14Higher-order chromatin organization such as A/B compartments, TADs and chromatin loops are 15 temporarily disrupted during mitosis. These structures are thought to organize aspects of gene 16 regulation, and thus it is important to understand how they are re-established after mitosis. We 17 examined the dynamics of chromosome reorganization by Hi-C at defined time points following 18 exit from mitosis in highly purified, synchronous cell populations. We observed that A/B 19 compartments are rapidly established and progressively gain in strength following mitotic exit. 20Contact domain formation occurs from the "bottom-up" with smaller sub-TADs forming initially, 21 followed by convergence into multi-domain TAD structures. CTCF is strongly retained at a 22 that ana/telophase cells display significantly reduced compartmentalization between genomic 64 regions separated by >12Mb (Extended Data Fig. 3h). However, higher levels of 65 compartmentalization are observed between more distal (>100Mb) genomic regions after G1 entry 66 (Extended Data Fig. 3h, i), confirming that compartmentalization expands throughout the entire 67 chromosome after mitosis. In summary, a major re-configuration of genome structure occurs 68 during the pro-M-G1 transition, with a rapid establishment, progressive strengthening, and 69 expansion of A/B compartments throughout the genome (Extended Data Fig. 1a). 70 71 Next, we examined formation after mitosis of TADs and nested sub-TADs 11,12 using 3DNetMod 72 13 . We identified 8073 contact domains progressively gained from pro-metaphase to mid-G1 ( Fig. 73 1b, Extended Data Fig. 4b, Extended Data Table3). We observed gradually increased insulation at 74 domain boundaries, suggesting strengthening of TADs over time (Extended Data Fig. 4a). 75Previous studies reported complete loss of domains in pro-metaphase 2,3,7 . However, notably, 76 despite significant attenuation of signal, residual domain-like structures are still qualitatively and 77 algorithmically detectable in pro-metaphase cells (Extended Data Fig. 4c). The presence of pro-78 metaphase TAD/subTADs is unlikely due to contamination from interphase cells given the high 79 purity of our mitotic preps and the fact that these residual structures are not homogenously 80 distributed across the genome (Extended Data Fig. 4c). Thus, pro-metaphase is not prohibitive to 81 the maintenance of chromatin structures. 82 83 Formation of TADs may occur via convergence of previously emerged sub-TADs (bottom-up), 84 the partitioning of initially formed TADs into sub-TADs (top-down), or simultaneous birth of both 85 contact domain types (Extended Data Fig. 4d). On average, contact domains called at time points 86 later in G1 are larger than those called at preceding cell stages. (Fig. 1b, c). In addition, an average 87 of ~1.5 boundaries per domain are found at pro-metaphase, whereas only ~0.58 boundary is created 88 per domain in mid-G1 ( Fig. 1d), suggesting that boundaries are being shared between sequentially 89 formed domains. These results support...