Type II topoisomerases shape multi-scale 3D chromatin folding in regions of positive supercoils (Ttchem-seq).
Type II topoisomerases (TOP2s) resolve torsional stress accumulated during various cellular processes and are enriched at chromatin loop anchors and TAD boundaries, where, when trapped, can lead to genomic instability promoting the formation of oncogenic fusions. Whether TOP2s relieve topological constraints at these positions and/or participate in 3D chromosome folding, remains unclear. Here, we combine 3D genomics, imaging and GapRUN, a method for the genome-wide profiling of positive supercoiling, to assess the role of TOP2s in shaping chromosome organization in human cells. Acute TOP2 depletion led to the emergence of new, large-scale contacts at the boundaries between active, positively supercoiled and lamina-associated domains. TOP2-dependent changes at the higher-order chromatin folding were accompanied by remodeling of chromatin-nuclear lamina interactions and of gene expression, while at the chromatin loop level, TOP2 depletion predominantly remodeled transcriptionally-anchored, positively supercoiled loops. We propose that TOP2s act as a fine-regulator of chromosome folding at multiple scales. Overall design: We used colorectal carcinoma HCT116 cells in which the two endogenous TOP2A alleles were tagged with the mini auxin-inducible degron protein domain (mAID) allowing elimination of up to 95% of endogenous TOP2A after addition of auxin. We used this TOP2A-degron line to also generate a TOP2B knockout using CRISPR/Cas9 deletion, and thus be able to acutely degrade TOP2A in the presence or absence of TOP2B. 8 hours of auxin treatment sufficed for the  quantitative depletion of TOP2A protein. Cells were synchronized in G1, TOP2A and/or TOP2B were perturbed and we pulsed cells with 4sU for TTChem-seq library preparation.