The regulatory nature and physical structure of chromatin are dynamically regulated by post- translational modifications of core histone proteins. The recent discovery of histone demethylases has initiated a rapid increase in our knowledge about the regulation of application and removal of histone modifications. Lysine-specific demethylase 1 (LSD1, also known as KDM1 and BCH110) is unique among known human histone demethylases in both its membership in the flavin-dependent amine oxidase family of enzymes and its ability to function as either a transcriptional activator or repressor, depending upon the identity of the lysine residue that is demethylated. LSD1 has been implicated in the regulation of a variety of genes, from nuclear hormone receptors to hematopoietic differentiation factors. In addition, the identification of the tumor-suppressor p53 as a non-histone substrate for LSD1-mediated demethylation adds further complexity to its physiological role. Preliminary indications that alterations in LSD1 activity may play a role in oncogenic transformation accentuate the need for a better understanding of the function of LSD1. However, little is known about the molecular details of both histone demethylase action and its interaction with protein cofactors. In this proposal we will establish an understanding of these molecular details of LSD1, with a long-term goal of designing modulators of LSD1 activity that will allow us to further probe its function and assess the potential for modifying LSD1 activity in vivo. In Aim 1 we will investigate the intriguingly diverse yet highly regulated substrate specificity of LSD1 through the use of peptides and defined nucleosomal substrates. In Aim 2 we will examine the effect on LSD1 activity by two of its key partner proteins, CoREST and HDAC1, and identify the interaction sites between CoREST, LSD1, and the nucleosome that facilitate nucleosomal demethylation. CoREST will serve as a model system for understanding how interacting proteins contribute to the specificity of LSD1 action. Together, the results of these studies will clarify the chemical basis for LSD1 function and provide a foundation to further probe the physiological function of LSD1 in a rational manner.