Project Summary Overview: During interphase, the cohesin complex extrudes chromatin loops to mediate the formation of chromatin domains and loops, which are elementary 3D structures of the human genome and guide enhancer- promoter interactions. Disruption to these structures can lead to many diseases including cancer. Our research has significantly advanced our understanding of how cohesin actively extrude native chromatin loops. Specifically, we have achieved the following milestones: 1) Established a simple yet very powerful system enabling mechanistic studies of loop extrusion in near-native nuclei under highly controlled manners. 2) Uncovered that cohesin engages with chromatin in distinct ways dependent on the stages of loop extrusion. 3) Discovered distinct biochemical and conformation states of cohesin during loop extrusion. Goals: Our preliminary results revealed a correlation between cohesin’s biochemical states and its role in genome folding, and also demonstrated the power of our semi-in vitro systems to study genome folding. We aim to use this system to capture and analyze distinct states of cohesin during the highly dynamic loop extrusion process. Our innovative approach also affords us the ability to analyze additional cohesin-dependent activities such as transcription. We plan to address four major gaps in our understanding of genome folding and its relevance in cancer. 1) how dose cohesin engage with chromatin? We will use synthetic variants of cohesin and CTCF to probe the molecular connection between CTCF binding and cohesin-chromatin interactions. 2) What changes occur in cohesin's composition and conformation during the loop extrusion process? We will establish a novel analytic framework to systematically explore dynamic changes of cohesin and identify novel regulators of cohesin. 3) how does cohesin interplay with transcription machinery? We will employ our methods to analyze cohesin’s states during transcription and probe the interplay between genome folding and transcription. 4) how do cancer-associated mutations within cohesin affect genome folding and functions. Cohesin mutations are strongly associated with cancer, but their mechanisms of action remain largely unclear. We will integrate our analyses with AlphaFold to construct a predictive model and assess the impact of clinically relevant mutations within cohesin on genome folding and transcription. Vision: Our innovative methodologies not only enable the functional analysis of cohesin's biochemical states (subunit composition and conformation such as its ring-like structure) during highly dynamic activities like loop extrusion and transcription, but also afford a comprehensive understanding of genome folding and regulation, particularly in the context of cancer.