Epigenetic regulation is a newly appreciated and fundamentally important set of gene control mechanisms that profoundly influences chromatin function. Histone lysine and arginine methylation, demethylation, and the detection of these methyl marks are components of a histone code that underlies epigenetic regulation. Epigenetic regulators modulate the structure, function, and access of the mammalian genome to regulate transcription. Hundreds of epigenetic effectors have been identified, many of which are enzymes that catalyze reversible chromatin modifications. Many of these enzymes contain distinct functional domains, within the same polypeptide, for both creating (or removing) and binding to a given methyl mark. To date, most if not all available structural knowledge of these chromatin (de)modifying enzymes comes from the structures of individual domains. To approach a more complete understanding of the mechanisms of epigenetic regulation, we need to understand how these different functional domains work, both individually and in concert. The central goal of this proposal is to understand the interactions and spatial relationships between such domains by determining structures spanning multiple domains of several complementary epigenetic regulators. Such information will help us to address whether the writer and reader domains act on the same histone, and whether there are any inter-domain interactions that can influence/regulate their target specificity. Importantly, broader themes may be recognized because several distinct epigenetic regulators will be studied in parallel. I propose here four new specific aims that are designed to answer four related questions. (1) How does a lysine methylation mark for repression spread? (2) How does an existing methyl mark prevent the modification of neighboring residues in histones? (3) How are the histone marks of repression connected to DNA methylation? (4) How are the local methyl marks of repression removed within a nucleosome? PUBLIC HEALTH RELEVANCE: Epigenetic regulation is a newly appreciated and fundamentally important set of gene control mechanisms that profoundly influences chromatin function, which has direct relevance to a large number of human diseases. An increasing number of chromatin modifying and de-modifying enzymes have been associated with neurodegenerative disorders, metabolic diseases, inflammation, and, most notably, cancer. Thus, structural and biochemical studies directed against this emerging class of gene regulatory enzymes may provide a method for the development of highly selective therapeutic agents that promise entirely novel approaches for the treatment of human diseases. In this proposal, we will explore questions of dynamic regulation (creating and removing) of histone lysine modifications, modification- and position-specific interactions, and biochemical crosstalk between modifications by several distinct epigenetic regulators.