Investigating the role of human topoisomerases in maintaining chromosome topology and preventing genomic instability

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Processes that traverse DNA, such as replication and transcription, both require and induce dramatic changes in DNA topology. As these changes occur in the context of chromatin, they must be coordinated with processes that shape chromosome organization per se, such as the formation of chromatin loops and higher-order chromosome folding. Changes in DNA topology are controlled by DNA topoisomerases, a family of potent enzymes that regulate torsional stress by relaxing, unknotting and decatenating constrained DNA. Seminal in vivo studies have uncovered fundamental roles of topoisomerases in regulating DNA topology in lower eukaryotes, however, low resolution experiments using inhibitors or long-term loss-of-function knockdowns in human cells have provided limited information on how topoisomerases control torsional stress in the genomic and chromatin context across the 3D genome. We propose to combine state-of-the-art high-throughput sequencing and imaging methodologies with acute loss-of-function strategies in human cells to systematically study the role of topoisomerases on chromosome topology and to understand how their functions control gene expression and prevent genomic instability across the 3D genome. Our efforts will be focusing on understanding how acute loss of topoisomerase function: (1) influences the supercoiling landscape across the 3D genome, the position of transcribing polymerase and gene expression; (2) the formation of non-B DNA structures such as R-loops; (3) the spatial genome organization and folding and, (4) promotes genomic instability across the 3D genome. Our ultimate goal is to comprehensively understand how the various types of topoisomerases regulate torsional stress in the context of chromatin and chromosome organization and to shed light on how their functions coordinate with fundamental cellular processes such as replication, transcription and loop extrusion, to regulate gene expression and prevent genomic instability.