The ?relocalization of chromatin modifiers? theory of aging postulates that the degenerative process of aging is the result of a lifetime accumulation of epigenetic noise, which transcriptionally dysregulates cellular signaling pathways, triggering symptoms of tissue dysfunction and health deterioration. Evidence suggests that DNA methylation, in particular, may set the pace of the aging clock in several mammalian tissues, yet it is unclear whether these changes are reversible. Skeletal muscle is a particular tissue of interest because of its high metabolic load, biomass, and susceptibility to age-related dysfunction, termed sarcopenia. My approach is to first survey of the epigenomic landscape of individual nuclei isolated from young and old mouse muscle using snATAC-seq and integrate snRNA-seq data to test whether regions of chromatin accessibility in each discreet cell type correlate with gene expression from those regions. I will integrate whole genome bisulfite sequencing with 5hmC capture data, to find CpG sites that are hydroxymethylated in young muscle but remain methylated in old and see if these correlate with gene expression. To test the hypothesis that age-associated muscle dysfunction is the result of changes in the epigenetic landscape that lead to a TET2-driven loss of cellular identity, I will use the single nucleus and 5hmC data to determine if age- associated changes to the epigenome are correlated with a loss of cellular identity, as measured by significant changes to the transcriptomes of old muscle cells. Next, I will focus on the role of TET2 dioxygenase in adult skeletal muscle from young and old animals. I will perform CHiP-seq to locate TET2 in healthy young muscle and then determine if this localization is altered in old skeletal muscle. I will also test TET2 activity, including 5mC oxidation, and stability in culture and in vivo in old and young muscle, using AMPK and electrical stimulation to increase TET2 activation. Finally, I will determine if age-associated muscle dysfunction is reversible in vivo using epigenetic reprogramming. Our preliminary results show that OSK epigenetic reprogramming dramatically enhances regeneration of terminally differentiated somatic cells (neurons) and improves visual acuity in aged mice in a TET-dependent manner. Because epigenetic aging is seen broadly in all tissues, I predict that the TET-dependent mechanism of OSK reprogramming is not limited to a single tissue type, and that it may benefit aged skeletal muscle. I will treat young and old muscle cells in culture and muscle tissue with OSK-AAV and measure myogenicity, repair after acute injury, muscle force and other hallmarks of muscle aging. If my aims are achieved the field will benefit from my newly developed methods and integrative analyses of single nucleus-based transcriptomic and epigenomic data. Moreover, the proposed experiments will define the role of TET2 demethylation dynamics in aging muscle and determine if TET2 dysregulation is a driver of sarcopenia. Finally, investigating the epigenetic mechanisms that drive muscle dysfunction in aging may provide novel targets for anti-aging therapies, such as OSK-epigenetic reprogramming.
Age-related muscle loss and dysfunction, sarcopenia, represents a major public health problem as it results in loss of mobility, lack of physical independence, and increased morbidity and mortality. The primary aim of this study is to use cutting-edge bioinformatic and molecular techniques to elucidate the epigenetic mechanisms that contribute to muscle wasting in aging and to test the ability of epigenetic reprogramming to prevent or reverse these symptoms.
