Language：English   Publishing type：Research paper (scientific journal)   Publisher：Research Square Platform LLC  

Abstract

In higher eukaryotic cells, a string of nucleosomes, where long genomic DNA is wrapped around core histones, are rather irregularly folded into a number of condensed chromatin domains, which have been revealed by super-resolution imaging and Hi-C technologies. Inside these domains, nucleosomes fluctuate and locally behave like a liquid. The behavior of chromatin may be highly related to DNA transaction activities such as transcription and repair, which are often upregulated in cancer cells. To investigate chromatin behavior in cancer cells and compare those of cancer and non-cancer cells, we focused on oncogenic-HRAS (Gly12Val)-transformed mouse fibroblasts CIRAS-3 cells and their parental 10T1/2 cells. CIRAS-3 cells are tumorigenic and highly metastatic. First, we found that HRAS-induced transformation altered not only chromosome structure, but also nuclear morphology in the cell. Using single-nucleosome imaging/tracking in live cells, we demonstrated that nucleosomes are locally more constrained in CIRAS-3 cells than in 10T1/2 cells. Consistently, heterochromatin marked with H3K9me3 and H3K27me3 was upregulated in CIRAS-3 cells. Finally, Hi-C analysis showed enriched interactions of the B-B compartment in CIRAS-3 cells, which likely represents transcriptionally inactive chromatin. Increased heterochromatin may play an important role in cell migration, as they have been reported to increase during metastasis. Our study also suggests that single-nucleosome imaging provides new insights into how local chromatin is structured in living cells.

DOI： 10.21203/rs.3.rs-3631068/v1

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Other Link： https://www.researchsquare.com/article/rs-3631068/v1.html

Language：English   Publishing type：Research paper (scientific journal)  

Genomic DNA is wrapped around a core histone octamer and forms a nucleosome. In higher eukaryotic cells, strings of nucleosomes are irregularly folded as chromatin domains that act as functional genome units. According to a typical textbook model, chromatin can be categorized into two types, euchromatin and heterochromatin, based on its degree of compaction. Euchromatin is open, while heterochromatin is closed and condensed. However, is euchromatin really open in the cell? New evidence from genomics and advanced imaging studies has revealed that euchromatin consists of condensed liquid-like domains. Condensed chromatin seems to be the default chromatin state in higher eukaryotic cells. We discuss this novel view of euchromatin in the cell and how the revealed organization is relevant to genome functions.

DOI： 10.1016/j.tcb.2023.05.007

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ajhg.2023.03.014

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

In eukaryotes, higher-order chromatin organization is spatiotemporally regulated as domains, for various cellular functions. However, their physical nature in living cells remains unclear (e.g., condensed domains or extended fiber loops; liquid-like or solid-like). Using novel approaches combining genomics, single-nucleosome imaging, and computational modeling, we investigated the physical organization and behavior of early DNA replicated regions in human cells, which correspond to Hi-C contact domains with active chromatin marks. Motion correlation analysis of two neighbor nucleosomes shows that nucleosomes form physically condensed domains with ~150-nm diameters, even in active chromatin regions. The mean-square displacement analysis between two neighbor nucleosomes demonstrates that nucleosomes behave like a liquid in the condensed domain on the ~150 nm/~0.5 s spatiotemporal scale, which facilitates chromatin accessibility. Beyond the micrometers/minutes scale, chromatin seems solid-like, which may contribute to maintaining genome integrity. Our study reveals the viscoelastic principle of the chromatin polymer; chromatin is locally dynamic and reactive but globally stable.

DOI： 10.1126/sciadv.adf1488

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Eukaryotic genome DNA is wrapped around core histones and forms a nucleosome structure. Together with associated proteins and RNAs, these nucleosomes are organized three-dimensionally in the cell as chromatin. Emerging evidence demonstrates that chromatin consists of rather irregular and variable nucleosome arrangements without the regular fiber structure and that its dynamic behavior plays a critical role in regulating various genome functions. Single-nucleosome imaging is a promising method to investigate chromatin behavior in living cells. It reveals local chromatin motion, which reflects chromatin organization not observed in chemically fixed cells. The motion data is like a gold mine. Data analyses from many aspects bring us more and more information that contributes to better understanding of genome functions. In this review article, we describe imaging of single-nucleosomes and their tracked behavior through oblique illumination microscopy. We also discuss applications of this technique, especially in elucidating nucleolar organization in living cells.

