Comprehensive mapping of 5-hmC epigenetic dynamics in axon regeneration

In contrast to CNS neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated, along with increased 5-hydroxymethylcytosine (5-hmC) in DRG neurons. We generated genome-wide 5-hmC maps in adult DRG, which demonstrated that peripheral and central axotomy (no regenerative effect) triggered differential 5-hmC changes that are associated with distinct signaling pathways. 5-hmC was altered in half of regeneration-associated genes (RAGs), supporting its role for RAG regulation. Our analyses predicted transcription factors HIF-1, STAT and IRF that may collaborate with Tet3 for 5-hmC modifications. Intriguingly, central axotomy lead to widespread 5-hmC modifications with little overlap with peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5-hmC as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury. 5-hmC profiles generated from DRGs at 3 conditions (PL, DCL, Ctrl) plus Input, performed in triplicate

与中枢神经系统（Central Nervous System, CNS）神经元不同，背根神经节（dorsal root ganglia, DRG）神经元在周围轴突切断术后可切换至再生状态。针对该条件损伤模型下的再生反应染色质调控因子开展筛选实验时，我们鉴定出四甲基胞嘧啶双加氧酶3（Tet methylcytosine dioxygenase 3, Tet3）表达上调，同时背根神经节神经元内的5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5-hmC）水平升高。我们构建了成年背根神经节的全基因组5-hmC图谱，结果显示周围轴突切断术与中枢轴突切断术（无再生效果）均可引发差异化的5-hmC水平变化，且此类变化与不同的信号通路密切相关。半数再生相关基因（regeneration-associated genes, RAGs）的5-hmC修饰状态发生改变，这为5-hmC参与再生相关基因调控提供了实验依据。我们通过分析预测，转录因子HIF-1、STAT与IRF可与Tet3协同参与5-hmC修饰过程。值得注意的是，中枢轴突切断术会引发广泛的5-hmC修饰，且与周围轴突切断术诱导的修饰几乎无重叠，这可能构成神经元再生的障碍。本研究揭示，5-hmC是此前未被发现的、介导轴突损伤后差异化应答反应的表观遗传调控机制。本实验针对3种处理条件（PL、DCL、Ctrl）及Input样本的背根神经节开展了三次生物学重复的5-hmC图谱检测。