Dynamic organization of chromatin domains revealed by super-resolution live-dell imaging

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here by permission of Cell Press for personal use, not for redistribution. The definitive version was published in Molecular Cell 67 (2017): 282-293, doi:10.1016/j.molcel.2017.06.018.The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy, PALM) and single nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ~160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome–nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.This work was supported by MEXT and JSPS grants (23115005 and 16H04746, respectively) and a JST CREST grant (JPMJCR15G2).2018-07-1

The RIF1-Long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress

Acknowledgements Thanks to members of the Aberdeen Chromosome Biology Group for helpful comments, and Ronan Broderick and Wojciech Niedzwiedz for advice on mitotic bridge analysis. We thank Raif Yuecel and his team at the Iain Fraser Cytometry Centre for assistance, and Kevin Mackenzie and his team at the Microscopy and Histology Core Facility. Work was supported by Cancer Research UK Studentship Award C1445/A20596 and CRUK Programme Award C1445/A19059; by JSPS KAKENHI Grants Numbers 17K15068, 18H02170 and 18H04719; by research grants from the Daiichi Sankyo’s Foundation of Life Science and the Takeda Science Foundation; and by the UK Medical Research Council (MC_UU_00007/13). Collaboration was supported by a 2017 JSPS Summer Programme Fellowship. Funding Cancer Research UK (C1445/A20596) Anne D Donaldson Cancer Research UK (C1445/A19059) Anne D Donaldson Japan Society for the Promotion of Science (17K15068) Masato T Kanemaki Japan Society for the Promotion of Science (18H02170) Masato T Kanemaki Japan Society for the Promotion of Science (18H04719) Masato T Kanemaki Medical Research Council (MC_UU_00007/13) Nick GilbertPeer reviewedPublisher PD

Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes

Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, Ran\u27s dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic depletion assays to dissect Ran\u27s mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle-pole focusing or for targeting TPX2. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-beta coordinately promote HURP\u27s dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes

UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G1 phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G2 phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G1 by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced "clean" DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced "dirty" DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ

Characterizing replisome disassembly in human cells

To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes

A pathway for mitotic chromosome formation

Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central spiral staircase condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding

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

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nagashima, R., Hibino, K., Ashwin, S. S., Babokhov, M., Fujishiro, S., Imai, R., Nozaki, T., Tamura, S., Tani, T., Kimura, H., Shribak, M., Kanemaki, M. T., Sasai, M., & Maeshima, K. Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II. Journal of Cell Biology, 218(5), (2019):1511-1530, doi:10.1083/jcb.201811090.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.We thank Dr. Y. Hiromi, Dr. S. Hirose, Dr. H. Seino, and Dr. S. Ide for critical reading of this manuscript. We thank Dr. S. Ide, Dr. D. Kaida, Dr. T. Nagai, Dr. V. Doye, Dr. G. Felsenfeld, and Dr. K. Horie for valuable help and materials. We also thank the Maeshima laboratory members for helpful discussions and support. R. Imai and T. Nozaki are Japan Society for the Promotion of Science Fellows. R. Nagashima was supported by 2017 SOKENDAI Short-Stay Study Abroad Program. This work was supported by a Japan Society for the Promotion of Science grant (16H04746), Takeda Science Foundation, RIKEN Pioneering Project, a Japan Science and Technology Agency Core Research for Evolutional Science and Technology grant (JPMJCR15G2), a National Institute of General Medical Sciences grant (R01-GM101701), and National Institute of Genetics JOINT (2016-A2 (6))

Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B

Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation