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Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is debatable. Using live-cell and super-resolved 3D microscopy, we demonstrate that cohesin depleted cells pass through an endomitosis and rebuild a single multilobulated nucleus (MLN) with chromosome territories (CTs) pervaded by interchromatin channels. CTs contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. Splicing speckles are located nearby within the lining channel system. These clusters form microscopically defined, active and inactive compartments, which correspond to A/B compartments, detected with ensemble Hi-C. Functionality of MLN despite continuous absence of cohesin was demonstrated by their ability to pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity. ### Competing Interest Statement The authors have declared no competing interest. * 3D FISH : 3D fluorescence in situ hybridization 3D SIM : 3D structured illumination microscopy AID : auxin inducible degron ANC / INC : active / inactive nuclear compartment CT : chromosome territory CD(C) : chromatin domain (cluster) CTCF : CCCTC binding factor DAPI : 4’,6-diamidino-2-phenylindole EdU : 5-Ethynyl-2’-deoxyuridine Hi-C : chromosome conformation capturing combined with deep sequencing IC : interchromatin compartment MLN : multilobulated nucleus NC : nucleosome cluster PBS : phosphate buffered saline PBST : phosphate buffered saline with 0.02% Tween PR : perichromatin region RD : replication domain RL : replication labeling TAD : topologically associating domain

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