Publications
2026
12.
Xiaohui Gao, Chaebeen Ko, Yuanchao Dong, Takeru Fujii, Satoshi Uchino, Yoshiaki Kobayashi, Akihito Harada, Hiroaki Ohishi, Yasuyuki Ohkawa, Hiroshi Kimura, Hiroshi Ochiai
Minute-scale coupling of chromatin marks and transcriptional bursts Journal Article
In: bioRxiv, 2026.
Abstract | Links | タグ: Kimura G, Ochiai G, Ohkawa G
@article{Gao2026.02.08.704500,
title = {Minute-scale coupling of chromatin marks and transcriptional bursts},
author = {Xiaohui Gao and Chaebeen Ko and Yuanchao Dong and Takeru Fujii and Satoshi Uchino and Yoshiaki Kobayashi and Akihito Harada and Hiroaki Ohishi and Yasuyuki Ohkawa and Hiroshi Kimura and Hiroshi Ochiai},
url = {https://www.biorxiv.org/content/early/2026/02/10/2026.02.08.704500},
doi = {10.64898/2026.02.08.704500},
year = {2026},
date = {2026-02-10},
urldate = {2026-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes. Whether chromatin marks around individual genes change on these rapid timescales remains unclear. Here we show that local chromatin modification signals around endogenous genes in mouse embryonic stem cells fluctuate reversibly with transcriptional state, using live imaging of individual genes together with fluorescent probes that report histone modifications. Activation-associated acetylation and methylation marks increased in association with transcriptional activation and decreased with inactivation, whereas a Polycomb-associated repressive mark behaved oppositely. Transcriptional coactivators and both histone acetyltransferase and deacetylase complexes were enriched during transcriptionally active state, consistent with opposing enzymatic activities shaping local acetylation levels. Inhibiting histone deacetylases altered the durations of active and inactive events, supporting a role for deacetylation in regulating transcriptional state transitions. Thus, histone modifications undergo reversible, minute-scale changes coupled to transcriptional activity. This framework helps explain how stochastic transcriptional bursts can occur with stable gene regulation over longer timescales.},
keywords = {Kimura G, Ochiai G, Ohkawa G},
pubstate = {published},
tppubtype = {article}
}
Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes. Whether chromatin marks around individual genes change on these rapid timescales remains unclear. Here we show that local chromatin modification signals around endogenous genes in mouse embryonic stem cells fluctuate reversibly with transcriptional state, using live imaging of individual genes together with fluorescent probes that report histone modifications. Activation-associated acetylation and methylation marks increased in association with transcriptional activation and decreased with inactivation, whereas a Polycomb-associated repressive mark behaved oppositely. Transcriptional coactivators and both histone acetyltransferase and deacetylase complexes were enriched during transcriptionally active state, consistent with opposing enzymatic activities shaping local acetylation levels. Inhibiting histone deacetylases altered the durations of active and inactive events, supporting a role for deacetylation in regulating transcriptional state transitions. Thus, histone modifications undergo reversible, minute-scale changes coupled to transcriptional activity. This framework helps explain how stochastic transcriptional bursts can occur with stable gene regulation over longer timescales.
11.
Gabriel Galindo, Daiki Maejima, Jacob DeRoo, Scott R. Burlingham, Gretchen Fixen, Tatsuya Morisaki, Hallie P. Febvre, Ryan Hasbrook, Ning Zhao, Soham Ghosh, E. Handly Mayton, Christopher D. Snow, Brian J. Geiss, Yasuyuki Ohkawa, Yuko Sato, Hiroshi Kimura, Timothy J. Stasevich
AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications Journal Article
In: Sci. Adv., vol. 12, no. 1, 2026, ISSN: 2375-2548.
