Project Summary/Abstract Dysfunction and death of neurons severely affect the quality of life, yet therapeutic options remain limited. Recent studies demonstrated clinic potential to replace lost neurons, e.g., transplanting of hESC- derived retinal cells improves disrupted visual acuity. However, research progress is hindered by limited mechanistic understanding of how to program cells into mature states for proper neurite outgrowth. Neurons undergo dramatic remodeling during development to become fully mature. This conserved process consists of precisely regulated pruning and outgrowth of neurites, facilitated by accurate transcriptome dynamics. How chromatin is organized to achieve the dynamic transcriptome during this process remains undefined. My long- term goal is to study epigenetic mechanisms underlying neuronal maturation and promote their application toward studies with clinic potential to improve life quality. This proposed project uses both Drosophila and mouse as model systems to study insulator regulation of 3D chromatin organization during neuronal outgrowth. CTCF is a conserved insulator protein that plays a central role in 3D chromatin organization. Shep is a conserved RNA/DNA-binding protein that is essential for proper neuronal outgrowth. My preliminary data indicate that Shep antagonizes CTCF insulator to promote neuronal outgrowth of fly. Here I am proposing three aims to investigate epigenetic mechanisms underlying my preliminary findings: 1) Determine neuron- specific Shep regulation of 3D chromatin organization at its target loci in fly. 2) Investigate neuron-specific mechanisms by which Shep antagonizes CTCF insulator function in fly. 3) Elucidate Shep and CTCF function in regulating morphological and physiological maturation of mouse neurons. This project will employ extensive neuron-specific high throughput sequencing coupled with physiological assays to reveal molecular mechanisms underlying neuronal remodeling in both fly and mouse. These data will provide novel insights into epigenetic regulation of neuronal maturation and suggest compelling mechanisms for studies with medical and clinic potential. My career goal is to create my independent research group, employing fly as an efficient tool to reveal mechanisms that are subsequently tested in the mouse model. My mentor Dr. Elissa Lei's group has long been studying insulator-mediated chromatin organization and gene expression. I will get extensive training in epigenetics, including scientific knowledge and technical skills to generate sequencing libraries in the Lei group. My co-mentor Dr. Jeff Diamond will provide essential training on mouse neuroscience and physiology. My second co-mentor Dr. Victor Corces will provide both scientific advice on epigenetic studies and guidance on career development once I establish my lab at a university. The K99/R00 award will assist me to achieve my goals by providing essential resource and funding at my mentored-stage and my earl...