Chromosomes are segmented into domains and compartments, but how these structures are spatially related in three dimensions (3D) is unclear. Here, we developed tools that directly extract 3D information from Hi-C experiments and integrate the data across time. With our “4DHiC” method, we use X chromosome inactivation (XCI) as a model to examine the time evolution of 3D chromosome architecture during large-scale changes in gene expression. Our modeling resulted in several insights. Both A/B and S1/S2 compartments divide the X chromosome into hemisphere-like structures suggestive of a spatial phase-separation. During the XCI, the X chromosome transits through A/B, S1/S2, and megadomain structures by undergoing only partial mixing to assume new structures. Interestingly, when an active X chromosome (Xa) is reorganized into an inactive X chromosome (Xi), original underlying compartment structures are not fully eliminated within the Xi superstructure. Our study affirms slow mixing dynamics in the inner chromosome core and faster dynamics near the surface where escapees reside. Once established, the Xa and Xi resemble glassy polymers where mixing no longer occurs. Finally, Xist RNA molecules initially reside within the A compartment but transition to the interface between the A and B hemispheres and then spread between hemispheres via both surface and core to establish the Xi.

染色体被分割成域和区室，但这些结构在三个维度 (3D) 中的空间相关性尚不清楚。在这里，我们开发了直接从 Hi-C 实验中提取 3D 信息并跨时间整合数据的工具。通过我们的“4DHiC”方法，我们使用 X 染色体失活 (XCI) 作为模型来检查基因表达大规模变化期间 3D 染色体结构的时间演化。我们的建模产生了一些见解。A/B 和 S1/S2 隔室都将 X 染色体分成半球状结构，暗示空间相分离。在 XCI 期间，X 染色体通过 A/B、S1/S2 和巨型结构域结构，仅经历部分混合以呈现新结构。有趣的是，当活性 X 染色体 (Xa) 重组为非活性 X 染色体 (Xi) 时，Xi 超结构内的原始底层隔室结构并未完全消除。我们的研究证实了内部染色体核心的缓慢混合动力学和逃逸者居住的表面附近更快的动力学。一旦建立，Xa 和 Xi 就类似于不再发生混合的玻璃状聚合物。最后，Xist RNA 分子最初位于 A 隔间内，但过渡到 A 和 B 半球之间的界面，然后通过表面和核心在半球之间传播以建立 Xi。一旦建立，Xa 和 Xi 就类似于不再发生混合的玻璃状聚合物。最后，Xist RNA 分子最初位于 A 隔间内，但过渡到 A 和 B 半球之间的界面，然后通过表面和核心在半球之间传播以建立 Xi。一旦建立，Xa 和 Xi 就类似于不再发生混合的玻璃状聚合物。最后，Xist RNA 分子最初位于 A 隔间内，但过渡到 A 和 B 半球之间的界面，然后通过表面和核心在半球之间传播以建立 Xi。