Biology subjects

Kimura, H.

Publications and source records attributed to Kimura, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

NELF coordinates Pol II transcription termination and DNA replication initiation

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD, a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTSO_LIExpression of NELFCD transcript is up-regulated in colorectal tumors C_LIO_LINELF-C protein is mandatory for the transition between G1-S phases during cell cycle C_LIO_LINELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausing C_LIO_LINELF-C loss leads Pol II to invade DNA replication initiation zones C_LI

molecular biology↗

bioRxiv2023

Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

Interpretation of variants identified during genetic testing is a significant clinical challenge. In this study, we developed a high-throughput CDKN2A functional assay and characterized all possible CDKN2A missense variants. We found that 17.7% of all missense variants were functionally deleterious. We also used our functional classifications to assess the performance of in silico models that predict the effect of variants, including recently reported models based on machine learning. Notably, we found that all in silico models performed similarly when compared to our functional classifications with accuracies of 39.5-85.4%. Furthermore, while we found that functionally deleterious variants were enriched within ankyrin repeats, we did not identify any residues where all missense variants were functionally deleterious. Our functional classifications are a resource to aid the interpretation of CDKN2A variants and have important implications for the application of variant interpretation guidelines, particularly the use of in silico models for clinical variant interpretation.

genetics↗

bioRxiv2023

Multiple HP1 binding motifs in SUV420H2 heterochromatic targeting module

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

cell biology↗

bioRxiv2023

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin

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 or BAZ2A could determine the localization of H3K36me2 in different cell types. To explore the physiological role of heterochromatic H3K36me2, we analyzed mouse tissues and embryos. As a result, H3K36me2 was found in heterochromatin at the 2- to 4-cell stages of mouse preimplantation embryos, suggesting its involvement in developmental regulation. SummaryThe authors discovered histone H3K36me2, which is believed to be enriched in potentially active genomic regions, is also located in transcriptionally inactive regions called heterochromatin in some cell types. The detailed molecular mechanism of its heterochromatin targeting is now revealed.

cell biology↗

bioRxiv2023

Precise immunofluorescence canceling for highly multiplexed imaging capturing specific cell state

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular 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 developed 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.

cell biology↗

bioRxiv2023

Comprehensive analysis of A-type lamin tail region for repairing the nuclear lamina

The nuclear lamina (NL) lines the nuclear envelope (NE) to maintain nuclear structure in metazoan cells. The major NL components, the nuclear lamins contribute to the protection against NE rupture induced by mechanical stress. Lamin A (LA) and a short form of the splicing variant lamin C (LC) are diffused from the nucleoplasm to sites of NE rupture in immortalized mouse embryonic fibroblasts (MEFs). LA localization to the rupture sites is significantly slow and weak compared to LC because of its relatively small pool in the nucleoplasm, but the precise mechanism remains unknown. In this study, we induce NE rupture in wild-type and LA/C-knockout MEFs, and Hutchinson-Gilford Progeria syndrome (HGPS) knock-in MEFs that express progerin, a LA mutant lacking the second proteolytic cleavage site, by laser microirradiation and AFM indentation. The farnesylation at the CaaX motif of unprocessed LA and the inhibition of the second proteolytic cleavage decrease the nucleoplasmic pool and slow the localization to the rupture sites in a long-time window (60-70 min) after the induction of NE rupture. Our data could explain the defective repair of NE rupture in HGPS through the farnesylation at the CaaX motif of unprocessed progerin. In addition, unique segments in LA-specific tail region cooperate with each other to inhibit the rapid accumulation within a short-time window (3 min) that is also observed with LC. Significance StatementNuclear lamins are the major components of the nuclear lamina (NL) that lies the nuclear envelope (NE). Lamin A (LA) is slowly localized to sites of nuclear envelope (NE) rupture compared to lamin C (LC). This study reveals that the farnesylation at the CaaX motif of unprocessed LA and the inhibition of the second proteolytic cleavage decrease the nucleoplasmic pool and slow the localization to the rupture sites within a long-time window (60-70 min) after the induction of NE rupture, which could explain the defective repair of NE rupture in Hutchinson-Gilford Progeria syndrome (HGPS). Additionally, unique segments in LA-specific tail region are critical for inhibiting the rapid accumulation within a short-time window (3 min).

cell biology↗

bioRxiv2023

Automated escape system: identifying prey's kinematic and behavioral features critical for predator evasion

