Project Summary/Abstract The overarching goal of Dr. Freeman’s research has been to understand how alterations of the genome/epigenome contribute to aging and how therapies could potentially target these changes to maintain a ‘youthful’ epigenome that retards aging, delays/prevents age-related diseases, and improves the health and quality of life of the elderly. Epigenetic dysregulation is a hallmark of aging, but exactly how this contributes to cellular dysfunction and disease is not known. To elucidate this contribution, Dr. Freeman’s VA research has focused on epigenomic regulation in astrocytes, microglia, and neurons in the hippocampus with aging. Using novel cell isolation and advanced sequencing approaches, cell-type specific base resolution maps of both methylation (mC) and hydroxymethylation (hmC) in CG and non-CG contexts are being generated. With paired RNA expression data, bioinformatic techniques are being used to understand the regulation of gene expression with aging. This type of base-specific, genome-wide, and cell-type specific studies have not been performed in aging research and will potentially offer the most in-depth view of epigenomic changes with aging to date. Further, to demonstrate the prevention of age-related epigenomic changes, caloric restriction is being used. In total, these studies will not only increase the understanding of the neuroepigenomics of aging but also provide targets for future interventional studies using epigenome editing. Importantly these studies examine both males and females to provide sex-informed insight as his prior studies have demonstrated that the majority of age- related epigenomic changes are sexually divergent. Dr. Freeman is also PI or co-PI on three NIH projects and one foundation funded project. These studies extend his VA research in aging through use of novel transgenic mouse models and anti-aging interventions in the brain, retina, and GI system. These models developed by his lab allow temporally controlled, cell-type specific tagging of nuclei and ribosomes to isolate DNA for epigenomic analysis and RNA for expression analysis without the need for cell sorting. In the brain and retina, heterochronic plasma transfer, in which plasma from young mice is administered to old mice for rejuvenation, is being used to determine if age-related epigenomic changes can be reversed after they have occurred. In parallel, using proteomic approaches, DNMT and TET interacting proteins are being identified that target them to specific genomic regions for epigenomic alteration. More broadly, Dr. Freeman also has an extensive collaborative portfolio with other OKC VAMC investigators. In these VA and NIH funded studies, Dr. Freeman is examining cell-type specific mechanisms of aging in muscle, brain, ovary, joints, and colon. In addition to bringing molecular and biochemical expertise to these studies, a common thread of epigenetics of aging allows comparisons and contrasts to be made across the organ...