Sasai
Masaki
Sasai
Masaki
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ArticleSingle nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.(Rockefeller University Press, 2019-03-01) Nagashima, Ryosuke ; Hibino, Kayo ; Ashwin, S. S. ; Babokhov, Michael ; Fujishiro, Shin ; Imai, Ryosuke ; Nozaki, Tadasu ; Tamura, Sachiko ; Tani, Tomomi ; Kimura, Hiroshi ; Shribak, Michael ; Kanemaki, Masato T. ; Sasai, Masaki ; Maeshima, KazuhiroAlthough 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.
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ArticleOrientation-independent-DIC imaging reveals that a transient rise in depletion attraction contributes to mitotic chromosome condensation(National Academy of Sciences, 2024-08-27) Iida, Shiori ; Ide, Satoru ; Tamura, Sachiko ; Sasai, Masaki ; Tani, Tomomi ; Goto, Tatsuhiko ; Shribak, Michael ; Maeshima, KazuhiroGenomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the “chromosome milieu” during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells.