PROJECT SUMMARY/ABSTRACT The development of technologies for genome wide mapping of chromatin interactions has yielded important insights into the organization of chromosomes. These studies have demonstrated that chromosomes are organized into topological domains, corresponding to genomic regions with high local interaction frequencies. Furthermore, studies of factors that contribute to higher order chromatin structure have indicated that certain regulatory elements and factors play key roles in establishing higher-order chromatin structure. What remains unclear at this time is how these structures, inferred from population averages of static chromatin interactions, are arranged in both space and time in live cell. We have recently developed live cell imaging and biophyiscal approaches to study the motion of individual loci. We will extend and use this strategy to validate finding of the structural models predicted by genome wide chromatin interaction experiments. In aim 1, we will develop methods for validating interactions between specific regulatory loci. Specifically, we will develop new experimental tools that enable tracking of multiple genomic loci with multi-color live cell imaging. We will used these tools to determine how the distributions and means of spatial distances in vivo relate to static chromatin interaction data, and identify the principles of chromatin organization. In aim 2, we will carry out experiments with multi-color live cell imaging to characterize the topological domains and individual elements using biophysical modeling. We will further use genome-editing technology to perturb the genome and determine the role of specific DNA elements in regulating chromatin dynamics and organization. The data and tools generated in this component will likely provide new insight into chromatin topology and dynamics in live cells.