA.Optic nerves were dissected and post-fixed in 4% PFA overnight at 4°C.On the next day, optic nerves were washed 3 times with PBS, dehydrated in an ascending series of tetrahydrofuran (50%, 70%, 80%, 100% and 100%, v/v in distilled water, 20 min each, MilliporeSigma 186562), and cleared in a 1:2 mixed solution of benzyl alcohol and benzyl benzoate (BABB, MilliporeSigma 305197 and B6630).Incubations were done on an orbital shaker at room temperature.Nerves were stored in BABB and protected from light at room temperature before imaging.
RNA-seq and data analysis.RNA was isolated from FACS-enriched RGCs 3 days after ONC using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific KIT0204) following the manufacture's manual.RNA quality was verified using the Agilent Fragment Analyzer (Agilent Technologies).RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Kit (Illumina) and quality checked by the Agilent Fragment Analyzer.Equimolar amounts of the finished libraries were then pooled and sequenced on an Illumina NextSeq 500 with 2×75 bp paired reads.
Raw FASTQ data were mapped to the mouse reference genome (GRCm38) using STAR aligner (version 2.7.0d) with default parameters.The number of counts per gene was estimated using the "quantMode" command in STAR.Quantified raw counts were used in DESeq2 (version 1.22.2) to obtain DEGs.Genes with less than 10 counts in total from six libraries were excluded from analysis.Genes with adjusted P < 0.05 and fold change > 1.5 were chosen as DEGs.Principal component analysis (PCA) and hierarchical clustering were also performed with the transformed count matrix in DESeq2.One library in the Ezh2 overexpression condition was excluded from further data analysis due to low repeatability with two other libraries in the same condition.Normalized counts were used to produce heatmaps and scatter plots.GO analysis (biological process) was done using DAVID Bioinformatics Resources 6.8.ATAC-seq and data analysis.ATAC-seq libraries were constructed from FACS-enriched RGCs (50,000 cells for each library) 3 days after ONC following a previously published protocol (2).Briefly, cells were pelleted by centrifugation (500 g, 5 min, 4°C), washed with ice-cold PBS, pelleted again by centrifugation (500 g, 5 min, 4°C), and lysed in 50 μl ice-cold lysis buffer.Immediately after lysis, nuclei were pelleted by centrifugation (500 g, 10 min, 4°C), resuspended in 50 μl transposase reaction mix (Tagment DNA TDE1 Enzyme and Buffer Kits, Illumina 20034197) and incubated at 37°C for 30 min.After the transposition reaction, the product was purified with the DNA Clean & Concentrator-5 Kit (Zymo Research D4003).20 μl tagmented DNA was PCR amplified with NEBNext High-Fidelity PCR Master mix (New England Biolabs M0541) and forward and reverse UDI primers.Amplification was first performed for 5 cycles, following which 5 μl of each partially amplified library was used to perform qPCR to determine the additional number of PCR cycles needed for each library.Final amplified libraries were purified using 1.1× Ampure XP bead purification (Beckman Coulter A63880).Equimolar amounts of the finished libraries were then pooled and sequenced on an Illumina NovaSeq 6000 with 2×100 bp paired reads.
After removing adapters of Illumina reads with Cutadapt, pair-end ATAC-seq reads were mapped to the mouse reference genome (GRCm38) using Bowtie2 with default parameters.Qualified properly paired reads (MAPQ score > 10) were assessed by SAMTools.Duplicate reads were removed with MarkDuplicates function in Picard.After using MACS2 to call peak regions of each sample, we used multiBamSummary function in deepTools to calculate read counts for all samples.ChIPseeker was used to annotate genomic context of identified peaks.Gene annotation information was accessed using TxDb.Mmusculus.UCSC.mm10.knownGene.Differential accessibility analysis was performed by DESeq2.GO analysis (biological process) was done using DAVID Bioinformatics Resources 6.8.Fragment size distribution and transcription start site enrichment were performed by ATACseqQC.