Abstract | Links | タグ: Kimura G, Ohkawa G
@article{Galindo2026,
title = {AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications},
author = {Gabriel Galindo and Daiki Maejima and Jacob DeRoo and Scott R. Burlingham and Gretchen Fixen and Tatsuya Morisaki and Hallie P. Febvre and Ryan Hasbrook and Ning Zhao and Soham Ghosh and E. Handly Mayton and Christopher D. Snow and Brian J. Geiss and Yasuyuki Ohkawa and Yuko Sato and Hiroshi Kimura and Timothy J. Stasevich},
doi = {10.1126/sciadv.adx8352},
issn = {2375-2548},
year = {2026},
date = {2026-01-02},
urldate = {2026-01-02},
journal = {Sci. Adv.},
volume = {12},
number = {1},
publisher = {American Association for the Advancement of Science (AAAS)},
abstract = {Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an artificial intelligence-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 of 26 antibody sequences into functional single-chain variable fragment intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost effective, and broadly accessible for biological research.},
keywords = {Kimura G, Ohkawa G},
pubstate = {published},
tppubtype = {article}
}
Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an artificial intelligence-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 of 26 antibody sequences into functional single-chain variable fragment intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost effective, and broadly accessible for biological research.
2025
10.
Takeru Fujii, Kosuke Tomimatsu, Michiko Kato, Miho Ito, Shoko Sato, Hitoshi Kurumizaka, Yuko Sato, Kazumitsu Maehara, Hiroshi Kimura, Akihito Harada, Yasuyuki Ohkawa
Reconstructing epigenomic dynamics through a single-cell multi-epigenome data integration framework Journal Article
In: Nat Commun, vol. 16, no. 1, 2025, ISSN: 2041-1723.
Abstract | Links | タグ: Kimura G, Kurumizaka G, Ohkawa G
@article{Fujii2025,
title = {Reconstructing epigenomic dynamics through a single-cell multi-epigenome data integration framework},
author = {Takeru Fujii and Kosuke Tomimatsu and Michiko Kato and Miho Ito and Shoko Sato and Hitoshi Kurumizaka and Yuko Sato and Kazumitsu Maehara and Hiroshi Kimura and Akihito Harada and Yasuyuki Ohkawa},
doi = {10.1038/s41467-025-67016-9},
issn = {2041-1723},
year = {2025},
date = {2025-12-17},
journal = {Nat Commun},
volume = {16},
number = {1},
publisher = {Springer Science and Business Media LLC},
abstract = {Transcriptional regulation arises from the dynamic and combinatorial actions of multiple regulatory factors on genomic DNA. Although many epigenomic regulators have been identified, the precise order in which these factors accumulate at individual gene loci to activate transcription remains unclear. Here we show a single-cell data integration framework that infers the binding order of multiple chromatin factors at single-cell resolution. Central to this framework is sci-mtChIL-seq, a scalable single-cell method that simultaneously profiles genome-wide binding of RNA polymerase II (RNAPII) and diverse epigenomic regulators. By defining transcriptional states through RNAPII occupancy and integrating multiple sci-mtChIL-seq datasets, we systematically link the combinatorial patterns of transcription factor binding, histone modifications and chromatin remodeling. This framework reveals the temporal coordination among chromatin factors during transcriptional activation, providing a powerful approach to uncover context-dependent epigenomic dynamics and the principles of gene regulation in complex cellular systems.},
keywords = {Kimura G, Kurumizaka G, Ohkawa G},
pubstate = {published},
tppubtype = {article}
}
Transcriptional regulation arises from the dynamic and combinatorial actions of multiple regulatory factors on genomic DNA. Although many epigenomic regulators have been identified, the precise order in which these factors accumulate at individual gene loci to activate transcription remains unclear. Here we show a single-cell data integration framework that infers the binding order of multiple chromatin factors at single-cell resolution. Central to this framework is sci-mtChIL-seq, a scalable single-cell method that simultaneously profiles genome-wide binding of RNA polymerase II (RNAPII) and diverse epigenomic regulators. By defining transcriptional states through RNAPII occupancy and integrating multiple sci-mtChIL-seq datasets, we systematically link the combinatorial patterns of transcription factor binding, histone modifications and chromatin remodeling. This framework reveals the temporal coordination among chromatin factors during transcriptional activation, providing a powerful approach to uncover context-dependent epigenomic dynamics and the principles of gene regulation in complex cellular systems.
