Visualization of loop extrusion by DNA nanoscale tracing in single human cells

Abstract

SummaryThe spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random structures such as chromatin compartments and topologically associating domains2,3. However, if the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a workflow for high-resolution reconstruction of 3D genome architecture without DNA denaturation. Classical fluorescence in situ hybridization approaches can link chromatin architecture to DNA sequence but disrupt chromatin structure at the critical nanoscale of individual loops. Our workflow employs non-denaturing enzymatic strand resection4,5, to conserve chromatin structure and can resolve the 3D-fold of chromosomal DNA with better than 5-kb-resolution in single human cells. Our results show that the chromatin fiber behaves as a random coil that can be further structured in a manner consistent with loop formation, explaining the emergence of topologically associated domain-like features in cell population averages. Mining a large amount of single-cell data computationally, we reveal chromatin folding intermediates consistent with progressive loop extrusion and stabilized loops, highlighting the power of our method to visualize the nanoscale features of genome organization in situ.