组蛋白H3K27me3及去甲基化酶UTX/JMJD3在视神经再生中的功能及分子机制研究

The regeneration and repair of the damaged mammalian central nervous system (CNS) has been a huge challenge in the field of neuroscience, the major reason is that neurons are no longer capable of dividing and proliferating, and their capacity of axonal regeneration is almost completely lost. Histone H3k27 methylation, which is widely existed in CNS, is one of the most important epigenetic regulations of gene expression. However, its functional roles in the regeneration and repair of the adult CNS are still largely unknown. Our previous studies have revealed that histone methyltransferase EZH2 and demethylases UTX/IMID3 are highly expressed in post-mitotic neurons, and knocking down EZH2 impairs sensory axon regeneration both in vitro and in vivo. In contrast, ectopically downregulation of UTX/JMID3 is significantly promote axon regeneration. More importantly, our preliminary study also demonstrates that knocking out UTX in retinal ganglion cells dramatically enhances their survivability and optic nerve regeneration, indicating that histone H3k27 methylation might play critical roles in CNS injury and repair. This proposed research project will utilize our own conditional knockout mice of UTX and JMJD3 to study the function of histone H3k27 methylation in CNS injury and repair, and to identify key molecules and molecular mechanisms that are responsible for nerve regeneration through advanced RNA-seq and Chip-seq technologies. With the aid of electrophysiological and visual behavioral analyses, we will examine the feasibility and potential of modulating histone methylation levels for visual functional recovery after optic nerve injury. This project will help to develop new theories for understanding adult CNS regeneration, and to shape novel strategies for promoting axon regeneration in the damaged CNS.

中枢神经系统（CNS）损伤后的再生与修复一直是神经科学领域的难题。组蛋白H3K27甲基化修饰是表观遗传调控的重要方式之一，但在CNS损伤修复中的研究尚属空白。我们前期工作表明H3K27甲基化转移酶EZH2和去甲基化酶UTX/JMJD3不但具有调节周围神经再生的能力，而且能显著影响视网膜神经节细胞（RGC）的存活和轴突再生，暗示着H3K27甲基化修饰在CNS损伤修复中扮演重要角色。本课题拟利用我们前期构建好的UTX与JMJD3的条件敲除鼠，研究UTX/JMJD3和H3K27me3在视神经再生和RGC存活中的功能；通过转录组测序与Chip-seq鉴定关键信号分子，解析UTX/JMJD3和H3K27me3调控神经再生的分子机制；借助视神经电生理和视觉行为学分析手段，探索干预H3K27me3对视觉功能重建的可行性与途径，为CNS损伤修复的治疗提供新靶点和新思路，具有重要的理论意义和临床转化前景。

中枢神经系统（CNS）损伤后的再生与修复一直是神经科学领域的难题。组蛋白H3K27甲基化修饰是表观遗传调控的重要方式之一，但在CNS损伤修复中的研究尚属空白。我们前期工作表明H3K27甲基化转移酶EZH2和去甲基化酶UTX/JMJD3不但具有调节周围神经再生的能力，而且能显著影响视网膜神经节细胞（RGC）的存活和轴突再生，暗示着H3K27甲基化修饰在CNS损伤修复中扮演重要角色。该项目利用我们前期构建好的UTX与JMJD3的条件敲除鼠，特异性敲除RGC中UTX/JMJD3基因，进而提高组蛋白H3K27me3的修饰水平，研究UTX/JMJD3和H3K27me3在视神经再生和RGC存活中的功能；通过转录组测序与Chip-seq鉴定关键信号分子，解析UTX/JMJD3和H3K27me3调控神经再生的分子机制。结果显示UTX在视神经节细胞中特异敲除后，损伤RGC的转录组图谱被逆转到了发育早期，从而促进了视神经节细胞的存活与再生,同时我们的结果显示UTX通过非编码RNA miR-124来调控RGC的再生能力，结果表明UTX/JMJD3介导的H3K27me3修饰是视神经再生修复的重要靶点。同时我们发现UTX/JMJD3酶活性的特异抑制剂GSK-J4可以显著促进RGC再生能力。此外我们发现染色质重塑复合体组分Arid1a在RGC中敲除后可以明显的提高损伤RGC的存活能力，进而发现Arid1a的敲除改变了染色质的可及性，从而改变了下游靶基因的转录水平；miR-26a的过表达可以显著的提高损伤RGC的存活和再生能力，其下有调控的靶基因为PTEN。上述结果为CNS损伤修复的治疗提供新靶点和新思路，具有重要的理论意义和临床转化前景。