9.
Chihiro Matsuda, Akane Ichiki, Yuko Sato, Yukino Kudo, Mika Saotome, Chihiro Takayama, Khoa Minh Le, Satoshi Uchino, Ryota Higuchi, Kazuhiko Kawata, Kosuke Tomimatsu, Manabu Ozawa, Masahito Ikawa, Yasuyuki Ohkawa, Yoshihiro Baba, Hiroshi Kimura
Organization and Dynamics of Transcription Elongation Foci in Mouse Tissues Journal Article
In: Journal of Molecular Biology, 2025, ISSN: 0022-2836.
Abstract | Links | タグ: Kimura G, Ohkawa G
@article{Matsuda2025,
title = {Organization and Dynamics of Transcription Elongation Foci in Mouse Tissues},
author = {Chihiro Matsuda and Akane Ichiki and Yuko Sato and Yukino Kudo and Mika Saotome and Chihiro Takayama and Khoa Minh Le and Satoshi Uchino and Ryota Higuchi and Kazuhiko Kawata and Kosuke Tomimatsu and Manabu Ozawa and Masahito Ikawa and Yasuyuki Ohkawa and Yoshihiro Baba and Hiroshi Kimura},
doi = {10.1016/j.jmb.2025.169395},
issn = {0022-2836},
year = {2025},
date = {2025-08-13},
journal = {Journal of Molecular Biology},
publisher = {Elsevier BV},
abstract = {RNA polymerase II (RNAP2) transcribes most genes in eukaryotic nuclei. During the transition from transcription initiation to productive elongation, and throughout the elongation phase, RNAP2 becomes phosphorylated at the Ser2 residue within the heptapeptide repeats of the carboxyl-terminal domain of its largest subunit. Antibodies specific to RNAP2 Ser2 phosphorylation (Ser2ph) have enabled visualization of active transcription sites in fixed cells and tissues. Here, we report the generation and characterization of knock-in mice ubiquitously expressing a fluorescent protein-tagged, modification-specific intracellular antibody (mintbody) targeting RNAP2 Ser2ph. Using these mice, we successfully visualized transcription elongation foci in mouse tissues and characterized their distribution and dynamics across diverse cell types. RNAP2 Ser2ph-mintbody formed hundreds to thousands of nuclear foci, which were excluded from heterochromatin and transcriptionally repressed domains, such as the XY body in pachytene spermatocytes. Quantitative analysis revealed tissue- and cell type-specific variation in both the number and mobility of transcription elongation foci. The mobility of transcription foci was more restricted in differentiated cells compared to differentiating and proliferating cells, likely reflecting a reduced number of actively transcribed genes and more limited open chromatin regions upon differentiation. These findings suggest that the spatial organization and dynamics of transcription elongation are closely associated with cell identity and differentiation status. The RNAP2 Ser2ph-mintbody knock-in mice provide a valuable tool for future studies of transcription organization and dynamics at the tissue level.},
keywords = {Kimura G, Ohkawa G},
pubstate = {published},
tppubtype = {article}
}
RNA polymerase II (RNAP2) transcribes most genes in eukaryotic nuclei. During the transition from transcription initiation to productive elongation, and throughout the elongation phase, RNAP2 becomes phosphorylated at the Ser2 residue within the heptapeptide repeats of the carboxyl-terminal domain of its largest subunit. Antibodies specific to RNAP2 Ser2 phosphorylation (Ser2ph) have enabled visualization of active transcription sites in fixed cells and tissues. Here, we report the generation and characterization of knock-in mice ubiquitously expressing a fluorescent protein-tagged, modification-specific intracellular antibody (mintbody) targeting RNAP2 Ser2ph. Using these mice, we successfully visualized transcription elongation foci in mouse tissues and characterized their distribution and dynamics across diverse cell types. RNAP2 Ser2ph-mintbody formed hundreds to thousands of nuclear foci, which were excluded from heterochromatin and transcriptionally repressed domains, such as the XY body in pachytene spermatocytes. Quantitative analysis revealed tissue- and cell type-specific variation in both the number and mobility of transcription elongation foci. The mobility of transcription foci was more restricted in differentiated cells compared to differentiating and proliferating cells, likely reflecting a reduced number of actively transcribed genes and more limited open chromatin regions upon differentiation. These findings suggest that the spatial organization and dynamics of transcription elongation are closely associated with cell identity and differentiation status. The RNAP2 Ser2ph-mintbody knock-in mice provide a valuable tool for future studies of transcription organization and dynamics at the tissue level.
