DNA Methylation and Hydroxymethylation During Retinal Development and Stem Cell Maintenance

摘要：DNA甲基化(5mC)的要求和功能已在极少数情况下得到解决 器官和组织，对DNA羟甲基化(5hmC)的作用更是知之甚少。的确，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 证明它们在视网膜神经发生和视网膜干细胞维持中发挥关键作用。 在这里，我们重点研究从视网膜前体细胞过渡过程中的DNA甲基化和羟甲基化 (RPC)诱导分化的视网膜神经元，并调节视网膜干细胞的增殖和分化。我们 假设DNA甲基化和羟甲基化对基因表达的调节对 视网膜发育和视网膜干细胞维持，以及甲基化和 RPC和视网膜干细胞中特定位点的羟甲基化影响其分化为AS的能力 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 测定早期和晚期纯群体全基因组5mC、5hmC和转录组图谱 以及斑马鱼视网膜的早期和晚期视网膜神经节细胞(RGC)。然后，这些数据被 整合以生成全基因组的甲基组、羟甲基组和转录组数据库 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 用有机类模型从日本血吸虫纯种群中确定全基因组5mC、5hmC和转录组图谱 早期和晚期人类RPC并利用这些数据识别具有保守甲基化的基因 在RPC成熟过程中的变化，从而确定下游功能分析的相关基因座的优先顺序。AIM2 重点是Ted介导的DNA羟甲基化，这是一种表观遗传过程，我们对此有有限的了解 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的早期和晚期。Aim3检查视网膜 干细胞的维持，并确定DNMT1介导的甲基化在视网膜干细胞中是如何被需要的 调节细胞的增殖和分化。这项提案的结果将提供 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，以及 在功能上询问视网膜发育和视网膜中这两个关键过程的要求 干细胞维护。这些数据将对眼睛、中枢神经系统和其他研究人员产生广泛的兴趣 器官和组织，以及更普遍的基因表达的表观遗传调控。此外，在给定 围绕干细胞和基于IPSC的方法开发再生疗法的速度， 我们的结果将提供关于DNA甲基化和羟甲基化变化的关键信息 RPC在决定是增殖还是差异化时，可以用来进一步开发这些 产生将在临床环境中有用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.