Publisher：American Chemical Society (ACS)  

The combination of post-translational modifications (PTMs) of histones, a major component of chromatin, defines “histone code” having distinct impact on chromatin structures and gene expression. Therefore, methods to introduce histone PTMs at desired positions are useful tools for investigating the functions of epigenetic marks, especially to understand consequences of specific modifications in histones. Chemical catalysts, such as lysine (Lys)-acetylating catalysts, have been emerged to regioselectively promote histone PTMs without relying on histone-modifying enzymes. However, it has been poorly under-stood what factors determine Lys-residue selectivity of the histone acetylating catalysts. Here we show that the intrinsic reac-tivity, distance from the catalytically active site, and conformational flexibility of Lys residues, are the critical factors for determining acetylation yield and regioselectivity. Combining molecular dynamics simulations of catalyst-nucleosome com-plexes and experimental optimization of the catalyst structure, we developed three catalysts that selectively acetylated K43, K108, and K120 of H2B, respectively. Biochemical analyses of the regioselectively acetylated nucleosomes revealed that each Lys acetylation showed distinct impacts on inter-nucleosomal interactions and the affinity to a nucleosome-binding molecule. Our data provide a guideline for developing regioselective histone acetylation catalysts and may further accelerate studies of epigenetics regulated by histone PTMs.

DOI： 10.26434/chemrxiv-2024-d1l1b

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.13109

Publisher：Cold Spring Harbor Laboratory  

Abstract

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into theRosa26locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X-chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

DOI： 10.1101/2024.03.13.584715

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

<jats:title>Abstract</jats:title><jats:p>Skeletal tissues including cartilage and bones are characteristic features of vertebrates that are crucial for supporting body morphology and locomotion. Studies mainly in mice have shown that osteoblasts and chondroblasts are supplied from several progenitors like the sclerotome cells in the embryonic stage, osteo‐chondroprogenitors in growing long bones, and skeletal stem cells of bone marrow in the postnatal period. However, the exact origins of progenitor cells, their lineage relationships, and their potential to differentiate into osteoblasts and chondroblasts from embryos to adult tissues are not well understood. In this study, we conducted clonal cell tracking in zebrafish and showed that <jats:italic>sox9a</jats:italic><jats:sup>+</jats:sup> cells are already committed to either chondrogenic or osteogenic fates during embryonic stages and that respective progenies are independently maintained as mesenchymal progenitor pools. Once committed, they never change their lineage identities throughout animal life, even through regeneration. In addition, we further revealed that only osteogenic mesenchymal cells replenish the osteoblast progenitor cells (OPCs), a population of reserved tissue stem cells found to be involved in the de novo production of osteoblasts during regeneration and homeostasis in zebrafish. Thus, our clonal cell tracking study in zebrafish firstly revealed that the mesenchymal progenitor cells that are fated to develop into either chondroblasts or osteoblasts serve as respective tissue stem cells to maintain skeletal tissue homeostasis. Such mesenchymal progenitors dedicated to producing either chondroblasts or osteoblasts would be important targets for skeletal tissue regeneration.</jats:p>

DOI： 10.1111/dgd.12908

Language：English   Publisher：John Wiley & Sons Australia, Ltd  

ゼブラフィッシュにおいて間葉系前駆細胞プールが骨形成原細胞と軟骨形成細胞の産生と維持に及ぼす影響について検討した。ゼブラフィッシュの遺伝子sox9aの5-kbプロモーターを用いてcreERt2を発現するTgsを産生することが可能であり、tyt230系はtyt229系と比較して高い組換え効率を示していた。このようなTgsは細胞系を追跡するのに有用であり、受精3日後においてsox9aのダウンレギュレーションに伴い標識効率の著明な減少が認められた。標識細胞の多くは高度の樹状形態を呈する鰭褶の間葉系細胞に寄与しており、後部領域の標識細胞の87%は間葉系細胞であった。sox9a+細胞は胚形成期に軟骨形成、骨形成のいずれかの運命を有しており、それぞれの子孫が間葉系前駆細胞プールとして保持されることが示された。骨形成間葉系細胞のみが骨芽細胞性前駆細胞(OPCs)を補充する役割を有していることが明らかになった。軟骨芽細胞や骨芽細胞の産生に関わるような間葉系前駆細胞は骨格組織の再生に重要な標的に成りうるものと考えられた。

DOI： 10.1101/2023.12.03.569804

DOI： 10.1101/2023.10.20.563387

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Current Opinion in Structural Biology  

Higher-order genomic structures play a critical role in regulating gene expression by influencing the spatial proximity of promoters and enhancers. Live-cell imaging studies have demonstrated that three-dimensional genome structures undergo dynamic changes over time. Transcription is also dynamic, with genes frequently switching between active and inactive states. Recent observations suggest that the formation of condensates, composed of transcription-related factors, RNA, and RNA-binding proteins, around genes can regulate transcription. Advancements in technology have facilitated the visualization of the intricate spatiotemporal relationship between higher-order genomic structures, condensate formation, and transcriptional activity in living cells.

DOI： 10.1016/j.sbi.2023.102615

Web of Science

Scopus

PubMed

Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

ABSTRACT

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

DOI： 10.1242/bio.059783

Other Link： https://journals.logists.com/bio/bio/article-pdf/doi/10.1242/bio.059783/2857331/bio059783.pdf

Language：English   Publisher：(一社)日本生理学会  

Language：English  

DOI： 10.1016/j.cub.2022.11.015

Language：English   Publisher：Nature Communications  

Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.

DOI： 10.1038/s41467-022-35286-2

Web of Science

Scopus

PubMed

Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

DOI： 10.1083/jcb.202201024

DOI： 10.1101/2022.11.06.515321

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

DOI： 10.1101/2022.06.13.495463

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

DOI： 10.1016/j.molcel.2022.03.009

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cell Biology  

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.

DOI： 10.1083/jcb.202104134

Scopus

PubMed

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

DOI： 10.1016/j.ceb.2022.01.002

Publisher：Cold Spring Harbor Laboratory  

Summary

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

DOI： 10.1101/2022.01.06.472721

Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry ({RSC})  

A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based on the Q-body technology. The technology was further applied to the live cell monitoring of p53 levels, and live cell sorting based on p53 expression.

DOI： 10.1039/D2SC02355E