8.
Tamiko Nozaki, Mayu Onoda, Misuzu Habazaki, Yuma Takeuchi, Hisashi Ishida, Yuko Sato, Tomoya Kujirai, Kayo Hanada, Kenzo Yamatsugu, Hitoshi Kurumizaka, Hiroshi Kimura, Hidetoshi Kono, Shigehiro A. Kawashima, Motomu Kanai
Designer Catalyst-Enabled Regiodivergent Histone Acetylation Journal Article
In: J. Am. Chem. Soc., 2025, ISSN: 1520-5126.
Abstract | Links | タグ: Kawashima G, Kimura G, Kurumizaka G
@article{Nozaki2025,
title = {Designer Catalyst-Enabled Regiodivergent Histone Acetylation},
author = {Tamiko Nozaki and Mayu Onoda and Misuzu Habazaki and Yuma Takeuchi and Hisashi Ishida and Yuko Sato and Tomoya Kujirai and Kayo Hanada and Kenzo Yamatsugu and Hitoshi Kurumizaka and Hiroshi Kimura and Hidetoshi Kono and Shigehiro A. Kawashima and Motomu Kanai},
url = {https://pubs.acs.org/doi/full/10.1021/jacs.5c01699},
doi = {10.1021/jacs.5c01699},
issn = {1520-5126},
year = {2025},
date = {2025-04-13},
urldate = {2025-04-13},
journal = {J. Am. Chem. Soc.},
publisher = {American Chemical Society (ACS)},
abstract = {The “histone code,” defined by the combinatorial patterns of post-translational modifications (PTMs) on histones, plays a pivotal role in chromatin structure and gene expression. Tools for the regioselective introduction of histone PTMs in living cells are critical for dissecting the functions of these epigenetic marks. Here, we report the design and development of three regioselective catalysts that acetylate distinct lysine residues (K43, K108, and K120) on histone H2B. Using a combination of molecular dynamics simulations of catalyst-nucleosome complexes and systematic experimental optimization of catalyst structures, we identified key design principles for achieving regioselectivity. Specifically, excluding highly reactive off-target lysine residues from the catalyst effective region (CER) while maintaining proximity to a target lysine residue proved crucial. Biochemical and cellular analyses of the catalytic histone acetylation revealed that each lysine acetylation elicited unique effects on the binding affinity and activity of nucleosome-interacting molecules, as well as on transcriptional programs and cellular phenotypes. These findings establish a framework for designing regioselective histone acetylation catalysts and advance our understanding of the regulatory mechanisms underlying histone PTMs.},
keywords = {Kawashima G, Kimura G, Kurumizaka G},
pubstate = {published},
tppubtype = {article}
}
The “histone code,” defined by the combinatorial patterns of post-translational modifications (PTMs) on histones, plays a pivotal role in chromatin structure and gene expression. Tools for the regioselective introduction of histone PTMs in living cells are critical for dissecting the functions of these epigenetic marks. Here, we report the design and development of three regioselective catalysts that acetylate distinct lysine residues (K43, K108, and K120) on histone H2B. Using a combination of molecular dynamics simulations of catalyst-nucleosome complexes and systematic experimental optimization of catalyst structures, we identified key design principles for achieving regioselectivity. Specifically, excluding highly reactive off-target lysine residues from the catalyst effective region (CER) while maintaining proximity to a target lysine residue proved crucial. Biochemical and cellular analyses of the catalytic histone acetylation revealed that each lysine acetylation elicited unique effects on the binding affinity and activity of nucleosome-interacting molecules, as well as on transcriptional programs and cellular phenotypes. These findings establish a framework for designing regioselective histone acetylation catalysts and advance our understanding of the regulatory mechanisms underlying histone PTMs.
