Packaging the Genome: the Structure of Mitotic Chromosomes

Mitotic chromosomes are essential structures for the faithful transmission of duplicated genomic DNA into two daughter cells during cell division. Although more than 100 years have passed since chromosomes were first observed, it remains unclear how a long string of genomic DNA is packaged into compact mitotic chromosomes. Although the classical view is that human chromosomes consist of radial 30 nm chromatin loops that are somehow tethered centrally by scaffold proteins, called condensins, cryo-electron microscopy observation of frozen hydrated native chromosomes reveals a homogeneous, grainy texture and neither higher-order nor periodic structures including 30 nm chromatin fibres were observed. As a compromise to fill this huge gap, we propose a model in which the radial chromatin loop structures in the classic view are folded irregularly toward the chromosome centre with the increase in intracellular cations during mitosis. Consequently, compact native chromosomes are made up primarily of irregular chromatin networks cross-linked by self-assembled condensins forming the chromosome scaffol

Chromosome structure: improved immunolabeling for electron microscopy

To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIα (topoIIα) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIα and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIα and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIα tether chromatin to loops according to a scaffolding-type mode

JAPANESE AND U.S. MEDIA COVERAGE OF THE IRAQ WAR: A COMPARATIVE ANALYSIS

This study investigates the relationship between the media and politics by analyzing the Iraq War coverage of two leading U.S. and Japanese newspapers: the New York Times and the Asahi. This dissertation reveals that these two print media, although both liberal in their general orientation, treated the Iraq War differently. First, it quantitatively finds that they are quite distinctive in their choice of main topics. During the run-up period to the war, the Asahi put more focus on the role of the United Nations while the majority of the stories appearing in the New York addressed U.S. decisions about Iraq. Second, the dissertation's qualitative analysis of editorials reveals that a different emphasis on who the "evil" doers in the war are. While the New York treated the oppressive Saddam Hussein regime and terrorists as the "evildoers," the Asahi portrayed the U.S. as the big evil doer. Further, content analysis of articles written by embedded journalists who were with coalition forces in Iraq revealed that the two newspapers' articles showed significant disparities in the degree of sympathy they showed to the forces. Numerous background factors have influenced this media content. Interviews with Japanese journalists and scholars revealed that the cultures of anti-militarism held by Japanese that originated from Japan's defeat in World War II remain firm within Japanese news organizations. Anti-militaristic sentiments and cultural factors, such as religion, appear to have influenced how their organizations portrayed the war in Iraq. Further, this dissertation statistically shows that the media's impact is significant in shaping the political agenda and public opinion. Poll data of Japanese sentiments about the United States show a decline in positive feelings towards the United States as the ratio of negative stories of U.S. Iraq policies carried by the Asahi rose. In addition, the Asahi's critical assessments of the Japanese government's Iraq policies showed a moderate negative congruence with public support for their Cabinet. Also, there was a moderate negative relationship between the New York Times' unfavorable coverage of the U.S. government's policies of Iraq and presidential approval ratings

Organization of fast and slow chromatin revealed by single-nucleosome dynamics

Understanding chromatin organization and dynamics is important since they crucially affect DNA functions. In this study, we investigate chromatin dynamics by statistically analyzing single-nucleosome movement in living human cells. Bi-modal nature of the mean squared displacement distribution of nucleosomes allows for a natural categorization of the nucleosomes as fast and slow. Analyses of the nucleosome-nucleosome correlation functions within these categories along with the density of vibrational modes show that the nucleosomes form dynamically correlated fluid regions, i.e., dynamic domains of fast and slow nucleosomes. Perturbed nucleosome dynamics by global histone acetylation or cohesin inactivation indicate that nucleosome-nucleosome interactions along with tethering of chromatin chains organize nucleosomes into fast and slow dynamic domains. A simple polymer model is introduced, which shows the consistency of this dynamic domain picture. Statistical analyses of single-nucleosome movement provide rich information on how chromatin is dynamically organized in a fluid manner in living cells

Flexible and dynamic nucleosome fiber in living mammalian cells

© Landes Bioscience, 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nucleus 4 (2013): 349-356, doi:10.4161/nucl.26053.Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.This work was supported by a grant-in-aid for a MEXT grant, JST CREST, Yamada Science Foundation and Takeda Science Foundation. Nozaki T and Hihara A are JSPS fellows

Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Molecular Biology of the Cell 28 (2017): 3349-3359, doi:10.1091/mbc.E17-06-0359.In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.This work was supported by MEXT and Japan Society for the Promotion of Science (JSPS) grants (Nos. 23115005 and 16H04746, respectively), as well as a Japan Science and Technology Agency (JST) CREST grant (No. JPMJCR15G2). R.I. and T.N. are JSPS Fellows. R.I. was supported by the SOKENDAI Short-Stay Study Abroad Program in fiscal year 2016

Importin-β and the small guanosine triphosphatase Ran mediate chromosome loading of the human chromokinesin Kid

Nucleocytoplasmic transport factors mediate various cellular processes, including nuclear transport, spindle assembly, and nuclear envelope/pore formation. In this paper, we identify the chromokinesin human kinesin-like DNA binding protein (hKid) as an import cargo of the importin-α/β transport pathway and determine its nuclear localization signals (NLSs). Upon the loss of its functional NLSs, hKid exhibited reduced interactions with the mitotic chromosomes of living cells. In digitonin-permeabilized mitotic cells, hKid was bound only to the spindle and not to the chromosomes themselves. Surprisingly, hKid bound to importin-α/β was efficiently targeted to mitotic chromosomes. The addition of Ran–guanosine diphosphate and an energy source, which generates Ran–guanosine triphosphate (GTP) locally at mitotic chromosomes, enhanced the importin-β–mediated chromosome loading of hKid. Our results indicate that the association of importin-β and -α with hKid triggers the initial targeting of hKid to mitotic chromosomes and that local Ran-GTP–mediated cargo release promotes the accumulation of hKid on chromosomes. Thus, this study demonstrates a novel nucleocytoplasmic transport factor–mediated mechanism for targeting proteins to mitotic chromosomes

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))

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole-Imidazole Polyamide Probes

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole-imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5′-(TTAGGG)n-3′) and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome

A Transient Rise in Free Mg 2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation

細胞分裂期の染色体凝縮はマグネシウムイオンの増加によって起こる --生細胞イメージングにより新たなメカニズムを検証--. 京都大学プレスリリース. 2018-01-19.For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [ 1–5 ], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [ 6–17 ], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [ 18–28 ], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like “beads on a string” by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [ 29 ]. However, technical limitations to measure intracellular free divalent cations, but not total cations [ 30 ], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [ 31 ] and adenosine triphosphate (ATP) [ 32 ] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [ 33 ]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance