Department of Biological Sciences, University at Albany, State University of New York, Albany, NY, 12222, USA.
Center for Neuroscience Research, University at Albany, State University of New York, Albany, NY, 12222, USA.
BMC Genomics. 2022 Jan 4;23(1):2. doi: 10.1186/s12864-021-08247-0.
Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS.
Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC.
Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.
由于一些中枢神经系统（CNS）神经元（例如视神经损伤后的视网膜神经节细胞（ONC））在整个生命过程中都能再生轴突，而其他神经元（例如脊髓损伤后的后脑神经元）在蝌蚪变成青蛙时会失去这种能力，因此南非爪蟾（Xenopus laevis）为探索同一生物体中再生和非再生对 CNS 损伤的反应之间的差异提供了独特的机会。早期的一项三向 RNA-seq 研究（青蛙 ONC 眼、蝌蚪 SCI 后脑、青蛙 SCI 后脑）鉴定了在 CNS 再生和非再生中差异表达的基因中调节染色质可及性的基因[11]。本研究使用来自这些相同动物的在轴突再生高峰期收集的 DNA 进行全基因组亚硫酸氢盐测序（WGBS），以研究 DNA 甲基化在多大程度上可能是再生和非再生 CNS 之间染色质可及性差异的基础。
与 DNA 甲基化水平与 CNS 再生能力呈负相关的假设一致，再生的蝌蚪后脑和青蛙眼的 DNA 甲基化程度低于非再生的青蛙后脑。此外，与哺乳动物 CNS 损伤的观察结果一致，非再生的青蛙后脑的 DNA 甲基化在 SCI 后减少。然而，与预期的在再生 CNS 中轴突切断后 DNA 甲基化水平会进一步降低相反，这些区域的 DNA 甲基化随着损伤而增加。在再生 CNS 中，与损伤相关的 CpG 甲基化差异在基因启动子区域特别丰富，而非 CpG 甲基化差异在启动子区域、基因间和基因内区域分布更为均匀。在非再生 CNS 中，组织相关（即再生与非再生 CNS）和损伤诱导的启动子区域 CpG 甲基化减少与 RNA 表达增加显著相关，但在再生 CNS 中观察到的损伤诱导的、跨启动子区域的 CpG 甲基化增加则不然，表明它与染色质可及性的增加而非减少有关。这一假设得到了以下观察结果的支持：在再生 CNS 中，许多表现出增加的、损伤诱导的、与启动子相关的 CpG 甲基化的基因也表现出增加的 RNA 表达，并与活跃启动子和增强子的组蛋白标记物相关联。视神经再生中的 5hmC DNA 免疫沉淀发现，在 CpG 甲基化中观察到的与损伤相关的增加与 5hmC 变化不同。
尽管看似矛盾，但在再生 CNS 中观察到的与损伤相关的 DNA 甲基化增加与干细胞和癌症有许多相似之处。因此，再生 CNS 中轴突切断引起的这些 DNA 甲基化变化为成功的 CNS 轴突再生提供了一种新的有利的表观遗传状态的证据。本研究中描述的数据集应有助于为未来研究涉及的分子和细胞机制奠定基础。所获得的见解反过来应该有助于为哺乳动物的 CNS 损伤治疗指明新的治疗方法。
