Project Summary The genome organization plays a critical role in control of gene activity. Over the past few years, membraneless condensates assembled through liquid-liquid phase separation have emerged as a new theme in organizing the genome, which is one of science's 2018 breakthroughs. Despite the importance of phase separation for the genome organization, the underlying molecular mechanisms remain enigmatic. Our general goal is to use Polycomb group (PcG) proteins as a paradigm to understand how liquid-liquid phase separation organizes the genome. We recently demonstrated that PcG proteins assemble into condensates through liquid- liquid phase separation. We have pioneered the development of in vitro and in vivo single-molecule techniques that enable addressing key questions in the genome organization through liquid-liquid phase separation. Based upon this conceptual and technical foundation, our specific goal is to identify the molecular factors that dictate the nucleation of the PcG condensates at specific genomic locations, to define the molecular mechanism that underpins how H3K27me3-marked genes are brought together into the PcG condensates, and to determine the functional roles of PcG condensates in reaction kinetics and transcriptional repression. We will achieve this goal by using a multi- disciplinary approach that combines our unique single-molecule tools with biochemical, genetic, and genomic analyses. Our studies will establish a paradigm-shifting model that underpins how PcG proteins phase separate to form condensates that organize Polycomb target genes to achieve efficient biochemical reactions and transcriptional repression. Moreover, we will add new single-molecule tools to study the genome organization through liquid-liquid phase separation. Thus, this study will have a deep and sustained impact on our understanding of how phase separation organizes the genome, which has implications far beyond the epigenetic field.