PROJECT SUMMARY: Reversible lysine methylation is well understood to regulate histone proteins and chromatin templated processes. Dysregulation of the enzymes responsible for adding (lysine methyltransferases) and removing (lysine demethylases) lysine methylation is directly associated with many human diseases. In addition to histone proteins, thousands of non-histone proteins in the human proteome contain lysine methylation, yet we still understand very little about the function of non-histone lysine methylation. Our long-term goal is to understand how lysine methylation regulates non-histone protein function and cellular processes. Within this broad framework, we strive to identify biological processes that are regulated by lysine methylation, the substrate specificity of KMTs and KDMs, and how dysregulation of lysine methylation signaling contributes to human disease and developmental disorders. Toward this goal, we recently developed a functional proteomics platform to profile the substrate selectivity of KMTs and successfully used it to identify the circadian regulator PER2 as a substrate of lysine methyltransferase SMYD2. We also recently optimized a mass spectrometry pipeline for high- resolution mapping of lysine methylation. Over the next five years, we will build on this progress and use these new methods in projects that seek to answer three fundamental questions in the context of the circadian clock and neuronal differentiation: How big is the lysine methylome? What are the physiologically relevant substrates for KMTs/KDMs? And what is the function of non-histone lysine methylation? We will answer these questions by leveraging our strengths in biochemistry, proteomics, genomics, and cell-based studies, to expand our basic mechanistic understanding of the function of lysine methylation in cellular processes.