研究実績
2025
6.
Nao Yonezawa, Yasushi Hiraoka, Tokuko Haraguchi, Kazuo Yamagata
T4 DNA-Induced Reconstruction of Artificial Nuclei in Living Mouse Oocytes Book Chapter
In: Methods in Molecular Biology, pp. 183–199, Springer US, 2025, ISBN: 9781071647141.
Abstract | Links | タグ: Yamagata G
@inbook{Yonezawa2025,
title = {T4 DNA-Induced Reconstruction of Artificial Nuclei in Living Mouse Oocytes},
author = {Nao Yonezawa and Yasushi Hiraoka and Tokuko Haraguchi and Kazuo Yamagata},
doi = {10.1007/978-1-0716-4714-1_13},
isbn = {9781071647141},
year = {2025},
date = {2025-08-21},
booktitle = {Methods in Molecular Biology},
pages = {183--199},
publisher = {Springer US},
abstract = {Fertilization involves a specialized nuclear formation process distinct from that of somatic cells, and reconstitution methods are useful for understanding its underlying mechanisms. Recently, we successfully reconstructed a nuclear-like structure, termed an “artificial nucleus,” in living mouse oocytes by microinjecting solutions of T4 DNA (~166 kbp). This achievement allowed us to identify the physicochemical properties necessary for nuclear formation. In this paper, we describe a method for constructing artificial nuclei using T4 DNA solutions, which will contribute to future studies on the mechanisms of nuclear formation.},
keywords = {Yamagata G},
pubstate = {published},
tppubtype = {inbook}
}
Fertilization involves a specialized nuclear formation process distinct from that of somatic cells, and reconstitution methods are useful for understanding its underlying mechanisms. Recently, we successfully reconstructed a nuclear-like structure, termed an “artificial nucleus,” in living mouse oocytes by microinjecting solutions of T4 DNA (~166 kbp). This achievement allowed us to identify the physicochemical properties necessary for nuclear formation. In this paper, we describe a method for constructing artificial nuclei using T4 DNA solutions, which will contribute to future studies on the mechanisms of nuclear formation.
5.
Atsushi Takasu, Toshiaki Hino, Osamu Takenouchi, Yasuki Miyagawa, Zhihua Liang, Shota Tanaka, Tomoya Mimura, Chisato Ida, Yuki Matsuo, Yuna Lee, Haruka Ikegami, Miho Ohsugi, Shogo Matoba, Atsuo Ogura, Kazuo Yamagata, Kazuya Matsumoto, Tomoya S Kitajima, Kei Miyamoto
Characterization of H3K4me3 in mouse oocytes at the metaphase II stage Journal Article
In: J Biol Chem, pp. 110308, 2025, ISSN: 1083-351X.
Abstract | Links | タグ: Miyamoto G, Yamagata G
@article{pmid40449591,
title = {Characterization of H3K4me3 in mouse oocytes at the metaphase II stage},
author = {Atsushi Takasu and Toshiaki Hino and Osamu Takenouchi and Yasuki Miyagawa and Zhihua Liang and Shota Tanaka and Tomoya Mimura and Chisato Ida and Yuki Matsuo and Yuna Lee and Haruka Ikegami and Miho Ohsugi and Shogo Matoba and Atsuo Ogura and Kazuo Yamagata and Kazuya Matsumoto and Tomoya S Kitajima and Kei Miyamoto},
doi = {10.1016/j.jbc.2025.110308},
issn = {1083-351X},
year = {2025},
date = {2025-05-29},
urldate = {2025-05-01},
journal = {J Biol Chem},
pages = {110308},
abstract = {Central functions of histone modifications in germ cell and embryonic development have been documented. Accumulating evidence suggests that oocytes possess unique profiles of histone modifications, among which histone H3 lysine 4 trimethylation (H3K4me3) is broadly spread on the mouse oocyte chromosomes at the metaphase II (MII) stage, unlike later embryonic stages. However, the characteristics and developmental roles of H3K4me3 on MII chromosomes are unclear. Here, we discovered that H3K4me3 was abundantly localized on some of the MII oocyte chromosomes facing the cortical side. Using multicolor FISH and CRISPR-Sirius-based labeling of chromosomes, we revealed that the X chromosome tended to be localized at the cortical side with strong H3K4me3 signals. Anchoring oocyte chromosomes to the cortex may play a role in the asymmetric H3K4me3 distribution. Furthermore, we found that the forced removal of H3K4me3 through the overexpression of a specific lysine demethylase in MII oocytes resulted in abnormal chromosome-spindle structure and impaired preimplantation development after in vitro fertilization. These findings highlight the developmental function of H3K4me3 in transcriptionally-silent MII oocytes.},
keywords = {Miyamoto G, Yamagata G},
pubstate = {published},
tppubtype = {article}
}
Central functions of histone modifications in germ cell and embryonic development have been documented. Accumulating evidence suggests that oocytes possess unique profiles of histone modifications, among which histone H3 lysine 4 trimethylation (H3K4me3) is broadly spread on the mouse oocyte chromosomes at the metaphase II (MII) stage, unlike later embryonic stages. However, the characteristics and developmental roles of H3K4me3 on MII chromosomes are unclear. Here, we discovered that H3K4me3 was abundantly localized on some of the MII oocyte chromosomes facing the cortical side. Using multicolor FISH and CRISPR-Sirius-based labeling of chromosomes, we revealed that the X chromosome tended to be localized at the cortical side with strong H3K4me3 signals. Anchoring oocyte chromosomes to the cortex may play a role in the asymmetric H3K4me3 distribution. Furthermore, we found that the forced removal of H3K4me3 through the overexpression of a specific lysine demethylase in MII oocytes resulted in abnormal chromosome-spindle structure and impaired preimplantation development after in vitro fertilization. These findings highlight the developmental function of H3K4me3 in transcriptionally-silent MII oocytes.
