Biology subjects

Ochiai, H.

Publications and source records attributed to Ochiai, H..

13 recordsLinked to original sources

bioRxiv2026

Senolytic targeting of CAF-induced KRT17+ colon cancer cells inhibits metastatic invasion

Metastasis remains the leading cause of cancer mortality, yet effective strategies to eliminate metastasis-initiating cells are lacking. Here, we dissected intratumoral heterogeneity in colon cancer using single-cell analyses and identified a KRT17+ slow-cycling cancer cell population that exhibits features of metastasis-initiating cells (L1CAM) and senescent-like cells (CDKN2A, BCL2L1). Spatial transcriptomics and immunostaining revealed that these cells localize at tumor-stroma interfaces, where they are closely associated with TGF-{beta}1-producing subset of cancer-associated fibroblasts (CAFs) and exhibit SMAD3 activation. Mechanistically, TGF-{beta}1 induces KRT17 expression in patient-derived cancer cells, while co-culture with CAFs drives the emergence of KRT17 migratory cells in a KRT17-dependent manner. Functionally, genetic ablation of KRT17 or senolytic targeting of BCL2L1 suppresses peritumoral invasion and liver metastasis in xenograft models. Clinically, KRT17 cells co-localize with TGF-{beta}1 CAFs, and their co-expression with L1CAM correlates with advanced disease stage. These findings support a model in which stromal TGF-{beta} signaling promotes the emergence of a KRT17 invasive cancer cell state with senescence-associated features and suggest that senolytic strategies may represent a potential approach to limit metastatic progression in colon cancer.

cancer biology↗

bioRxiv2026

Minute-scale coupling of chromatin marks and transcriptional bursts

Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses 1-5. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes 6-10. 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 modifications11-16. 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 17,18. 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.

molecular biology↗

bioRxiv2024

MEGA-FISH: Multi-omics Extensible GPU-Accelerated FISH Processing Framework for Huge-Scale Spatial Omics

Spatial omics enables comprehensive mapping of cell types and states in their spatial context, providing profound insights into cellular communication and tissue organization. However, analyzing large tissue sections, especially crucial for clinical applications, remains a significant challenge due to the computational demands of current image processing methods. To overcome these limitations, we developed MEGA-FISH, a flexible, GPU-accelerated Python framework optimized for large-scale spatial omics image analysis. Benchmarking on simulated and tissue images demonstrated that MEGA-FISH achieved high accuracy in spot detection while significantly reducing processing times compared with established tools. The frameworks adaptable computational capabilities optimize resource allocation (e.g., GPU or multi-core CPU) for diverse tasks, and its scalable architecture enables integration with advanced imaging and segmentation techniques. By bridging cutting-edge imaging methods and single-cell analysis, MEGA-FISH provides an efficient platform for multi-modal analysis and advances research and clinical applications of spatial omics at organ and organism scales.

bioinformatics↗

bioRxiv2024

In silico nanoscope to study the interplay of genome organization and transcription regulation

In eukaryotic genomes, regulated access and communication between cis-regulatory elements (CREs) are necessary for enhancer-mediated transcription of genes. The molecular framework of the chromatin organization underlying such communication remains poorly understood. To better understand it, we develop a multiscale modeling pipeline to build near-atomistic models of the 200 kb Nanog gene locus in mouse embryonic stem cells comprising nucleosomes, transcription factors, co-activators, and RNA polymerase II-Mediator complexes. By integrating diverse experimental data, including protein localization, genomic interaction frequencies, cryo-electron microscopy, and single-molecule fluorescence studies, our model offers novel insights into chromatin organization and its role in enhancer-promoter communication. The models equilibrated by high-performance molecular dynamics simulations span a scale of [~]350 nm, revealing an experimentally consistent local and global organization of chromatin and transcriptional machinery. Our models elucidate that the sequence-regulated chromatin accessibility facilitates the recruitment of transcription regulatory proteins exclusively at CREs, guided by the contrasting nucleosome organization compared to other regions. By constructing an experimentally consistent near-atomic model of chromatin in the cellular environment, our approach provides a robust framework for future studies on nuclear compartmentalization, chromatin organization, and transcription regulation.

