Systematic analysis of the epigenetic events driving axonal regeneration

Abstract

Axonal regeneration after injury to the peripheral branch of the dorsal root ganglia (DRG) is associated with injury-induced signalling, regenerative gene expression and increased histone acetylation. These events do not occur after injury to the central branch, which is associated with regenerative failure. Whilst previous studies have assessed differential gene expression within the DRG after axonal injury, to date no systematic study has been performed to understand how histone acetylation is associated with differential gene expression. To address this, RNA sequencing (RNAseq) and chromatin immunoprecipitation sequencing (ChIPseq) for histone acetylation (H3K27ac and H3K9ac) was performed in the DRG 24 hours after injury to the peripheral (Sham vs sciatic nerve axotomy [SNA]) or central branch (Laminectomy [Lam] vs dorsal column axotomy [DCA]). From the RNAseq analysis, 3222 genes were identified to be differentially expressed within the DRG after peripheral branch injury (n=1560 upregulated; n=1662 downregulated, P<0.05). In contrast, after central branch injury, 948 genes were identified to be differentially expressed (n=525 upregulated; n=423 downregulated, P<0.05). Combined RNA- and ChIP-seq analyses demonstrated that increased H3K27ac and H3K9ac was significantly associated with increased gene expression after peripheral but not central branch injury. Moreover, after peripheral branch injury, 27.5% (n=430) of all upregulated genes had increased enrichment for H3K27ac and/or H3K9ac, whilst 2.6% (n=40) had decreased enrichment for H3K27ac and/or H3K9ac (TSS ± 2Kb, P<0.05). After central branch injury, only 4.6% (n=24) of all upregulated genes had increased enrichment for H3K27ac and/or H3K9ac, whilst 8.0% (n=34) had decreased enrichment for H3K27ac and/or H3K9ac (TSS ± 2Kb, P<0.05). Amongst the top upregulated genes after peripheral but not central branch injury, with increased enrichment for H3K27ac across its TSS ± 2Kb, was CITED2, a transcriptional adaptor that recruits transcription factors to sites of histone acetylation (Sham vs SNA: 2.9-fold, <P<0.0001; Lam vs DCA, -0.7-fold, P=0.1). In vitro studies demonstrated that the overexpression of CITED2 was associated with increased DRG neurite outgrowth. Lastly, the histone deacetylase (HDAC) inhibitor, panobinostat, was identified as a small molecule that could drive CITED2 expression and enhance H3K27ac and H3K9ac in vitro and it was shown that treatment with panobinostat could enhance regeneration and improve sensorimotor recovery after SCI in mice. These findings warrant further investigation in pre-clinical models of SCI.