Expanding retinal regenerative potential through chromatin biology in single cells

在哺乳动物中，神经元在损伤后基本上是非增殖的，这导致了针对 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 Müler胶质细胞的神经元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Ascl14。有趣的是，在小鼠中通过基因工程过表达Ascl1会导致 产生一些神经细胞类型，如双极细胞，但不产生光感受器或神经节细胞。 目的开发和使用单细胞技术，将Müller细胞推向更多样化的细胞类型，并 确定哪些顺式调控元件驱动细胞命运的这些变化。从神经胶质细胞中生产光感受器 这将标志着失明再生疗法的发展取得了非凡的进步。 单细胞测序革命现在使人们能够在一个单一的实验中识别 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，在一个单一的 CEL11-24，现在是应用和开发旨在解码的单细胞技术的最佳时机 异质细胞群中命运变化的调控，例如那些正在经历重新编程的细胞。 在目标1中，我将着手揭示推动穆勒胶质细胞转变为小说的条件 重新编排命运。最近的一项研究表明，Müler神经胶质细胞向神经元转化的效率极高 通过用组蛋白脱乙酰酶抑制剂(HDACi)处理细胞而得到改善6.我假设染色质 在不受HDACi处理干扰的情况下，修饰可防止细胞类型在 Ascl1的诱导。通过利用特拉普内尔实验室最近开发的SCI-Plex技术，我将培养 细胞在数百种不同的小分子处理下，每种处理都针对表观基因组的各个方面，以及 然后读出单细胞转录本10。通过对染色质生物学的系统扰动，我希望 扩展Müler胶质细胞的重新编程潜力，并更好地了解视网膜细胞的规格。 在目标2中，我建议设计一种既能读出转录组又能读出基因组的检测方法。 组蛋白修饰在单个细胞中的定位。多模式单细胞技术是一种强有力的方法 评估基因调控过程的各个方面是如何相互作用的。化验的结果，如 我的目标是开发，通过使用回归分析，允许构建链接的地图 基因的调控站点。这可以用来确定顺式监管要素对每个 米勒·格里亚重新编程轨迹。如果成功，类似的技术可以用来改进无数其他技术 差异化协议和更好地了解推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.