Loop stacking organizes genome folding from TADs to chromosomes

While population level analyses reveal significant roles for CTCF and cohesin in mammalian genome organization, their contribution to chromatin structure and gene regulation at the single-cell level remain incompletely understood 1–4 . Here, we use a super-resolution microscopy approach, Optical Reconstruction of Chromatin Architecture (ORCA) 5 to measure the effects of removal of CTCF or cohesin on genome folding across genomic scales. In untreated embryonic stem cells, we observe intricate, frequently stacked loops of chromatin which are largely dissolved upon cohesin removal. The loops compact chromatin at the < 3 Mb scale, increasing proximity between sequences not only within but also between TADs. We find multi-way contacts among loop anchors, preferentially at TAD borders, and these hubs largely dissolve upon CTCF degradation. CTCF-hubs bridge intervening TAD boundaries while keeping border distal regions from neighboring TADs apart outside the hub. Cohesin dependent loops at the < 3 Mb scale impede mixing at larger chromosomal scales through steric effects of loop stacking, dramatically reducing genomic cross-talk. Disruption of this ordered chromosomal structure led to increased cell-cell variability in gene expression, exceeding changes to average expression. Together our data revise the TAD-centric understanding of CTCF and cohesin, and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.