DOI： 10.1002/bies.202200043

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Background</title>
Knowing chromatin components at a DNA regulatory element at any given time is essential for understanding how the element works during cellular proliferation, differentiation and development. A region-specific chromatin purification is an invaluable approach to dissecting the comprehensive chromatin composition at a particular region. Several methods (e.g., PICh, enChIP, CAPTURE and CLASP) have been developed for isolating and analyzing chromatin components. However, all of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements.


</sec><sec>
<title>Results</title>
We have developed a new approach for affinity purification of specific chromatin segments employing an <italic>N</italic>-methyl pyrrole (P)-<italic>N</italic>-methylimidazole (I) (PI) polyamide probe, which binds to a specific sequence in double-stranded DNA via Watson–Crick base pairing as a minor groove binder. This new technique is called proteomics and RNA-omics of isolated chromatin segments (PI-PRICh). Using PI-PRICh to isolate mouse and human telomeric components, we found enrichments of shelterin proteins, the well-known telomerase RNA component (TERC) and telomeric repeat-containing RNA (TERRA). When PI-PRICh was performed for alternative lengthening of telomere (ALT) cells with highly recombinogenic telomeres, in addition to the conventional telomeric chromatin, we obtained chromatin regions containing telomeric repeat insertions scattered in the genome and their associated RNAs.


</sec><sec>
<title>Conclusion</title>
PI-PRICh reproducibly identified both the protein and RNA components of telomeric chromatin when targeting telomere repeats. PI polyamide is a promising alternative to simultaneously isolate associated proteins and RNAs of sequence-specific chromatin regions under native conditions, allowing better understanding of chromatin organization and functions within the cell.


</sec>

DOI： 10.1186/s13072-021-00421-8

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Other Link： https://link.springer.com/article/10.1186/s13072-021-00421-8/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

Liquid droplets formed inside the cell by liquid-liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein-protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was "frozen" by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.

DOI： 10.26508/lsa.202001005

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The nucleolus is a nuclear body with multiphase liquid droplets for ribosomal RNA (rRNA) transcription. How rRNA transcription is regulated in the droplets remains unclear. Here, using single-molecule tracking of RNA polymerase I (Pol I) and chromatin-bound upstream binding factor (UBF), we reveal suppression of transcription with phase separation. For transcription, active Pol I formed small clusters/condensates that constrained rDNA chromatin in the nucleolus fibrillar center (FC). Treatment with a transcription inhibitor induced Pol I to dissociate from rDNA chromatin and to move like a liquid within the nucleolar cap that transformed from the FC. Expression of a Pol I mutant associated with a craniofacial disorder inhibited transcription by competing with wild-type Pol I clusters and transforming the FC into the nucleolar cap. The cap droplet excluded an initiation factor, ensuring robust silencing. Our findings suggest a mechanism of rRNA transcription suppression via phase separation of intranucleolar molecules governed by Pol I.

DOI： 10.1126/sciadv.abb5953

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

Alternative lengthening of telomeres, or ALT, is a recombination-based process that maintains telomeres to render some cancer cells immortal. The prevailing view is that ALT is inhibited by heterochromatin because heterochromatin prevents recombination. To test this model, we used telomere-specific quantitative proteomics on cells with heterochromatin deficiencies. In contrast to expectations, we found that ALT does not result from a lack of heterochromatin; rather, ALT is a consequence of heterochromatin formation at telomeres, which is seeded by the histone methyltransferase SETDB1. Heterochromatin stimulates transcriptional elongation at telomeres together with the recruitment of recombination factors, while disrupting heterochromatin had the opposite effect. Consistently, loss of SETDB1, disrupts telomeric heterochromatin and abrogates ALT. Thus, inhibiting telomeric heterochromatin formation in ALT cells might offer a new therapeutic approach to cancer treatment.

DOI： 10.1126/sciadv.aav3673

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DOI： 10.1016/j.ceb.2019.02.003

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1091/mbc.E17-06-0359

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

DOI： 10.1021/jacs.6b09023

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

DOI： 10.1038/srep29261

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Language：English   Publisher：Elsevier Ltd  

DOI： 10.1016/j.gde.2015.11.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

DOI： 10.1039/c4sc03755c

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/ncomms7674

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DOI： 10.1021/ja506058e

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DOI： 10.1016/j.cub.2013.07.048

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pgen.1003410

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/science.1179044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Inc.  

DOI： 10.1002/0471143030.cb2214s49

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2009.07.012

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1128/MCB.00731-06

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Language：Japanese  

J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Authorship：Last author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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Language：English   Publishing type：Research paper, summary (international conference)  

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