Project Summary: Post-translational modification of N-terminal α-amino (Nα) groups is a highly conserved and vastly utilized process seen in all species from bacteria to mammals. Dr. Schaner Tooley pioneered the field of Nα- methylation by identifying the first two eukaryotic Nα-methyltransferases, NRMT1 and NRMT2 (also known as METTL11A and METTL11B). She went on to characterize the consensus sequence of these enzymes, identify dozens of their substrates, and show Nα-methylation regulates protein/DNA interactions. Her lab was also the first to show that, like histone PTMs, Nα-PTMs are part of a functional code. They identified the first protein known to exist in both Nα-acetyl and Nα-methyl forms, Myosin Light Chain 9 (MYL9). They demonstrated that these two Nα-PTMs create distinct proteoforms of MYL9, with unique protein binding partners, internal PTM patterns, and cell compartment-specific functions. They have also demonstrated important roles for NRMT1 in oncogenesis and aging. NRMT1 loss in breast cancer cells promotes proliferation, migration, colony formation, and xenograph growth. NRMT1 knockout in mice promotes phenotypes associated with premature aging, including, early graying and hair loss, kyphosis, skin and liver fibrosis, neurodegeneration, memory loss, and decreased lifespan. They have shown that NRMT1 regulates both neural and muscle stem cell development, and hypothesize a general role for NRMT1 in stem cell differentiation drives many of the premature aging phenotypes. They now want to 1.) expand understanding of the biochemistry of NRMT1 regulation, 2.) identify additional substrates regulated by the Nα-PTM code, and 3.) mechanistically characterize the newly discovered role for NRMT1 in stem cell fate determination. The worked proposed in this diversity supplement will address the first point and examine the regulation of NRMT1 through binding of its METTL family homologs, NRMT2 and METTL13. While it is becoming apparent that a common method of methyltransferase regulation is through complex formation with close homologs, NRMT1 is the first methyltransferase known to be opposingly regulated by two different homologs. Here we will study both the biochemistry and biological relevance of these interactions. Successful completion of these experiments will not only further the development of NRMT1 as a potential therapeutic target for human cancers and age-related disorders, but also complete Haley Parker's training in the technical and professional skills necessary to continue a research career in drug design.