Remodeling of chromatin and transcriptomic landscape to enhance optic nerve regeneration

摘要 远距离轴突再生是最重要的方面之一，也是成功的前提。 视神经损伤后的功能恢复。尽管在加强国家安全方面取得了很大的进展 多种途径的固有轴突再生能力，远距离视神经再生可达 大脑中的原始靶点仍然是一个重大挑战。我们认为扩大足够多的伤者 从不同RGC亚型进入大脑的RGC轴突应该是功能恢复的主要任务 在视力受损后。因此，需要一个新的策略来1)提高RGC存活率，2)识别 其他基因靶点能够促进不同亚型RGC的再生，以及3)促进 广泛的远距离视神经再生，受抑制环境的影响较小。在.期间 RGC成熟时，其染色质结构会发生时间上的变化，导致转录水平的变化 这是支持轴突再生的内在能力丧失的原因。相反，目前发现的基因 这种促进视神经再生的作用可能改变了在转录水平上的发育变化 某种程度上。因此，揭示视网膜节细胞的染色质和转录图谱是非常重要的。 用于轴突再生，并确定关键的转录因子和/或染色质调节剂 再生视网膜节细胞的这种染色质状态。在目标1中，通过执行RNA-SEQ、ATAC-SEQ和CHIP-SEQ 纯化的视网膜神经节细胞在不同的成熟阶段和不同的再生状态，我们将使用先进的 综合生物信息学分析揭示视网膜节细胞染色质和转录组的有利格局 用于轴突再生，并确定关键的转录因子和/或染色质调节剂 再生视网膜节细胞的染色质状态。在目标2中，我们将进行功能筛选实验，以 确定它们在调节RGC存活和/或视神经再生中的作用及其潜在的 机械装置。我们最近的工作表明，删除非肌肉肌球蛋白IIA/B或组蛋白去甲基酶UTX， 当与增强的固有轴突再生能力相结合时，可能导致广泛的远距离光学 神经再生。基于这些结果，在目标3中，我们将探索是否将新识别的 带有UTX和肌球蛋白IIA/B基因敲除的转录因子可诱导远距离视神经再生 进入大脑。拟议的研究不仅将产生转录组分变化的详细图景， 视网膜节细胞成熟和再生过程中染色质的可及性和表观遗传景观 确定新的分子靶点和优化方法以重建视觉回路。

Summary Long distance axon regeneration is one of the most important aspects and a prerequisite for successful functional recovery after optic nerve injuries. Although great progress has been made to enhance the intrinsic axon regeneration ability via various approaches, long distance optic nerve regeneration reaching the original targets in the brain remains a major challenge. We think that extending sufficient number of injured RGC axons from different RGC subtypes into the brain should be the major tasks for functional recovery after visual injuries. Therefore, a new strategy is needed to 1) enhance RGC survival rate, 2) identify additional gene targets capable of enhance regeneration from a diverse subtypes of RGCs, and 3) promote extensive long-distance optic nerve regeneration that is less affected by the inhibitory environment. During RGC maturation, their chromatin structures change temporally, leading to changed transcriptomics underlying the loss of intrinsic ability to support axon regeneration. Conversely, the current identified genes that act to enhance optic nerve regeneration presumably alter the developmental changes in transcriptomics in some way. Thus, it is important to reveal the chromatin and transcriptomics landscape of RGCs favorable for axon regeneration, and identify key transcription factors and/or chromatin modulators underlying such chromatin state of regenerating RGCs. In Aim 1, by performing RNA-seq, ATAC-seq and ChIP-seq of purified RGCs at different maturation stages, and different regenerative states, we will use advanced integrative bioinformatics analyses to reveal the chromatin and transcriptomics landscape of RGCs favorable for axon regeneration, and identify key transcription factors and/or chromatin modulators underlying such chromatin state of regenerating RGCs. In Aim 2, we will perform functional screening experiments to determine their roles in regulation of RGC survival and/or optic nerve regeneration, and their underlying mechanisms. Our recent work showed that deleting non-muscle myosin IIA/B or histone demethylase UTX, when combined with enhanced intrinsic axon regeneration ability, could lead to extensive long-distance optic nerve regeneration. Based on these results, in Aim 3, we will explore if combining the newly identified transcription factors with UTX and myosin IIA/B knockout could induce long distance optic nerve regeneration into the brain. The proposed studies will not only generate a detailed picture of changes in transcriptomics, chromatin accessibility and epigenetic landscape of RGCs during maturation and regeneration, but also identify novel molecular targets and optimized approaches to re-establish visual circuity.