PROJECT SUMMARY The nucleosome is the fundamental unit of chromatin and a vibrant signaling hub for our genomes. Combinatorial patterns of histone post-translational modifications (PTMs) and chemical modifications to DNA establish local epigenetic landscapes that regulate genomic processes, including gene expression, replication, and DNA damage repair. As genomic processes are critical to the maintenance of cellular identity and genomic integrity, they are frequently misregulated in diverse diseases like cancer and developmental disorders. Despite clear relevance to fundamental molecular biology and human health, the molecular mechanisms through which histone PTMs are curated by nuclear enzymes that write and erase the PTMs in a nucleosome context are still largely unknown. Moreover, outside of a small number of test cases, it is unclear how combinations of histone PTMs recruit readers of histone PTMs to specific genomic loci. These knowledge gaps limit the development of therapeutics targeting chromatin signaling for cancer or other diseases. We address these fundamental questions using designer nucleosomes with chemically defined sets of histone PTMs which allow us to explore how epigenetic signatures tune chromatin interactions proteome wide. In addition, to answering long-standing questions about chromatin signaling, these nucleosome interactome screens uncover new biologic processes that lead our research program in unexpected directions. In parallel, we solve near atomic resolution structures of writers, readers, and erasers of histone PTMs bound to nucleosomes. These studies provide molecular snapshots of the regulators of genomic processes in action in a physiologic chromatin context. Our structures enable mechanistic hypothesis testing in cellular models with precision structure-guided mutations. Currently, we are exploring how large multi-subunit complexes coordinate multiple enzymatic activities to regulate gene expression by pairing structural biology with biochemistry, biophysics, cell biology, and genomics. Finally, we are determining how the Anaphase Promoting Complex, a megadalton ubiquitin ligase that is a master regulator of the cell cycle, functions on chromatin to control gene expression and histone homeostasis. Overall, we expect to define universal patterns of chromatin recruitment through epigenetic landscapes and elucidate molecular mechanisms through which chromatin proteins function to regulate gene expression and other genomic processes. These studies will enable hypothesis-driven functional studies in disease model systems and uncover new avenues for drug discovery.