Project Summary/Abstract Cellular responses to available macronutrients and extra-cellular signals rely on the unique epigenetic state of the cell, defined by a layer of biochemical information above the genome that dictates specific gene expression. The epigenome consists of DNA sequence-dependent proteins, non-coding RNAs, DNA methylation and histone post-translation modifications (PTMs) such as lysine acetylation and methylation. The latter two mechanisms are catalyzed by enzymes that must ‘interpret’ incoming signals, ‘read’ the existing epigenetic landscape and ‘respond’ appropriately. Enzymes that modify histones and non-histone proteins such as methyltransferases, demethylases, acetyltransferases and deacetylases use central metabolites (S- adenosyl methionine, SAM; α-ketoglutarate, αKG; acetyl-CoA and nicotinamide adenine dinucleotide, NAD+, respectively) as co-substrates. New evidence suggests that fluctuation in such epi-metabolites caused by diet, environment, microbiota and genetics can drive PTM dynamics, however the relevant mechanisms remain unclear in most cases. Are changes in epi-metabolites sensed by signaling pathways or by substrate-level driven catalysis or both? Also, does local production of epi-metabolites enable/accelerate gene expression mechanisms on chromatin? Non-histone protein acetylation is a major PTM that can regulate many aspects of cellular function and occurs in all cellular compartments. But despite broad knowledge of what gets modified, the most pressing challenge is to understand the how, the why and the when, which constitutes an overarching theme of this proposal. A major portion of the research to understand reversible protein acetylation as a regulatory PTM will involve knowledge of how pathway-specific acetyl-CoA (and other acyl-CoAs) production leads to dynamic acetylation after extra-cellular stimulation. Also, this work will focus on the detailed molecular mechanisms by which nuclear NAD+-dependent deacetylases SIRT6/7 (Sirtuins 6 & 7) are regulated and how these enzymes perform such exquisite deacetylation of nucleosomes. A sub-theme of this proposal that connects these two projects is to understand the fundamental principles that govern PTM enzymes acting on chromatin/nucleosomes. This proposal is uniquely poised to make major advances to these salient questions. To accomplish these goals, the projects synergistically employ in vitro biochemistry/biophysics, complementary genetics and pharmacology, and cell- and animal-based models. Results from these investigations will provide i.) insight into the etiology of diseases resulting from the link between metabolism and the epigenome, ii.) foundations for drug development against the enzymes described here, and iii.) a fundamental understanding of how the cell ‘interprets’ incoming signals in the context of existing epigenetic information and ‘responds’ appropriately.