DNA Methylation and Hydroxymethylation During Retinal Development and Stem Cell Maintenance

视网膜发育和干细胞维护过程中的 DNA 甲基化和羟甲基化

• 批准号: 9903328
• 负责人: Jeffrey Gross
• 金额: $ 37.71万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903328
• 关键词: Address; Adult; Affect; Bioinformatics; Biological Models; Cell Differentiation process; Cell Maintenance; Cell Maturation; Cell Therapy; Cells; Clinical; Competence; DNA; DNA Methylation; DNA analysis; Data; Databases; Decision Making; Development; Disease; Disease Progression; Embryonic Development; Epigenetic Process; Event; Eye; Gene Expression; Gene Expression Regulation; Generations; Genes; Genomics; Growth; Growth and Development function; Human; Injury; Knowledge; Maintenance; Mediating; Methylation; Modeling; Molecular; Multipotent Stem Cells; Natural regeneration; Neurons; Organ; Organoids; Pattern; Play; Population; Process; Proliferating; Regulation; Retina; Retinal Diseases; Retinal Ganglion Cells; Role; Signal Transduction; Source; Stem Cell Development; Techniques; Tetanus Helper Peptide; Tissues; Transgenic Organisms; Validation; Zebrafish; base; epigenetic regulation; experimental study; genetic manipulation; genome editing; genome-wide; induced pluripotent stem cell; innovation; interest; methylome; neurogenesis; next generation sequencing; progenitor; protein function; regenerative therapy; repaired; retinal neuron; retinal progenitor cell; stem; stem cell niche; stem cells; transcriptome

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.

摘要：DNA甲基化（5MC）的要求和功能已在极少数情况下解决 对DNA羟甲基化的作用（5HMC）的作用，器官和组织甚至更少。确实，DNA 在视网膜中尚未对甲基化和羟甲基化进行很好的研究，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过程中起着关键作用。 在这里，我们专注于视网膜祖细胞过渡期间的DNA甲基化和羟甲基化 （RPC）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 假设通过DNA甲基化和羟甲基调节基因表达对 视网膜发育和视网膜干细胞维持，以及甲基化和 在RPC和视网膜干细胞中特定基因座的羟甲基化会影响其分化的能力 视网膜神经元。 AIM1利用最新的下一代测序和生物信息学技术 确定来自早期和晚期纯种群的全基因组5MC，5HMC和转录组轮廓 RPC，以及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 集成以生成细胞的全基因组甲基甲基甲基，羟甲基和转录组数据库 在RPC成熟和RPC到RGC过渡期间。然后，我们利用创新的人IPSC衍生 确定全基因组5MC，5HMC和转录组曲线的器官模型 早期和晚期人RPC并利用这些数据来识别具有保守甲基化的基因 RPC成熟期间的变化，从而优先考虑下游功能分析的相关基因座。 AIM2 专注于TED介导的DNA羟甲基化，这是一种表观遗传过程，我们对此有限 在视网膜发育过程中了解。在这里，我们将确定TET2-和TET3介导的DNA如何 羟甲基促进斑马鱼视网膜发育的早期和晚期。 AIM3检查视网膜 干细胞维持并确定在视网膜干细胞中如何需要DNMT1介导的甲基化 调节该细分市场中细胞的增殖和分化。该提案的结果将提供 迄今为止，视网膜发育过程中，大多数详细的DNA甲基化和羟甲基的分析 在视网膜开发和视网膜期间功能上询问这两个关键过程的要求 干细胞维护。这些数据将引起眼睛，中枢神经系统和其他工作的人的广泛关注 器官和组织，以及在基因表达的表观遗传调节中。而且，给予 围绕干细胞和基于IPSC的方法发展再生疗法的速度， 我们的结果将提供有关DNA甲基化和在 RPC在做出决定是扩散还是区分可以进一步开发这些的决定 在临床环境中有用的产生细胞或组织的方法。