Project Summary/Abstract: The dynamic regulation of chromatin is critical for proper gene expression, and dysregulation leads to diseases including cancer and metabolic disorders. The basic subunit of chromatin is the nucleosome, which is formed by a histone protein core around which DNA wraps. The unstructured histone termini, referred to as histone tails, protrude from this core and are key players in chromatin regulation. Incorporation of histone post-translational modifications (PTMs, chemical or protein modification of histone residues) and histone variants (histones with distinct sequences that substitute for canonical histones) are two major mechanisms of chromatin regulation, and they act in part through the histone tails. Despite great strides being made in understanding the chromatin landscape, large gaps remain in our knowledge of chromatin structure, gene regulation, and the histone language. The long-term goal of the laboratory is to define the chromatin landscape (the complex molecular interactions within and between nucleosomes along with their genetic and epigenetic factors) that underlies gene expression so that dysregulation that causes human disease can be corrected. The objective is to develop a molecular-level understanding of the conformation and dynamics of the histone tails within the nucleosome along with their bridging interactions (altogether, the histone tail landscape) and how these translate to the level of chromatin structure and accessibility. This includes determining how histone tail landscapes are perturbed by histone post-translational modifications and histone variants and how these landscapes are interconnected with each other and those of other nucleosome components. In this five-year MIRA proposal, the focus will be on: 1) determining how lysine acylations differentially regulate the histone tail landscape to contribute to metabolically- linked differential gene regulation, and 2) the variant-specific role of the centromeric H3 variant in regulating chromatin structure during mitosis and chromosome stability. A pairing of solution NMR spectroscopy with other biophysical approaches and phase separation studies, as proposed here, is essential to decipher this highly dynamic and heterogeneous system. Understanding the structural and dynamic effects of histone PTMs and variants on regulatory control of gene expression and chromatin structure will lead to a significant enhancement in our understanding of fundamental mechanisms of gene regulation and epigenetically-linked diseases.