DNA methylation and hydroxymethylation have distinct genome-wide profiles related to axonal regeneration

Alterations in environmentally sensitive epigenetic mechanisms (<i>e.g</i>., 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.

环境敏感性表观遗传机制（epigenetic mechanisms）的改变，会影响锐性损伤后脊髓内的轴突再生。传统的基于亚硫酸氢钠（sodium bisulphite）处理的DNA甲基化（DNA methylation）检测方法，无法区分胞嘧啶（cytosine）的甲基化与羟甲基化形式，这意味着过往研究报道的是5-甲基胞嘧啶（5-methylcytosine, 5mC）与5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）的复合信号。为明确DNA甲基化修饰对轴突再生的独立贡献，我们分别从经叶酸或水处理的F0世系衍生而来的未处理成年雄性F3大鼠中，采集锐性损伤后的脊髓组织：其中一组为脊髓损伤轴突再生能力增强的实验组，另一组为对照组。我们对基因组DNA开展全基因组水平的5hmC水平谱分析，共鉴定出658个差异羟甲基化区域（differentially hydroxymethylated regions, DhMRs）。通过全基因组亚硫酸氢盐测序（whole genome bisulphite sequencing）进行基因组分析后发现，在2260个差异甲基化区域（differentially methylated regions, DMRs）中，复合的5mC+5hmC DNA甲基化水平出现了与再生相关的改变。尽管通路分析显示，差异羟甲基化基因与差异甲基化基因均富集于具有生物学相关性的轴突发育通路，但仅有22个基因同时存在DhMR与DMR。由于这些差异修饰位点之间的平均间距超过60千碱基（kilobases, kb），绝大多数差异羟甲基化区域与差异甲基化区域彼此独立，且在轴突再生表型中发挥独特功能。本研究数据凸显了区分中枢神经系统（central nervous system, CNS）中5mC与5hmC水平独立贡献的重要性，并阐明了跨代遗传（transgenerational inheritance）背景下，DNA甲基化修饰在脊髓损伤与再生过程中的独立作用。