7.
Konrad Chudzik, Yuko Sato, Xingchi Yan, Simon Ullrich, Watanya Trakarnphornsombat, Lothar Schermelleh, Geoffrey Fudenberg, Hiroshi Kimura, Michael I. Robson, Irina Solovei
Ab-trapping - a peripheral staining artifact in antibody-based microscopy and genomics Unpublished
bioRxiv, 2025.
Abstract | Links | タグ: Kimura G
@unpublished{Chudzik2025,
title = {Ab-trapping - a peripheral staining artifact in antibody-based microscopy and genomics},
author = {Konrad Chudzik and Yuko Sato and Xingchi Yan and Simon Ullrich and Watanya Trakarnphornsombat and Lothar Schermelleh and Geoffrey Fudenberg and Hiroshi Kimura and Michael I. Robson and Irina Solovei},
url = {http://biorxiv.org/lookup/doi/10.1101/2025.04.09.648027},
doi = {10.1101/2025.04.09.648027},
year = {2025},
date = {2025-04-10},
urldate = {2025-04-10},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Antibodies (Ab) are essential for detecting specific epitopes in microscopy and genomics, but can produce artifacts leading to erroneous interpretations. Here, we characterize a novel artifact, Ab-trapping, in which antibodies bind at the periphery of a cellular structure and do not diffuse further into its interior. This causes anomalous peripheral staining for multiple critical targets, including endogenous or ectopically expressed nuclear proteins like nucleolar proteins, histone variants and their modifications like H3K9me2. Ab-trapping can affect any assay relying on Ab diffusion, including immunofluorescence microscopy and recent genomics approaches like CUT&Tag. Critically, computational modeling and experimental validation reveal that Ab-trapping is caused by high epitope abundance, high Ab affinity, and low diffusion rates. Consequently, its effects can be mitigated by using alternative Abs and optimizing incubation conditions. Ab-trapping is therefore a considerable artifact that should be considered when designing experiments and interpreting results.},
howpublished = {bioRxiv},
keywords = {Kimura G},
pubstate = {published},
tppubtype = {unpublished}
}
Antibodies (Ab) are essential for detecting specific epitopes in microscopy and genomics, but can produce artifacts leading to erroneous interpretations. Here, we characterize a novel artifact, Ab-trapping, in which antibodies bind at the periphery of a cellular structure and do not diffuse further into its interior. This causes anomalous peripheral staining for multiple critical targets, including endogenous or ectopically expressed nuclear proteins like nucleolar proteins, histone variants and their modifications like H3K9me2. Ab-trapping can affect any assay relying on Ab diffusion, including immunofluorescence microscopy and recent genomics approaches like CUT&Tag. Critically, computational modeling and experimental validation reveal that Ab-trapping is caused by high epitope abundance, high Ab affinity, and low diffusion rates. Consequently, its effects can be mitigated by using alternative Abs and optimizing incubation conditions. Ab-trapping is therefore a considerable artifact that should be considered when designing experiments and interpreting results.
6.
Tomoya Kujirai, Kenta Echigoya, Yusuke Kishi, Mai Saeki, Tomoko Ito, Junko Kato, Lumi Negishi, Hiroshi Kimura, Hiroshi Masumoto, Yoshimasa Takizawa, Yukiko Gotoh, Hitoshi Kurumizaka
Structural insights into how DEK nucleosome binding facilitates H3K27 trimethylation in chromatin Journal Article
In: Nature Structural & Molecular Biology, 2025, ISSN: 1545-9993.