O_LIIdentifying the kinematic and behavioral variables of prey that influence the evasion from predator attacks is essential not only for comprehending the determinants of successful predator evasion but also for shedding light on the evolution of specific traits and the dynamics of predator-prey relationships on a larger scale. However, quantifying the relationship between these variables and the success or failure of predator evasion is challenging, particularly for variables with small variations within prey species. One promising approach to address this challenge is the use of a simulated prey system, which allows us to manipulate the kinematic and behavioral features of prey and expose them to real predators. Nevertheless, creating a system that moves comparably to real prey animals remains difficult, especially for invertebrate and lower vertebrate species that respond quickly to predators and escape rapidly. C_LIO_LIIn this study, we have developed an automated escape system that is comparable to real prey species, responding to a predator within tens of milliseconds and escaping at over 1.0 m/s. The system automatically detects an approaching predator and pulls the prey away from the predator once the predator reaches a predetermined threshold distance. Reaction distance, response latency, as well as escaping speed, duration, and direction can be adjusted in the system. C_LIO_LIBy repeatedly measuring the response latency and escaping speed of the system, we demonstrated the systems ability to exhibit fast and rapid responses while maintaining consistency across successive trials. As a case study, we manipulated the escape speed and reaction distance of the prey to expose them to a predatory fish, Coreoperca kawamebari. The results show that both variables significantly affect the outcome of predator-prey interactions. C_LIO_LIThese findings indicate that the developed escape system is useful for identifying kinematic and behavioral features of prey that are critical for predator evasion. Moreover, due to its relatively low cost and customizability, we propose that this system can be applied to investigate various aspects of animal behaviors (e.g., eliciting escape responses by artificial stimuli) in different animal species. C_LI

animal behavior and cognition↗

bioRxiv2023

Histone methyltransferase SUV39H1 regulates the Golgi complex via the nuclear envelope-spanning LINC complex

Cell motility is related to the higher-order structure of chromatin. Stimuli that induce cell migration change chromatin organization; such stimuli include elevated histone H3 lysine 9 trimethylation (H3K9me3). We previously showed that depletion of histone H3 lysine 9 methyltransferase, SUV39H1, suppresses directional cell migration. However, the molecular mechanism underlying this association between chromatin and cell migration remains elusive. The Golgi apparatus is a cell organelle essential for cell motility. In this study, we show that loss of H3K9 methyltransferase SUV39H1 but not SETDB1 or SETDB2 causes dispersion of the Golgi apparatus throughout the cytoplasm. The Golgi dispersion triggered by SUV39H1 depletion is independent of transcription, centrosomes, and microtubule organization, but is suppressed by depletion of any of the following three proteins: LINC complex components SUN2, nesprin-2, or microtubule plus-end-directed kinesin-like protein KIF20A. In addition, SUN2 is closely localized to H3K9me3, and SUV39H1 affects the mobility of SUN2 in the nuclear envelope. Further, inhibition of cell motility caused by SUV39H1 depletion is restored by suppression of SUN2, nesprin-2, or KIF20A. In summary, these results show the functional association between chromatin organization and cell motility via the Golgi organization regulated by the LINC complex.

cell biology↗

bioRxiv2023

Live-cell imaging uncovers the relationship between histone acetylation, transcription initiation, and nucleosome mobility

Post-translational protein modifications play an important role in the regulation of gene dynamics. Certain modifications, such as histone acetylation and RNA polymerase II phosphorylation, are associated with transcriptionally active chromatin. However, the spatial and temporal relationship between chromatin and post-translational protein modifications, and how these dynamics facilitate selective gene expression, remain poorly understood. In this study, we address this problem by developing a general methodology for quantifying in live cells the dynamics of chromatin across multiple time and length scales in the context of residue-specific protein modifications. By combining Fab-based labeling of endogenous protein modifications with single-molecule imaging, we track the dynamics of chromatin enriched with histone H3 Lysine-27 acetylation (H3K27ac) and RNA polymerase II Serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals chromatin enriched with H3K27ac is separated from chromatin enriched with RNAP2-Ser5ph. Furthermore, in these separated sites, we show the presence of the two modifications are inversely correlated with one another on the minutes timescale. We then track single nucleosomes in both types of sites on the sub-second timescale and again find evidence for distinct and opposing changes in their diffusive behavior. While nucleosomes diffuse [~]15% faster in chromatin enriched with H3K27ac, they diffuse [~]15% slower in chromatin enriched with RNAP2-Ser5ph. Taken together, these results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each modification marks distinct sites that are transcriptionally poised or active, respectively.

molecular biology↗

bioRxiv2022

Targeted disruption of transcription bodies causes widespread activation of transcription

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how they affect gene expression. Here, we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-Seq and live-cell imaging. We find that the disruption of transcription bodies results in downregulation of hundreds of genes, providing experimental support for a model in which transcription bodies increase the efficiency of transcription. We also find that a significant number of genes are upregulated, counter to the suggested stimulatory effect of transcription bodies. These upregulated genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that the disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9, the kinase that releases paused polymerase II, is highly enriched in the two large transcription bodies. Importantly, overexpression of CDK9 in wild type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription genome-wide: the accumulation of transcriptional machinery creates a favourable environment for transcription locally, while depriving genes elsewhere in the nucleus from the same machinery.

