PROJECT SUMMARY Microglia perform essential roles in central nervous system homeostasis with diverse functions including effects on memory and learning as well as refinement of synaptic networks. Emerging evidence indicates that dysregulation of microglia contributes to the pathogenesis of numerous neurodegenerative and neurodevelopmental diseases. Our recent observation that brain environment strongly influences microglial- specific gene expression and regulatory landscapes has implications for understanding the pathogenic response of microglia. The aims in this grant are devised to expand on these data to investigate the transcriptional networks and environmental interactions that influence and maintain the microglial phenotype. Scientific investigations will focus on understanding microglial transcriptional networks and gene- environment interactions using in vitro induced pluripotent stem cell modeling together with cutting edge genomic technologies to provide unbiased molecular outcomes. Specific Aim 1 will investigate the brain- specific signals necessary to maintain the transcriptional network through coculture systems. Not only will this inform gene-environment interactions but will improve iPSC disease modeling to incorporate microglia. Specific Aim 2 will characterize environmentally dependent transcription factors (TFs) through targeted overexpression to understand how these TFs interact with lineage determining TFs and one another to establish functional enhancers and control gene expression. Specific Aim 3 will complement SA2 by reducing expression of an environmentally dependent TF and delineating the microglial contribution to neurodevelopmental pathology. Studies in this grant will utilize the expertise of two excellent and proven mentors, Dr. Christopher Glass who is a leader in genetic and epigenetic analyses and investigating the effect of enhancer landscapes on gene expression and Dr. Fred Gage, a renowned leader in stem cell modeling of disease. The combined training from these two leaders in the field will allow for comprehensive in-depth studies of microglia and contribute to the understanding of how microglia contribute to both the healthy and diseased state as well as improve modeling for neuroinflammatory disease. Together with my mentors and the support of Dr. Blurton- Jones for iPSC microglial differentiations and Dr. Brennand for CRISPR techniques I have a mentoring team that will ensure that I remain on track for career advancement. This mentored award will provide specific training in genomics and epigenetics and allow for a thoughtful combination of stem cell disease modeling with next generation sequencing. In addition, intensive workshops, seminars, journal clubs, and specific laboratory technique training will accompany protected research time. These aims will inform my long-term career objects to model and study pediatric genetic and idiopathic neuroinflammatory disease, and allow for a structured foundation on which to do so. The proposed research and career development plan will benefit greatly from the intellectually rich and collaborative environments at UCSD and the Salk Institute.