Abstract | Links | タグ: Gotoh G, Kimura G, Kurumizaka G
@article{10.1038/s41594-025-01493-w,
title = {Structural insights into how DEK nucleosome binding facilitates H3K27 trimethylation in chromatin},
author = {Tomoya Kujirai and Kenta Echigoya and Yusuke Kishi and Mai Saeki and Tomoko Ito and Junko Kato and Lumi Negishi and Hiroshi Kimura and Hiroshi Masumoto and Yoshimasa Takizawa and Yukiko Gotoh and Hitoshi Kurumizaka},
doi = {10.1038/s41594-025-01493-w},
issn = {1545-9993},
year = {2025},
date = {2025-02-21},
urldate = {2025-02-21},
journal = {Nature Structural & Molecular Biology},
abstract = {Structural diversity of the nucleosome affects chromatin conformations and regulates eukaryotic genome functions. Here we identify DEK, whose function is unknown, as a nucleosome-binding protein. In embryonic neural progenitor cells, DEK colocalizes with H3 K27 trimethylation (H3K27me3), the facultative heterochromatin mark. DEK stimulates the methyltransferase activity of Polycomb repressive complex 2 (PRC2), which is responsible for H3K27me3 deposition in vitro. Cryo-electron microscopy structures of the DEK–nucleosome complexes reveal that DEK binds the nucleosome by its tripartite DNA-binding mode on the dyad and linker DNAs and interacts with the nucleosomal acidic patch by its newly identified histone-binding region. The DEK–nucleosome interaction mediates linker DNA reorientation and induces chromatin compaction, which may facilitate PRC2 activation. These findings provide mechanistic insights into chromatin structure-mediated gene regulation by DEK.},
keywords = {Gotoh G, Kimura G, Kurumizaka G},
pubstate = {published},
tppubtype = {article}
}
Structural diversity of the nucleosome affects chromatin conformations and regulates eukaryotic genome functions. Here we identify DEK, whose function is unknown, as a nucleosome-binding protein. In embryonic neural progenitor cells, DEK colocalizes with H3 K27 trimethylation (H3K27me3), the facultative heterochromatin mark. DEK stimulates the methyltransferase activity of Polycomb repressive complex 2 (PRC2), which is responsible for H3K27me3 deposition in vitro. Cryo-electron microscopy structures of the DEK–nucleosome complexes reveal that DEK binds the nucleosome by its tripartite DNA-binding mode on the dyad and linker DNAs and interacts with the nucleosomal acidic patch by its newly identified histone-binding region. The DEK–nucleosome interaction mediates linker DNA reorientation and induces chromatin compaction, which may facilitate PRC2 activation. These findings provide mechanistic insights into chromatin structure-mediated gene regulation by DEK.
5.
Gabriel Galindo, Daiki Maejima, Jacob DeRoo, Scott R. Burlingham, Gretchen Fixen, Tatsuya Morisaki, Hallie P. Febvre, Ryan Hasbrook, Ning Zhao, Soham Ghosh, E. Handly Mayton, Christopher D. Snow, Brian J. Geiss, Yasuyuki Ohkawa, Yuko Sato, Hiroshi Kimura, Timothy J. Stasevich
bioRxiv, 2025.
