Biology subjects

Sanbonmatsu, K.

Publications and source records attributed to Sanbonmatsu, K..

5 recordsLinked to original sources

bioRxiv2025

AI-Assisted Cryo-ET Workflow for 3D Visualization of Chromatin during Cellular Differentiation

Understanding how chromatin architecture changes during cellular differentiation requires structural methods that can resolve native genomic organization at high resolution. Here, we present an AI-assisted cryo-electron tomography (cryo-ET) and segmentation workflow to quantify chromatin compaction across various stages of motor neuron differentiation from induced pluripotent stem cells (iPSC). By directly imaging extracted and vitrified chromatin, we preserve native structure and avoid artifacts from heavy metal staining and resin embedding. Using three-dimensional (3D) density analysis, we measure chromatin density and capture the progressive increase in chromatin compaction with lineage commitment. This is then correlated with population-averaged Hi-C experiments, observing consistency between the microscale higher order structure of chromatin and global contact patterns. Our approach enables direct visualization of chromatin organization under near-physiological conditions, bridging the gap between structural imaging and genome-wide contact mapping. This platform therefore establishes an AI-assisted experimental framework for linking chromatin architecture to regulatory mechanisms during differentiation.

genomics↗

bioRxiv2025

From 2D to 4D: a Containerized Workflow and Browser to Explore Dynamic Chromatin Architecture

BackgroundCharacterizing the physical organization of the genome is essential for understanding long-range gene reg- ulation, chromatin compartmentalization, and epigenetic accessibility. Hi-C experiments generate two- dimensional (2D) genome-wide contact maps of chromatin interactions by capturing the spatial proximity between genomic loci, which reveal interaction frequencies but lack the spatial resolution needed to interpret the three-dimensional (3D) genome structure(s). Emerging evidence suggests that epigenetic regulation is closely linked to 3D genome architecture, and that structural changes over time (4D) drive key biological pro- cesses in development, disease, and environmental response. Thus, integrating 3D structure with functional data is critical for a more complete understanding of genome regulation. Previous work, most notably the 4DHiC chromosome modeling framework, has shown that physical multi-dimensional modeling approaches rooted in polymer physics and molecular dynamics can resolve these structures at biologically meaningful resolutions by integrating temporal Hi-C data with physical constraints to uncover dynamic chromosome reorganization. Thus, molecular dynamics simulations, constrained by Hi-C contact matrices, can resolve fine-scale structural changes and reveal functionally significant transitions in chromatin conformation. ResultsHerein, we present the 4D Genome Browser Workflow (4DGBWorkflow) and the 4D Genome Browser (4DGB). The algorithm is based on the 4DHiC method and the containerized tool is an end-to-end workflow that can transform, filter, and view 4D epigenomics and chromatin datasets, allowing non-specialists to apply three-dimensional modeling principles to diverse datasets and experimental conditions. The software executes on a laptop running macOS, Linux or Windows. From input Hi-C files (.hic), the 4DGBWorkflow produces 3D reconstructions of chromosomes, integrates the reconstruction with track data (e.g., epigenetic marks, transcriptome profiles), and provides comparative visualization of the results in a single workflow. ConclusionsThe 4DGBWorkflow and 4D Genome Browser are open-source tools for comparative analysis and visual- ization of 4D chromosome datasets, including chromatin architecture and epigenomic signals. Automatic integration of Hi-C data with molecular dynamics democratizes the construction of time resolved 3D genome structures, simplifying complex simulations and data integration schemes.

bioinformatics↗

bioRxiv2025

Human Protein Synthesis Requires aminoacyl-tRNA Pivoting During Proofreading

Rigorous studies have characterized the aa-tRNA selection mechanism in bacteria, which is essential for maintaining translational fidelity. Recent investigations have identified critical distinctions in humans, such as the requirement of subunit rolling and a tenfold slower proofreading step. Although these studies captured key intermediates involved in tRNA selection, they did not elucidate the transitions of aa-tRNA between intermediates. Here, we simulated 1,856 aa-tRNA accommodation events into the human ribosomal A site, revealing the requirement of a distinct [~]30{degrees} pivoting of aa-tRNA about the anticodon stem within the accommodation corridor. This pivoting is crucial for navigating the crowded accommodation corridor, which becomes more constrained due to subunit rolling. Subunit rolling-dependent crowding increases the steric contributions of the accommodation corridor during aa-tRNA accommodation, consistent with the 10-fold reduction in the rate of proofreading. The pivoting of the aa-tRNA enables precise alignment within the accommodation corridor, allowing it to traverse the narrower passage. Furthermore, we found that domain III of eEF1A interacts with the accommodating aa-tRNA through conserved basic residues, providing a steric block to prevent dissociation from the A site. Together, these findings provide a structural framework for understanding the distinctions between bacterial and human aa-tRNA selection and demonstrate that the alignment of the aa-tRNA relative to the ribosomal catalytic sites is a critical determinant of translational fidelity.

biophysics↗

bioRxiv2024

Cleavage region organizes the structural architecture of the B2 SINE ribozyme

The SINE-encoded B2 retrotransposon is an RNA Polymerase III transcript that gets upregulated during various cellular stress responses. The B2 noncoding RNA can directly inhibit RNA Polymerase II, leading to a significant downregulation of transcripts during stress. Our recent findings have shown that B2 is a self-cleaving epigenetic ribozyme and that cleavage can be induced by interactions with epigenetic factors, co-regulating its function across distinct chromatin-binding target loci. Here, by integrating RNA chemical probing, small angle X-ray scattering, and 3D motif modeling, we determine structural ensemble-to-function relations for the B2 SINE ribozyme RNA. Perturbations of the RNA suggest that the B2 SINE ribozyme has a well-defined secondary structure and dynamic tertiary structure that both critically depend on the presence of the active site of cleavage. In an RNA engineering approach, we examine the effect of point mutations, deletions of the main cleavage site and deletions of the cleavage domain on the structural ensemble of the RNA. By combining this with functional data, we obtain relations of structural ensembles to various functional states. This perturbative approach serves as a template to unravel the relation of structural ensembles to functional states for other ncRNA and mRNA systems.

biophysics↗

bioRxiv2021

4D chromosome reconstruction elucidates the spatial reorganization of the mammalian X-chromosome

Chromosomes are segmented into domains and compartments; yet, how these structures are spatially related in 3D is unclear. Here, by directly integrating Hi-C capture experiments and 3D modeling, we use X-inactivation as a model to examine the time evolution of 3D chromosome architecture during substantial changes in gene expression. First, we show that gene expression A/B compartments are consistent with phase separation in 3D space. Second, we show that residuals of smaller scale structures persist through transitions, despite further large-scale reorganization into the final inactive configuration, comprising two "megadomains". Interestingly, these previously hidden residual structures were not detectable in 2D Hi-C maps or principal component analyses. Third, time-dependent reaction-diffusion simulations reveal how Xist RNA particles diffuse across the 3D X-superstructure as it reorganizes. Our 4DHiC pipeline helps satisfy the growing demand for methodologies that produce 3D chromosome reconstructions directly from 2D datasets, which are consistent with the empirical data.

biophysics↗