4.
Tatsuma Yao, Hisato Kobayashi, Tatsuki Hirai, Yuta Tokuoka, Mikiko Tokoro, Yuta Asayama, Yuka Suzuki, Yu Hatano, Hiroki Ikeda, Satoshi Sugimura, Takuya Yamamoto, Takahiro G Yamada, Yoshihiko Hosoi, Akira Funahashi, Noritaka Fukunaga, Yoshimasa Asada, Kazuki Kurimoto, Kazuo Yamagata
Zinc eluted from glassware is a risk factor for embryo development in human and animal assisted reproduction Journal Article
In: Biology of Reproduction, 2025, ISSN: 1529-7268.
Abstract | Links | タグ: Yamagata G
@article{Yao2025,
title = {Zinc eluted from glassware is a risk factor for embryo development in human and animal assisted reproduction},
author = {Tatsuma Yao and Hisato Kobayashi and Tatsuki Hirai and Yuta Tokuoka and Mikiko Tokoro and Yuta Asayama and Yuka Suzuki and Yu Hatano and Hiroki Ikeda and Satoshi Sugimura and Takuya Yamamoto and Takahiro G Yamada and Yoshihiko Hosoi and Akira Funahashi and Noritaka Fukunaga and Yoshimasa Asada and Kazuki Kurimoto and Kazuo Yamagata},
doi = {10.1093/biolre/ioaf050},
issn = {1529-7268},
year = {2025},
date = {2025-04-02},
urldate = {2025-04-02},
journal = {Biology of Reproduction},
publisher = {Oxford University Press (OUP)},
abstract = {In assisted reproduction, many factors in the culture environment, including light, temperature, pH, and culture media, can reduce preimplantation embryo viability. Laboratory glassware is also a known risk factor for in vitro embryos; however, the underlying mechanisms that disrupt embryonic development remain unclear. We identified Zn eluted from glassware as an embryotoxic substance. In mouse embryos, Zn induced delayed development, abnormalities in chromosome segregation, cytokinesis, zygotic gene activation (e.g. Zscan4a and murine endogenous retrovirus with leucine, also known as MERVL), and aberrantly upregulated developmental gene expression (e.g. Hoxa1, Hoxb9, T, and Fgf8) that could be mediated through metal regulatory transcription factors (e.g. Mtf1). Subsequently, Zn exposure led to significantly reduced blastocyst formation. Post-implantation, Zn-exposed embryos were associated with normal birth rates, however, the birth weight increased by an average of 18% compared with embryos cultured without Zn. Furthermore, Zn exposure affected the development of bovine and human embryos, with species-based variation in the strength and timing of these effects. To mitigate these embryotoxic effects, we identified a method to prevent glass toxicity using chelating agents. This research not only highlights the importance of risk control in embryo culture but also facilitates the development of safe and effective methods for assisted reproduction.},
keywords = {Yamagata G},
pubstate = {published},
tppubtype = {article}
}
In assisted reproduction, many factors in the culture environment, including light, temperature, pH, and culture media, can reduce preimplantation embryo viability. Laboratory glassware is also a known risk factor for in vitro embryos; however, the underlying mechanisms that disrupt embryonic development remain unclear. We identified Zn eluted from glassware as an embryotoxic substance. In mouse embryos, Zn induced delayed development, abnormalities in chromosome segregation, cytokinesis, zygotic gene activation (e.g. Zscan4a and murine endogenous retrovirus with leucine, also known as MERVL), and aberrantly upregulated developmental gene expression (e.g. Hoxa1, Hoxb9, T, and Fgf8) that could be mediated through metal regulatory transcription factors (e.g. Mtf1). Subsequently, Zn exposure led to significantly reduced blastocyst formation. Post-implantation, Zn-exposed embryos were associated with normal birth rates, however, the birth weight increased by an average of 18% compared with embryos cultured without Zn. Furthermore, Zn exposure affected the development of bovine and human embryos, with species-based variation in the strength and timing of these effects. To mitigate these embryotoxic effects, we identified a method to prevent glass toxicity using chelating agents. This research not only highlights the importance of risk control in embryo culture but also facilitates the development of safe and effective methods for assisted reproduction.
