Project Summary: Post-translational modification of N-terminal α-amino groups is a highly conserved and vastly utilized process seen in all species from bacteria to mammals. Dr. Schaner Tooley pioneered the field of N-terminal methylation by identifying the first two eukaryotic N-terminal methyltransferases, NRMT1 and NRMT2. She went on to characterize the consensus sequence of these enzymes, identify dozens of their substrates, and show N-terminal 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, kyphosis, and dermal fibrosis. They now want to expand understanding of the biochemistry of NRMT1 regulation, identify additional substrates regulated by the Nα-PTM code, and mechanistically characterize a newly discovered role for NRMT1 in stem cell fate determination. Successful completion of these goals will provide a more global understanding of NRMT1 function and help better develop NRMT1 as a therapeutic for human cancers and age-related disorders.