PROJECT SUMMARY Mammalian genomes are organized into loops and topologically associated domains by the cohesin protein complex. The formation of these structures has suggested models for how DNA regulatory elements, such as enhancers and promoters, are spatially arranged to coordinate gene expression. However, testing these models and their implications in cellular function and fate has been challenging due to the lack of systems for interrogating the activity of cohesin in vivo. Using mouse olfactory sensory neurons and serotonergic neurons from the raphe nuclei, post-mitotic cells in which the role of cohesin in genome function can be uncoupled from its role in sister chromatin cohesion, we recently uncovered that cohesin and its unloader WAPL play a critical role in the development of the central nervous system by regulating the expression of clustered Protocadherin (Pcdh) genes. In this proposal, we aim to leverage the Pcdh gene cluster as a new paradigm to study the molecular underpinnings of cohesin activity and its dynamics in governing the generation of neuronal cell surface diversity during brain development. Specifically, we propose to understand the role of auxiliary proteins to the cohesin complex in regulating cohesin dynamics and Pcdh gene expression in vivo. Finally, we aim to link the molecular operations of cohesin and its protein regulators to neural wiring and neural circuit assembly. The findings from our proposed studies are poised to generate new hypotheses for how genome architecture is coupled to gene expression in mammalian cells. They will provide an unprecedented view of neural wiring across scales, linking cell-type-specific regulation of genome structure to neural wiring during brain development. Finally, they will generate insights into developmental and intellectual disorders known as cohesinopathies, where dysregulation of the Pcdh genes is linked to improper cohesin actions.