TY - JOUR T1 - Visualization of loop extrusion by DNA nanoscale tracing in single human cells JF - bioRxiv DO - 10.1101/2021.04.12.439407 SP - 2021.04.12.439407 AU - KS Beckwith AU - Ø Ødegård-Fougner AU - NR Morero AU - C Barton AU - F Schueder AU - W Tang AU - S Alexander AU - JM Peters AU - R Jungmann AU - E Birney AU - J Ellenberg Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/05/02/2021.04.12.439407.abstract N2 - The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensation and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random chromatin structures such as topologically associating domains2, 3. However, whether the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we developed LoopTrace, a fluorescence imaging workflow for high-resolution reconstruction of 3D genome architecture that conserves chromatin structure at the nanoscale and can resolve the 3D-fold of chromosomal DNA with better than 5-kb precision in single human cells. Our results show that the chromatin fibre behaves as a random coil up to the megabase scale and is further structured by contacts between sites that anchor loops. Our single cell folds reveal that such looping interactions are sparse and lead to a large heterogeneity of folds with one or two dynamically positioned loop bases as the main reproducible feature of megabase scale chromosomal regions. Clustering folds by their 3D conformations revealed a series of structures consistent with progressive loop extrusion between major anchor sites. Consistently, the looping interactions and their non-random positioning depend on the presence of the loop extrusion enzyme cohesin and its anchor protein CTCF, respectively. Our approach is scalable and will be instrumental to image the functional 3D architecture of the genome directly at the nanoscale.Competing Interest StatementThe authors have declared no competing interest. ER -