molecular biology↗

bioRxiv2024

Anti-phase clustering of regulatory factors shapes gene bursting

Stem cell self-renewal relies on finely tuned transcriptional oscillations of pluripotency genes, yet the sequence by which transcription factors (TFs) govern "on" and "off" states remains unclear. Here, we integrate trimodal single-molecule imaging - SOX2 mobility, Nanog locus diffusion, and real-time Nanog mRNA synthesis using the STREAMING-tag reporter - to visualise endogenous regulatory dynamics in living cells. We identify two SOX2 binding modes linked to transcriptional priming and termination and show coordinated temporal activity of SOX2, OCT4, BRD4, and MED22 at the Nanog locus. Live-cell and fixed-cell measurements reveal that local RNA fluctuations modulate SOX2 visiting-frequency rates. During the active elongation, high nascent enhancer and mRNA levels transiently sequester SOX2 through non-specific interactions, reducing its effective repetitive rebinding and limiting interference with elongating polymerase. After transcript release, declining RNA density permits SOX2 re-engagement, with high-visiting frequency with longer residency and increased locus mobility. These dynamics reveal RNA-dependent feedback that alternately buffers and primes transcriptional re-initiation.

molecular biology↗

bioRxiv2024

Inter-cellular mRNA Transfer Alters Human Pluripotent Stem Cell State

Inter-cellular transmission of mRNA is being explored in mammalian species using immortal cell lines (1-3). Here, we uncover an inter-cellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells (mESCs) under the condition impermissible for human primed PSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis show the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 days of mESC culture, surface marker analysis, and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naive-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1, Tfap2c, and Klf4 are indispensable for human naive-like conversion. Thus, inter-species mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes(4), which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and inter-species cellular communications.

cell biology↗

bioRxiv2023

Transcription-coupled changes in higher-order genomic structure and transcription hub viscosity prolong enhancer-promoter connectivity

The orchestration of our genes heavily relies on a coordinated communication between enhancers and promoters, yet how this dynamic interplay remains elusive while transcription is active. Here, we investigated enhancer-promoter (E-P) interactions and relative to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence (IF)-FISH analyses and computational simulations. Our data reveal that the active state of specific genes is characterized by higher-order genomic structures and local condensates of transcriptional regulatory factors, leading to an elevation in local viscosity that highly stabilizes the duration of E-P interactions. Our study underscores the pivotal role of viscosity in transcriptional dynamics and paves the way for a more nuanced understanding of gene-specific regulatory mechanisms.

molecular 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

Label-free ghost cytometry for manufacturing of cell therapy products

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs, T cells and non-T cells in white blood cells (WBCs), activated T cells and quiescent T cells in PBMCs, and particulate impurities in PBMCs. The data support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers, assess viability, identify specific cell subsets or phenotypic states, and remove impurities during cell therapy manufacturing. Thus, LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency.

cell 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↗

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗

bioRxiv2019

Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

Transcriptional bursting is stochastic activation and inactivation of promoters, leading to discontinuous production of mRNA, and is considered to be a contributing factor to cell-to-cell heterogeneity in gene expression. However, it remains elusive how the kinetic properties of transcriptional bursting (e.g., burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. In this study, we performed a genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs) using single-cell RNA-sequencing. We found that the kinetics of transcriptional bursting was determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors. Furthermore, large-scale CRISPR-Cas9-based screening and functional analysis revealed that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncover key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

systems biology↗

bioRxiv2019

PHi-C: deciphering Hi-C data into polymer dynamics

Computational modelling methods for Hi-C data have revealed averaged and static features of the 3D genome in cell nuclei. Here, we describe a 4D simulation method, PHi-C (Polymer dynamics deciphered from Hi-C data), that depicts dynamic 3D genome features through polymer modelling. This method allows for demonstrations of dynamic characteristics of genomic loci and chromosomes, as observed in live-cell imaging experiments, and provides physical insights into Hi-C data.

bioinformatics↗