Abstract | Links | タグ: Kimura G, Ohkawa G
@unpublished{Galindo2025,
title = {AI-assisted protein design to rapidly convert antibody sequences to intrabodies targeting diverse peptides and histone modifications},
author = {Gabriel Galindo and Daiki Maejima and Jacob DeRoo and Scott R. Burlingham and Gretchen Fixen and Tatsuya Morisaki and Hallie P. Febvre and Ryan Hasbrook and Ning Zhao and Soham Ghosh and E. Handly Mayton and Christopher D. Snow and Brian J. Geiss and Yasuyuki Ohkawa and Yuko Sato and Hiroshi Kimura and Timothy J. Stasevich},
url = {http://biorxiv.org/lookup/doi/10.1101/2025.02.06.636921},
doi = {10.1101/2025.02.06.636921},
year = {2025},
date = {2025-02-08},
urldate = {2025-02-08},
publisher = {Cold Spring Harbor Laboratory},
abstract = {<jats:title>ABSTRACT</jats:title><jats:p>Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an AI-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 out of 26 antibody sequences into functional single-chain variable fragment (scFv) intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost-effective, and broadly accessible for biological research.</jats:p>},
howpublished = {bioRxiv},
keywords = {Kimura G, Ohkawa G},
pubstate = {published},
tppubtype = {unpublished}
}
<jats:title>ABSTRACT</jats:title><jats:p>Intrabodies are engineered antibodies that function inside living cells, enabling therapeutic, diagnostic, and imaging applications. While powerful, their development has been hindered by challenges associated with their folding, solubility, and stability in the reduced intracellular environment. Here, we present an AI-driven pipeline integrating AlphaFold2, ProteinMPNN, and live-cell screening to optimize antibody framework regions while preserving epitope-binding complementarity-determining regions. Using this approach, we successfully converted 19 out of 26 antibody sequences into functional single-chain variable fragment (scFv) intrabodies, including a panel targeting diverse histone modifications for real-time imaging of chromatin dynamics and gene regulation. Notably, 18 of these 19 sequences had failed to convert using the standard approach, demonstrating the unique effectiveness of our method. As antibody sequence databases expand, our method will accelerate intrabody design, making their development easier, more cost-effective, and broadly accessible for biological research.</jats:p>
2024
4.
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.
3.
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.
2.
Kosuke Tomimatsu, Takeru Fujii, Ryoma Bise, Kazufumi Hosoda, Yosuke Taniguchi, Hiroshi Ochiai, Hiroaki Ohishi, Kanta Ando, Ryoma Minami, Kaori Tanaka, Taro Tachibana, Seiichi Mori, Akihito Harada, Kazumitsu Maehara, Masao Nagasaki, Seiichi Uchida, Hiroshi Kimura, Masashi Narita, Yasuyuki Ohkawa
Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states Journal Article
In: Nat Commun, vol. 15, no. 1, pp. 3657, 2024.
Abstract | Links | タグ: Kimura G, Ochiai G, Ohkawa G
@article{10.1038/s41467-024-47989-9,
title = {Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states},
author = {Kosuke Tomimatsu and Takeru Fujii and Ryoma Bise and Kazufumi Hosoda and Yosuke Taniguchi and Hiroshi Ochiai and Hiroaki Ohishi and Kanta Ando and Ryoma Minami and Kaori Tanaka and Taro Tachibana and Seiichi Mori and Akihito Harada and Kazumitsu Maehara and Masao Nagasaki and Seiichi Uchida and Hiroshi Kimura and Masashi Narita and Yasuyuki Ohkawa},
doi = {10.1038/s41467-024-47989-9},
year = {2024},
date = {2024-05-08},
urldate = {2024-05-08},
journal = {Nat Commun},
volume = {15},
number = {1},
pages = {3657},
abstract = {Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes. Multiplexed imaging to study cellular pathways can be hampered by lack of antibody specificity, especially when assessing signal activation. Here, the authors present Precise Emission Canceling Antibodies (PECAbs), which enable high-specificity sequential imaging and the study of signaling pathways.},
keywords = {Kimura G, Ochiai G, Ohkawa G},
pubstate = {published},
tppubtype = {article}
}
Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes. Multiplexed imaging to study cellular pathways can be hampered by lack of antibody specificity, especially when assessing signal activation. Here, the authors present Precise Emission Canceling Antibodies (PECAbs), which enable high-specificity sequential imaging and the study of signaling pathways.
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.
