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.
Functional studies of DNA methylation and hydroxymethylation during development are few, and we have a limited understanding of how these processes influence tissue and organ formation. This is particularly true in the retina, where, despite several recent studies, we do not yet understand the importance of DNA methylation or hydroxymethylation during normal development or stem cell maintenance, nor do we understand how epigenetic changes can influence disease states in the eye. Experiments in this proposal focus on these issues and when completed, the results of our studies will inform our understanding of normal development processes, how these processes are affected during disease progression, and how epigenetic regulation can contribute to the development of regenerative therapies.