Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.

Significance DNA functions in living cells are crucially affected by the three-dimensional genome structure and dynamics. We analyze the whole genome of human cells by developing a polymer model of interphase nuclei. The model reveals the essential importance of the unfolding process of chromosomes from the condensed mitotic state for describing the interphase nuclei; through the unfolding process, heterogeneous repulsive interactions among chromatin chains induce phase separation of chromatin, which quantitatively explains the experimentally observed various genomic data. We can use this model structure as a platform to analyze the relationship among genome structure, dynamics, and functions.

Three-dimensional genome organization plays a critical role in DNA function. Flexible chromatin structure suggested that the genome is phase-separated to form A/B compartments in interphase nuclei. Here, we examined this hypothesis by developing a polymer model of the whole genome of human cells and assessing the impact of phase separation on genome structure. Upon entry to the G1 phase, the simulated genome expanded according to heterogeneous repulsion among chromatin chains, which moved chromatin heterogeneously, inducing phase separation. This repulsion-driven phase separation quantitatively reproduces the experimentally observed chromatin domains, A/B compartments, lamina-associated domains, and nucleolus-associated domains, consistently explaining nuclei of different human cells and predicting their dynamic fluctuations. We propose that phase separation induced by heterogeneous repulsive interactions among chromatin chains largely determines dynamic genome organization.

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