DNA Methylation and Hydroxymethylation During Retinal Development and Stem Cell Maintenance

摘要：DNA甲基化（5mC）的要求和功能已经在很少的研究中得到解决。 器官和组织，而对DNA羟甲基化（5hmC）的作用知之甚少。事实上，DNA 甲基化和羟甲基化在视网膜中还没有得到很好的研究，我们的初步研究 表明它们在视网膜神经发生和视网膜干细胞维持中起关键作用。 在此，我们重点研究了视网膜前体细胞转化过程中的DNA甲基化和羟甲基化， (RPC)分化的视网膜神经元，调节视网膜干细胞的增殖和分化。我们 假设通过DNA甲基化和羟甲基化调节基因表达对于 视网膜发育和视网膜干细胞的维持，以及甲基化和 RPCs和视网膜干细胞中特定基因座的羟甲基化影响其分化能力， 视网膜神经元Aim1利用最先进的下一代测序和生物信息学技术， 从早期和晚期的纯群体中确定全基因组5mC、5hmC和转录组谱 RPCs，以及来自斑马鱼视网膜的早期和晚期视网膜神经节细胞（RGC）。这些数据是 整合以生成细胞的全基因组甲基化组、羟甲基化组和转录组数据库 在RPC成熟和RPC向RGC过渡期间。然后，我们利用创新的人类iPSC衍生的 类器官模型，以确定全基因组5mC，5hmC和转录组谱从纯群体的 早期和晚期人类RPC，并利用这些数据来鉴定具有保守甲基化的基因 在RPC成熟过程中的变化，从而优先考虑下游功能分析的相关基因座。AIM2 重点是Ted介导的DNA羟甲基化，这是一个表观遗传过程，我们对它有一个有限的了解。 在视网膜发育过程中的理解。在这里，我们将确定tet2和tet3介导的DNA 羟甲基化促进斑马鱼视网膜发育早期和晚期方面。Aim3检查视网膜 干细胞的维持，并确定了视网膜干细胞如何需要dnmt 1介导的甲基化， 调节该生态位内细胞的增殖和分化。该提案的结果将提供 迄今为止对视网膜发育过程中DNA甲基化和羟甲基化的最详细分析， 在视网膜发育和视网膜发育过程中， 造血干细胞这些数据将对那些在眼睛、CNS和其他领域工作的人产生广泛的兴趣。 器官和组织，以及更普遍地在基因表达的表观遗传调节中。此外，鉴于 围绕干细胞和iPSC方法开发再生疗法的步伐， 我们的研究结果将提供关于DNA甲基化和羟甲基化变化的关键信息， RPC在决定是增殖还是分化时，可用于进一步开发这些 产生可用于临床环境的细胞或组织的方法。

SUMMARY: The requirements and functions for DNA methylation (5mC) have been resolved in very few organs and tissues, and even less is known about the roles of DNA hydroxymethylation (5hmC). Indeed, DNA methylation and hydroxymethylation have not been well studied in the retina, and our preliminary studies demonstrate that they play critical roles during retinal neurogenesis and in retinal stem cell maintenance. Here, we focus on DNA methylation and hydroxymethylation during the transition from retinal progenitor cell (RPC) to differentiated retinal neuron, and in regulating proliferation and differentiation of retinal stem cells. We hypothesize that regulation of gene expression by DNA methylation and hydroxymethylation is critical for retinal development and retinal stem cell maintenance, and that changes in methylation and hydroxymethylation of specific loci in RPCs and retinal stem cells influence their abilities to differentiate as retinal neurons. Aim1 utilizes state of the art next-generation sequencing and bioinformatics techniques to determine genome-wide 5mC, 5hmC and transcriptome profiles from pure populations of early and late stage RPCs, and early and late stage retinal ganglion cells (RGCs) from the zebrafish retina. These data are then integrated to generate a genome-wide methylome, hydroxymethylome and transcriptome database for cells during RPC maturation and the RPC to RGC transition. We then utilize an innovative human iPSC-derived organoid model to determine genome-wide 5mC, 5hmC and transcriptome profiles from pure populations of early and late stage human RPCs and leverage these data to identify genes with conserved methylation changes during RPC maturation and thereby prioritize relevant loci for downstream functional analyses. Aim2 focuses on Ted-mediated DNA hydroxymethylation, an epigenetic process about which we have a limited understanding during retinal development. Here, we will determine how tet2- and tet3-mediated DNA hydroxymethylation facilitate early and late aspects of retinal development in zebrafish. Aim3 examines retinal stem cell maintenance and determines how dnmt1-mediated methylation is required in retinal stem cells to modulate proliferation and differentiation of cells within this niche. The results of this proposal will provide the most detailed analyses of DNA methylation and hydroxymethylation during retinal development to date, and functionally interrogate the requirements for these two key processes during retinal development and in retinal stem cell maintenance. These data will be of broad interest to those working in the eye, CNS, and in other organs and tissues, as well as more generally in epigenetic regulation of gene expression. Moreover, given the pace at which regenerative therapies are being developed around stem cell and iPSC-based approaches, our results will provide critical information about DNA methylation and hydroxymethylation changes occurring in RPCs as they make decisions whether to proliferate or differentiate that can be utilized to further develop these approaches towards generating cells or tissue that will be useful in a clinical setting.