PROJECT SUMMARY/ABSTRACT PROBLEM: Polycomb repressive complex 1 (PRC1) is a multi-protein assembly that epigenetically regulates chromatin, which when misregulated, results in cancer. Originally identified in Drosophila as a four-component complex, PRC1 has expanded its membership and functional repertoire over evolution. An important unanswered question concerning human PRC1 is how certain combinations of PRC1 proteins assemble while others do not, and how the types of PRC1s formed change during cellular differentiation. PRC1 assembly is currently understood to occur through a series of 1:1 protein-protein interactions. This view, unfortunately, does not explain how the protein-protein interaction of one PRC1 domain can somehow influence the binding selectivity of another, seemingly unrelated protein-protein interaction. We have identified novel interactions secondary to the known 1:1 direct interactions that provide selective checkpoints for including specific protein combinations thereby allowing selective PRC1 assemblies. OBJECTIVE: We will systematically characterize these secondary protein-protein interactions then investigate its role in the assembly and function of the distinct PRC1s. METHODS: We will use biophysical methods (X-ray crystallography, analytical ultracentrifugation, biolayer interferometry) to dissect the molecular basis of these selective assemblies. The functional consequences of the secondary interactions that form these assemblies will be assessed using a novel FRET- based histone modification assay. SIGNIFICANCE: Gene regulation is performed by many different proteins, all of which work within multi-component systems. Current genomic approaches to investigating gene regulation, while informative, lack a molecular perspective of protein-protein interactions necessary for a complete understanding of these systems. We propose to fill this gap by determining how specific PRC1s assemble and function. By defining the molecular choreography underlying PRC1 assembly, we will provide a new perspective on the regulation and function of these complexes in genome regulation. Our results may also be broadly applicable for understanding other gene regulatory systems, many of which utilize multi- component systems with different homolog combinations.