PROJECT SUMMARY Neonatal hypoxic ischemic encephalopathy (HIE), a common brain injury from loss of oxygen and nutrients immediately prior to birth, results in long-term neurologic deficits even in the absence of significant perinatal cell death. A major gap in the perinatal injury field is the limited mechanistic understanding of how transient hypoxia results in persistent cellular deficits. A compelling mechanism linking prenatal hypoxia to persistent deficits is that prenatal hypoxia permanently alters the epigenome because the epigenome integrates development with response to the environment. To study the mechanisms underlying the pathology of HIE, I developed a novel mouse model of transient late gestation prenatal hypoxic injury that phenocopies mild HIE. Prenatal hypoxia leads to persistent behavioral and structural deficits in adult mice despite no significant increase in cell death in the fetal brain. My preliminary data showed decrease in dendritic spine density in corticothalamic neurons at postnatal day 28 (P28), weeks after hypoxia. Prenatal hypoxia was associated with upregulation of genes associated with the epigenome in single nucleus RNA and assay for transposase-accessible chromatin sequencing (snRNA/ATAC-seq) in the fetal neocortex within one hour of exposure. One of the most upregulated genes, the histone variant H3f3b, is a candidate for protecting the brain from more severe brain injury. However, the epigenetic trajectories of corticothalamic neurons seemed to be shifted even immediately after hypoxic exposure. Therefore, my central hypothesis is that H3f3b upregulation is necessary and sufficient to protect neurons from severe injury after prenatal hypoxia and distinct epigenetic regulators contribute to lasting neuronal injury. To test my hypothesis, I propose three aims. In Aim 1, I will use conditional knockout mice to test if H3f3b depletion increases cell death immediately after hypoxia and worsens the persistent deficits in spine density in P28 mice and behaviors in adult mice. In Aim 2, I will use in utero electroporation to test if overexpression of H3f3b improves deficits from hypoxia in spine density in P28 mice and behaviors in adult mice. Lastly, in Aim 3, I propose to use snRNA/ATAC-seq in the cortex of P28 mice to determine if prenatal hypoxia has a permanent effect on the epigenome of corticothalamic neurons. My long-term goal is to be a child neurologist who leverages my research expertise in molecular biology and clinical skills in perinatal brain injury both to develop targeted therapies towards this frequently devastating injury. My mentors, Drs. Eric Marsh and Michal Elovitz, are excellent physician scientists devoted to using translational research to improving outcomes in children with neurological disorders. Under their guidance for this K08 proposal, I will gain additional skills in bioinformatics, a deeper expertise in translational animal studies, and lab management skills necessary to become an...