ABSTRACT

Alterations in environmentally sensitive epigenetic mechanisms (e.g., DNA methylation) influence axonal regeneration in the spinal cord following sharp injury. Conventional DNA methylation detection methods using sodium bisulphite treatment do not distinguish between methylated and hydroxymethylated forms of cytosine, meaning that past studies report a composite of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To identify the distinct contributions of DNA methylation modifications to axonal regeneration, we collected spinal cord tissue after sharp injury from untreated adult F3 male rats with enhanced regeneration of injured spinal axons or controls, derived from folate- or water-treated F0 lineages, respectively. Genomic DNA was profiled for genome-wide 5hmC levels, revealing 658 differentially hydroxymethylated regions (DhMRs). Genomic profiling with whole genome bisulphite sequencing disclosed regeneration-related alterations in composite 5mC + 5hmC DNA methylation levels at 2,260 differentially methylated regions (DMRs). While pathway analyses revealed that differentially hydroxymethylated and methylated genes are linked to biologically relevant axon developmental pathways, only 22 genes harbour both DhMR and DMRs. Since these differential modifications were more than 60 kilobases on average away from each other, the large majority of differential hydroxymethylated and methylated regions are unique with distinct functions in the axonal regeneration phenotype. These data highlight the importance of distinguishing independent contributions of 5mC and 5hmC levels in the central nervous system, and denote discrete roles for DNA methylation modifications in spinal cord injury and regeneration in the context of transgenerational inheritance.

摘要

环境敏感的表观遗传机制的改变（例如., DNA 甲基化）影响脊髓中锐器损伤后的轴突再生。使用亚硫酸氢钠处理的传统 DNA 甲基化检测方法不区分甲基化和羟甲基化形式的胞嘧啶，这意味着过去的研究报告了 5-甲基胞嘧啶 (5mC) 和 5-羟甲基胞嘧啶 (5hmC) 的复合物。为了确定 DNA 甲基化修饰对轴突再生的不同贡献，我们收集了未经治疗的成年 F3 雄性大鼠锐器损伤后的脊髓组织，这些大鼠的脊髓轴突或对照组的再生增强，分别来自叶酸或水处理的 F0 谱系。对基因组 DNA 进行全基因组 5hmC 水平分析，揭示了 658 个差异羟甲基化区域 (DhMR)。全基因组亚硫酸氢盐测序的基因组分析揭示了 2,260 个差异甲基化区域 (DMR) 复合 5mC + 5hmC DNA 甲基化水平的再生相关改变。虽然通路分析显示差异羟甲基化和甲基化基因与生物学相关的轴突发育通路有关，但只有 22 个基因同时具有 DhMR 和 DMR。由于这些差异修饰平均彼此相距超过 60 千碱基，因此绝大多数差异羟甲基化和甲基化区域在轴突再生表型中具有独特的功能。这些数据强调了区分 5mC 和 5hmC 水平在中枢神经系统中的独立贡献的重要性，