Project Summary During transcription, enhancers need to contact gene promoters across large chromosome domains. Chromosome folding can assist enhancer-promoter communication by bringing them close together inside the nucleus. Mutations that alter genome folding underlie many human diseases, including developmental disorders and some cancers. Yet, even after decades of research, we do not understand the causal links between chromatin architecture and transcription regulation. Specifically, we do not understand why DNA looping can correlate positively, negatively, or sometimes not at all with gene activity. To move beyond correlation to causation, we need to better understand the molecular processes that couple genome folding and gene regulation. The cohesin complex has emerged as a key player in DNA looping, because it can hold two chromatin fibers together and extrude DNA loops as it translocates on DNA. Cohesin is loaded on chromatin by its co-factor NIPBL. Cohesin then translocates until it is blocked at binding sites for the CTCF transcription factor. We recently developed tools that allow controlling various aspects of cohesin loop extrusion by manipulating NIPBL and CTCF in mouse embryonic stem cells. These tools provide a novel approach to investigate the relationship between loop extrusion by cohesin and transcription. In this proposed study, we will determine how transcription and loop extrusion are molecularly coupled and elucidate the mechanisms that explain why only some genes rely on loop extrusion to function. We will also address how cohesin loop extrusion contributes to the functions of CTCF during cell differentiation. In addition, we will identify novel pathways that regulate enhancer-promoter communication by modulating loop extrusion. To achieve these goals, we will combine gene editing, epigenomic assays, biochemical assays and novel epigenome engineering modalities with high-throughput reporter assays in mouse embryonic stem cells and their differentiated derivatives. These investigations will substantially deepen our understanding of how genome folding by cohesin proteins influences gene transcription. Completion of this project will open new avenues to explore how these processes go awry in disease, a question our group is interested to investigate in the future.