Project Summary/Abstract Overview: The methylation of DNA and histone tail residues tunes chromatin structure to control gene expression programs. Methionine supplies one-carbon (1C) units for this S-adenosyl-methionine (SAM)-mediated methylation. These 1C units can come 1) directly from dietary methionine or 2) indirectly from serine, glycine or another 1C donor to regenerate methionine. Both 1C metabolism and this epigenetic landscape are critical in the development of and for healthy maintenance of differentiated tissues. Importantly, a common hallmark of aging, cancer and other diseases is altered levels of these amino acids that provide 1C units and/or DNA and histone methylation. Goals: Critically, what is not understood is how homeostasis of SAM metabolism is achieved across mammalian tissue types and in proliferating cells through the dynamic interplay between production from available nutrients versus consumption by methylation reactions. Further, it is not known how much SAM consumption is required to support epigenetic regulation. Our objective is to apply modern quantitative methods to understand the nutrient sources of the 1C units used for SAM synthesis and the contribution of SAM consumption to global DNA and histone methylation. This will be accomplished by addressing the following questions: 1) What are SAM production and consumption rates? We will use liquid chromatography-mass spectrometry (LC-MS) paired with stable isotope tracing to measure SAM turnover fluxes to understand how SAM levels are sustained in each major tissue types in vivo and in proliferating cells in vitro. 2) What are the nutrient sources of the 1C units used for SAM synthesis upon altered methionine and 1C unit availability? Using tracing, we will quantify contribution of the 1C unit for SAM synthesis from exogenous methionine versus endogenous production from other nutrients. 3) How much SAM consumption supports DNA and histone methylation? We will adapt established approaches in quantitative metabolic analysis to measure rates of global SAM-consuming methylation of DNA and histones. 4) How does SAM availability alter DNA and histone methylation dynamics? We will measure the influence of altered SAM production on DNA and histone methylation fluxes. Vision: Through the studies proposed in this R35 MIRA grant, our team will advance quantitative tools to interrogate novel roles for 1C metabolism in controlling SAM homeostasis and its influence on the epigenetic landscape across tissue types and in proliferating cells. This comprehensive knowledge will provide fundamental insights into how metabolic homeostasis integrates supply of available nutrients to support epigenetic-driven gene expression. Such knowledge is critical for developing therapeutic strategies to manipulate SAM metabolism in diseases with aberrant changes in epigenetic methylation.