Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. We have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were related to epigenomic status. Gene expression changes were highly correlated with direct cohesin binding and gain of TAD boundaries than with the loss of boundaries. Moreover, cohesin was broadly enriched in active compartment A chromosomes, which were retained after CTCF depletion. Our results demonstrate context-specific roles of cohesin for gene expression and chromatin folding.