cell biology↗

bioRxiv2022

Actin filaments accumulate in the nucleus during interphase and remain in the vicinity of condensing chromosomes during prophase to metaphase in zebrafish early embryos

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 -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. Summary statementFilamentous actin accumulates in the nucleus of zebrafish early embryos and forms patches associating with condensing chromosomes during prophase.

cell biology↗

bioRxiv2022

Live-Cell Tracking of γ-H2AX Kinetics Reveals the Distinct Modes of ATM and DNA-PK in Immediate Response to DNA Damage

DNA double-strand break (DSB) is a serious form of DNA damage that can cause genetic mutation. On the induction of DSBs, histone H2AX becomes phosphorylated by kinases, including ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). Phosphorylated H2AX ({gamma}-H2AX) can be a platform to recruit DNA repair machinery. Here we analyzed the immediate early kinetics of {gamma}-H2AX upon laser-induced DNA damage in ATM-proficient and -deficient living cells by using fluorescently labeled antigen-binding fragments specific for {gamma}-H2AX. The accumulation kinetics of {gamma}-H2AX were similar in both ATM-proficient and -deficient cells. However, {gamma}-H2AX accumulation was delayed when the cells were treated with a DNA-PK inhibitor, suggesting that DNA-PK rapidly phosphorylates H2AX at DSB sites. Ku80, a DNA-PK subunit, diffused freely in the nucleus without DNA damage, whereas ATM repeatedly bound to and dissociated from chromatin. The H2AX phosphorylation activity of ATM, but not DNA-PK, depended on a histone H4K16 acetyltransferase, males absent on the first (MOF). These results suggest distinct actions of ATM and DNA-PK that plays a primary role in immediate early {gamma}-H2AX accumulation.

cell biology↗

bioRxiv2022

Nanog organizes transcription bodies

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis, to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription, and independently of RNA accumulation. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies. HIGHLIGHTSO_LITranscription factors cluster prior to, and independently of transcription C_LIO_LINanog organizes transcription bodies: it is required for the clustering of Sox19b as well as RNA polymerase II C_LIO_LIThis organizing activity requires its DNA binding domain as well as one of its intrinsically disordered regions C_LIO_LITranscription elongation results in the disassembly of transcription factor clusters C_LI

cell biology↗

bioRxiv2022

Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

molecular biology↗

bioRxiv2022

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

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.

molecular biology↗

bioRxiv2022

Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

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 lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). 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 repair. SummaryKono et al. show the rapid recruitment of nucleoplasmic lamin C to sites of nuclear envelope rupture with Barrier-to-autointegration factor. Lamin A/C is also involved in nuclear DNA sensing with cytoplasmic cGAS at the ruptured sites.

cell biology↗

bioRxiv2021

Fractal scaling of C. elegans behavior is shaped by insulin signaling

Fractal scaling governs the complex behavior of various animal species and, in humans, can be altered by neurodegenerative diseases and aging1. However, the mechanism underlying fractal scaling remains unknown. Here, we videorecorded C. elegans that had been cultured in a microfluidic device for 3 days and analyzed temporal patterns of C. elegans actions by fractal analyses. The residence-time distribution of C. elegans shared a common feature with those of human and mice2-4. Specifically, the residence-time power-law distribution of the active state changed to an exponential-like decline at a longer time scale, whereas this change did not occur in the inactive state. The exponential-like decline disappeared in starved C. elegans but was restored by culturing animals with glucose. The exponential-like decline similarly disappeared in insulin-signaling daf-2 and daf-16 mutants. Therefore, we conclude that insulin signaling regulates fractal scaling of C. elegans behavior. Our findings indicate that neurosensory modulation of C. elegans behavior by insulin signaling is achieved by regulation of fractal scaling. In humans, diabetes mellitus is associated with depression, bipolar disorder, and anxiety disorder5, which affect daily behavioral activities. We hypothesize that comorbid behavioral defects in patients with diabetes may be attributed to altered fractal scaling of human behavior.

animal behavior and cognition↗

bioRxiv2021

Single-cell profiling of transcriptome and histone modifications with EpiDamID

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, including various combinations of transcriptome, genome and epigenome. However, comprehensive profiling of the histone post-translational modifications that influence gene expression at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of genetically engineered proteins. By fusing Dam to single-chain variable fragment antibodies, engineered chromatin reader domains, or endogenous chromatin-binding proteins, we render the DamID technology and all its implementations compatible with the genome-wide identification of histone post-translational modifications. Importantly, this enables the joint analysis of chromatin marks and transcriptome in a variety of biological systems at the single-cell level. In this study, we use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. We further demonstrate the applicability of this method to in vivo systems by mapping H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specifically in the notochord. Overall, EpiDamID is a new addition to a vast existing toolbox for obtaining systematic insights into the role of chromatin states during dynamic cellular processes.

genomics↗