Expanding retinal regenerative potential through chromatin biology in single cells

神经元在损伤后在哺乳动物中基本上是非增殖的，这导致了针对神经元损伤的强烈兴趣。 将邻近的神经胶质细胞重新编程为神经元1。在许多鱼类和两栖动物的视网膜中 神经元从Müller神经胶质的再生在损伤后自然发生2，3。这种再生能力依赖于 高度保守的转录因子Ascl 14。有趣的是，小鼠中Ascl 1的工程过表达导致了 产生一些神经元细胞类型，如双极细胞，但不产生光感受器或神经节细胞5 -7。我 旨在开发和使用单细胞技术，将Müller神经胶质推向更多样化的细胞类型， 确定哪些顺式调节元件驱动细胞命运的变化。从神经胶质产生光感受器 将标志着再生治疗失明的发展取得了非凡的进展。 单细胞测序革命现在使人们有可能在一个单一的实验中， 细胞群体对多达数千种处理条件的个体转录反应8 -10。 结合日益增长的趋势，即在一个单一的环境中同时测定多个监管步骤， cell 11 -24，现在是应用和开发单细胞解码技术的最佳时机 调节异质细胞群体中的命运变化，例如那些经历重编程的细胞。 在目标1中，我将着手揭示驱动穆勒神经胶质转化为新型神经胶质细胞的条件。 重新编程的命运最近的一项研究表明，穆勒胶质细胞向神经元转化的效率非常高 通过用组蛋白去乙酰化酶抑制剂（HDACi）处理细胞来改善。我假设染色质 HDACi治疗没有破坏的修饰，阻止了细胞类型在细胞分裂后更多样化的重新连接。 Ascl 1的诱导。通过利用Trapnell实验室最近开发的一种技术， 在数百种不同的小分子治疗下的细胞，每种治疗针对表观基因组的各个方面，以及 然后读出单细胞转录组10。通过染色质生物学的系统扰动，我希望 扩大Müller胶质细胞的重编程潜力，并更好地了解视网膜细胞的特化。 在目标2中，我建议设计一种能够读出转录组和基因组的检测方法， 在单个细胞中定位组蛋白修饰。多模式单细胞技术是强大的方法 评估基因调控过程的各个方面是如何相互作用的。分析的输出，例如 我的目标是通过使用回归分析，开发可以允许构建连接地图的 基因的调控位点。这可以用来确定顺式调控元件最负责每一个 缪勒神经胶质重新编程轨迹。如果成功的话，类似的技术可以用来改善无数其他 差异化协议，并更好地了解驱动差异化的监管格局。

Neurons are largely non-proliferative in mammals after injury leading to intense interest aimed at reprogramming the neighboring glial cells into neurons1. In the retina of many fish and amphibians regeneration of neurons from Müller glia occurs naturally after injury2,3. This regenerative capacity relies on the highly conserved transcription factor Ascl14. Intriguingly, engineered overexpression of Ascl1 in mice leads to the production of some neuronal cell types, such as bipolar cells, but not photoreceptors or ganglion cells5–7. I aim to develop and use single-cell technologies to push Müller glia towards more diverse cell types and to define which cis-regulatory elements drive those changes in cell fate. Production of photoreceptors from glia would mark exceptional progress towards the development of regenerative treatments for blindness. The single-cell sequencing revolution now makes it possible, within a single experiment, to recognize the individual transcriptional responses of cell populations to up to thousands of treatment conditions8–10. Combined with the growing movement towards assaying multiple regulatory steps simultaneously in a single cell11–24, there has never been a better time to apply and develop single-cell technologies aimed at decoding the regulation of fate changes in heterogeneous cell populations, such as those undergoing reprogramming. In Aim 1, I will set out to uncover conditions which drive the conversion of Müller glia to novel reprogramed fates. A recent study indicated that the efficiency of Müller glia to neuronal transition is vastly improved by the treatment of cells with a histone deacetylase inhibitor (HDACi)6. I hypothesize that chromatin modifications, not disrupted by HDACi treatment, prevent a more diverse rewiring of cell types upon the induction of Ascl1. By harnessing sci-Plex, a technique recently developed in the Trapnell lab, I will culture cells under hundreds of different small molecule treatments, each targeting aspects of the epigenome, and then readout single-cell transcriptomes10. Through the systematic perturbation of chromatin biology, I hope to expand the reprogramming potential of Müller glia and gain a better understanding of retinal cell specification. In Aim 2, I propose engineering an assay able to read out both the transcriptome and the genomic localization of histone modifications in a single cell. Multimodal single-cell technologies are powerful methods to assess how aspects of the gene regulatory process interact with each other. The output of an assay such as what I aim to develop could, through the use of regression analysis, allow the construction of maps linking regulatory sites to genes. This can be used to determine the cis-regulatory elements most responsible for each Müller glia reprogramming trajectory. If successful, similar techniques could be used to improve countless other differentiation protocols and to better understand the regulatory landscape driving differentiation.