2024
3.
Naoki Goto, Kazuma Suke, Nao Yonezawa, Hidenori Nishihara, Tetsuya Handa, Yuko Sato, Tomoya Kujirai, Hitoshi Kurumizaka, Kazuo Yamagata, Hiroshi Kimura
ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin Journal Article
In: J Cell Biol, vol. 223, no. 8, 2024, ISSN: 1540-8140.
Abstract | Links | タグ: Kimura G, Kurumizaka G, Yamagata G
@article{pmid38709169,
title = {ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin},
author = {Naoki Goto and Kazuma Suke and Nao Yonezawa and Hidenori Nishihara and Tetsuya Handa and Yuko Sato and Tomoya Kujirai and Hitoshi Kurumizaka and Kazuo Yamagata and Hiroshi Kimura},
doi = {10.1083/jcb.202310084},
issn = {1540-8140},
year = {2024},
date = {2024-08-01},
urldate = {2024-08-01},
journal = {J Cell Biol},
volume = {223},
number = {8},
abstract = {Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.},
keywords = {Kimura G, Kurumizaka G, Yamagata G},
pubstate = {published},
tppubtype = {article}
}
Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.
2.
Nao Yonezawa, Tomoko Shindo, Haruka Oda, Hiroshi Kimura, Yasushi Hiraoka, Tokuko Haraguchi, Kazuo Yamagata
Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes Journal Article
In: Genes Cells, 2024, ISSN: 1365-2443.
Abstract | Links | タグ: Kimura G, Yamagata G
@article{pmid39140385,
title = {Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes},
author = {Nao Yonezawa and Tomoko Shindo and Haruka Oda and Hiroshi Kimura and Yasushi Hiraoka and Tokuko Haraguchi and Kazuo Yamagata},
doi = {10.1111/gtc.13149},
issn = {1365-2443},
year = {2024},
date = {2024-08-01},
urldate = {2024-08-01},
journal = {Genes Cells},
abstract = {In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/μL of T4 DNA, but not 20 ng/μL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.},
keywords = {Kimura G, Yamagata G},
pubstate = {published},
tppubtype = {article}
}
In eukaryotes, DNA is housed within the cell nucleus. Molecules required for the formation of a nucleus have been identified using in vitro systems with frog egg extracts and in vivo imaging of somatic cells. However, little is known about the physicochemical factors and conditions required for nuclear formation in mouse oocytes. In this study, using a reconstitution approach with purified DNA, we aimed to determine factors, such as the amount and timing of DNA introduction, required for the formation of nuclei with nuclear transport activity in mouse oocytes. T4 phage DNA (~166 kbp) was microinjected into strontium-activated oocytes to evaluate the conditions appropriate for nuclear formation. Microinjection of 100-500 ng/μL of T4 DNA, but not 20 ng/μL, was sufficient for the formation of nucleus-like structures. Furthermore, microinjection of DNA during metaphase II to telophase II, but not during interphase, was sufficient. Electron and fluorescence microscopy showed that T4 DNA-induced nucleus-like structures had nuclear lamina and nuclear pore complex structures similar to those of natural nuclei, as well as nuclear import activity. These results suggest that exogenous DNA can form artificial nuclei with nuclear transport functions in mouse oocytes, regardless of the sequence or source of the DNA.
1.
Yuko Sato, Maoko Takenoshita, Miku Ueoka, Jun Ueda, Kazuo Yamagata, Hiroshi Kimura
Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody Journal Article
In: Histochem Cell Biol, 2024, ISSN: 1432-119X.
Abstract | Links | タグ: Kimura G, Yamagata G
@article{pmid38762823,
title = {Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody},
author = {Yuko Sato and Maoko Takenoshita and Miku Ueoka and Jun Ueda and Kazuo Yamagata and Hiroshi Kimura},
doi = {10.1007/s00418-024-02296-8},
issn = {1432-119X},
year = {2024},
date = {2024-05-01},
urldate = {2024-05-01},
journal = {Histochem Cell Biol},
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 the Rosa26 locus. 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.},
keywords = {Kimura G, Yamagata G},
pubstate = {published},
tppubtype = {article}
}
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 the Rosa26 locus. 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.
