Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II

Abstract

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.

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

“…In quiescent cells, the share of quickly diffusing RNA Pol II complexes was reduced compared to actively transcribing cells and, upon elongation inhibition using 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), the slowly diffusing fraction was greater than in untreated cells, indicating tenacious immobilization of RNA Pol II on the DNA template after initiation ( Figure 4c). The average diffusion constants in serum starved and DRB treated cells are similar for the three populations, suggesting that RNA Pol II not phosphorylated in absence of serum coherent with published data identifying serum starved cells in the first place [25]. We then compared the effect of transcriptional activity on chromatin dynamics in serum-starved and -stimulated cells.…”

“…adding serum to the medium brings cells back into the cell cycle and stimulates transcriptional activity [20,24,25]. Diffusion constants were calculated for each pixel based on the model selected by Bayesian inference.…”

Macromolecular and cytological changes in fission yeast G0 nuclei

“…For example, G0 B cells and mitotic yeast cells have more compact chromatin than proliferating and interphase cells, respectively, but they also have lower short-range Hi-C contact probabilities 56,57 . Time-resolved single-nucleosome tracking of RPE-1 cells has also shown that G0 phase chromatin is more dynamic than proliferating cell chromatin 58 . These counter-intuitive observations reflect our incomplete understanding of the many factors that influence chromatin structure and function in situ 59 .…”

Compartment-dependent chromatin interaction dynamics revealed by liquid chromatin Hi-C

“…In addition, their mobility is modulated by attractive interactions with other loci by factors that themselves are dynamic, and by local chromatin density. Live cell imaging experiments have examined locus motion extensively and found differences in mobility and constrained diffusion dependent on sub-nuclear position and chromatin state and activity (Bronshtein et al, 2009;Bronshtein et al, 2015;Hediger et al, 2002;Marshall et al, 1997;Nagashima et al, 2019;Shinkai et al, 2016;Thakar et al, 2006;Therizols et al, 2010). In such experiments the movement detected is strongly constrained by the fact that loci are part of very long chromosomes.…”

“…heterochromatic loci and loci tethered to the nuclear periphery vs. loci located in the nuclear interior (Bronshtein et al, 2009;Bronshtein et al, 2015;Hediger et al, 2002;Marshall et al, 1997;Nagashima et al, 2019;Shinkai et al, 2016;Thakar et al, 2006;Therizols et al, 2010).…”