25 research outputs found
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
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
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))
Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging
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
Tight associations between transcription promoter type and epigenetic variation in histone positioning and modification
Abstract
Background
Transcription promoters are fundamental genomic cis-elements controlling gene expression. They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture.
Results
Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones.
Conclusion
These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture