Biology subjects

Nagano, M.

Publications and source records attributed to Nagano, M..

12 recordsLinked to original sources

bioRxiv2026

Systematic mapping of enhancers, silencers, and topological elements with prime editing deletion screens

Noncoding regulatory elements in the human genome can control gene expression through distinct mechanisms, including acting as enhancers, silencers, or topological elements. However, we lack tools to identify all such classes systematically and quantify their effects on gene expression in an endogenous genomic context. Here we develop Swap-seq, a method that combines pooled twin prime editing with flow-sorting to delete hundreds of endogenous regulatory sequences and measure their quantitative effects on the expression of a nearby gene. We apply Swap-seq to dissect the regulatory elements that control PPIF expression in THP-1 monocytes. We quantify the effects of distal and intronic enhancers, characterize CTCF insulators, and discover a silencer that regulates distal genes through histone deacetylase (HDAC)-mediated repression of neighboring enhancers. Hundreds of other genomic elements share its chromatin signatures and transcription factor binding sites, suggesting that this silencer represents a broader class of repressive elements. We leverage Swap-seq data to benchmark sequence-to-function models and show that they often capture the local activity of regulatory elements but fail to link them to distal target genes. Swap-seq thus provides a generalizable tool to discover regulatory elements across mechanistic classes, characterize their effects on gene expression, and generate large-scale datasets for evaluating sequence-to-function models.

genomics↗

bioRxiv2026

CycloCross: Adversarial single-cell RNA-seq data translation across species

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular heterogeneity, yet most datasets are limited to a handful of model organisms, leaving critical gaps in cross-species biology. Existing computational methods for integrating multi-species scRNA-seq data often rely on one-to-one ortholog mapping, which fails to account for gene duplications, losses, or functional divergences. To address these challenges, we introduce CycloCross, an adversarial method based on the CycleGAN architecture, designed to translate scRNA-seq data between species without requiring a priori gene correspondences. Unlike generative models that synthesize data from noise, CycloCross leverages real data from a source species, enabling more realistic and data-efficient translations. We demonstrate its effectiveness in germ cell development, translating data between mouse, macaque, and human with conserved yet divergent transcriptional programs. CycloCross not only preserves temporal dynamics but also enables interpolation and extrapolation of gene expression at missing time points, offering predictions for experimentally inaccessible states. Furthermore, CycloCross generates biologically plausible samples even with limited data, outperforming traditional generative models.

bioinformatics↗

bioRxiv2026

Cohesin loading at regulatory elements shapes 3D genome folding during erythropoiesis

During erythropoiesis, differentiating cells silence much of the genome while maintaining high expression of select genes. It remains unclear whether this transcriptional specialization is linked to specific changes in 3D genome organization. We generated deep Micro-C maps across human erythropoiesis and identified erythroid cis-regulatory elements (CREs) that function as "matchmakers" by strengthening loops between neighboring elements. Matchmakers are associated with high H3K4me1, increase in strength during differentiation, and are linked to the expression of key erythropoiesis genes. Simulations suggest that a moderate cohesin loading bias across broad enhancer regions can explain the features of matchmakers, particularly under chromatin compaction. Targeted transcription factor and cohesin perturbations reduced matchmaker-associated structures and gene expression. We propose that matchmakers represent a subset of CREs that concentrate loop extrusion near key genes during erythroid differentiation.

genomics↗

bioRxiv2026

Structural basis for CTCF-mediated chromatin organization

Eukaryotic DNA is organized across multiple scales to support genome compaction, appropriate gene expression, and DNA recombination. A central player in these processes is the CCCTC binding factor (CTCF), which defines specific chromatin loop structures and insulates enhancer elements from promoters. Chromatin is organized in a distinct pattern around CTCF-bound sites; however, the role of this patterning remains unclear. Here, we report cryo-electron microscopy structures of reconstituted CTCF-nucleosome complexes, revealing that CTCF dimerization promotes the oligomerization of nucleosomes into defined higher-order assemblies involving specific histone-histone and CTCF-CTCF interactions. Notably, CTCF does not oligomerize efficiently on non-chromatinized DNA substrates. Disruption of identified CTCF interaction interfaces in cells results in a marked decrease in chromatin looping and functionally impairs cellular differentiation. These results indicate that chromatin structure at CTCF sites plays an important role in supporting higher-order interactions between distal regions of the genome and that these interactions are important for supporting cell-type-specific gene expression.

molecular biology↗

bioRxiv2026

Disruption of the NTCP-EGFR receptor complex as a strategy for selective inhibition of hepatitis B and D virus entry

Hepatitis B and D virus (HBV, HDV) enter hepatocytes through a coordinated process mediated by a receptor complex consisting of sodium taurocholate co transporting polypeptide (NTCP) and its entry cofactors, including epidermal growth factor receptor (EGFR). Here, we established an in vitro assay to evaluate the NTCP-EGFR interaction and identified Oxy229, an oxysterol-based compound that disrupted this molecular interaction. Oxy229 selectively inhibited HBV and HDV infection to HepG2-NTCP cells and primary human hepatocytes. Mechanistic analysis revealed that Oxy229 impaired the relocalization of the HBV-NTCP complex from plasma membrane to intracellular vesicles. Notably, Oxy229 did not compromise the physiological functions of NTCP and EGFR, i.e., bile acid transport and activation of downstream EGFR signaling pathways including Ras-MAPK and PI3K-Akt pathways, indicating selective inhibition of viral entry. Compound derivative analysis identified Oxy283, which acquired dual inhibitory activity against both NTCP-EGFR interaction and NTCP multimerization, resulting in enhanced anti-HBV potency. These findings establish the functional significance of the NTCP-receptor complex formation in HBV/HDV entry and highlight this machinery as a potential target for antiviral intervention.

microbiology↗

bioRxiv2025

Sphingolipid-driven interleaflet coupling orchestrates Rho-GTPase recruitment to nanodomains for signal activation in plants

Biological membranes are both laterally heterogeneous and asymmetrical across leaflets, yet how this asymmetry contributes to signal transduction remains unclear. Here we show that sphingolipid-driven interleaflet coupling coordinates nanodomain organization and Rho-GTPase activation in plants. Using molecular dynamics simulations, super-resolution and single-molecule imaging, quantitative genetics, and biochemistry, we find that very long acyl chain (VLCFA)-containing sphingolipids in the outer leaflet interdigitate with phosphatidylserine (PS) in the inner leaflet, forming a vertical molecular bridge that organizes PS into nanodomains. This coupling promotes recruitment and activation of the Rho-GTPase ROP6 in response to auxin, whereas disruption of VLCFA synthesis or sphingolipid composition disperses PS and ROP6 nanodomains, impairing cytoskeletal reorganization and directional growth. Our findings reveal interleaflet coupling as a fundamental organizing principle linking membrane asymmetry to signaling, providing a conceptual framework for spatial and temporal control of signal transduction across eukaryotic membranes.

plant biology↗

bioRxiv2025

Geometric inference of cross-species transcriptome correspondence using Gromov-Wasserstein optimal transport

Sequence homology reveals evolutionary gene correspondences, but identifying functionally corresponding genes within specific cell types or during cell fate specification remains non-trivial. Here, frameworks that translate insights from model organisms into other species would accelerate wet-lab studies, so we develop Species-OT, a cross-species transcriptome analysis framework using Gromov-Wasserstein optimal transport, which quantitatively compares the geometry of transcriptome distributions. Given a pair of bulk or single-cell RNA-sequencing datasets, Species-OT returns a gene-to-gene correspondence capturing probabilistic alignments of regulatory roles, and a transcriptomic distance quantifying overall divergence. Applied pairwise, Species-OT yields a transcriptomic discrepancy array and a hierarchical clustering tree analogous to a phylogenetic tree. We validate Species-OT using bulk RNA-seq data for cell fate specification from pluripotent stem cells in mice, monkeys, and humans and scRNA-seq data from pluripotent stem cells of six mammalian species. Species-OT identifies gene correspondences within cell fate specification, while transcriptomic discrepancies recapitulate expected species relationships.

bioinformatics↗

bioRxiv2023

Distinct role of TGN-resident clathrin adaptors for Rab5 activation in the TGN-endosome trafficking pathway

Clathrin-mediated vesicle trafficking plays central roles in the post-Golgi transport pathways from the trans-Golgi network (TGN) to endosomes. In yeast, two clathrin adaptors - AP-1 complex and GGA proteins (GGAs) - are predicted to generate distinct transport vesicles at the TGN, and epsin-related Ent3p/Ent5p act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN, rather than from the plasma membrane, is crucial for Rab5-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGAs are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Rab5 activity remains ambiguous. Here we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Rab5 activation. We were able to show that AP-1 disruption in ent3{Delta}/5{Delta} mutant impairs Rab5-GEF Vps9p transport to the Rab5 compartment, and severely reduces Rab5 activity. Additionally, GGAs, Golgi-resident PI4 kinase Pik1p and Rab11 GTPases Ypt31p/32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Rab5 activation in the TGN-endosome trafficking pathway.

cell biology↗

bioRxiv2022

The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment, and function independently of the Sec7p-residing region within the TGN.

cell biology↗

bioRxiv2021

Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

BackgroundEnvironmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. MethodsDNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples. ResultsNo PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

microbiology↗

bioRxiv2020

Purification, characterization and influence on membrane properties of the plant-specific sphingolipids GIPC

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment. The PM is crucial for signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols and phospholipids. The most abundant sphingolipids in the plant PM are the Glycosyl Inositol Phosphoryl Ceramides (GIPCs), representing up to 40% of total sphingolipids, assumed to be almost exclusively in the outer leaflet of the PM. In this study, we investigated the structure of GIPCs and their role in membrane organization. Since GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of different long chain bases and fatty acids. The glycan head groups of the different GIPC series from monocots and dicots were analysed by GC-MS showing different sugar moieties. Multiple biophysics tools namely Langmuir monolayer, {zeta}-Potential, light scattering, neutron reflectivity, solid state 2H-NMR and molecular modelling were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the phytosterols species and regulate the gel-to-fluid phase transition during temperature variations.

plant biology↗

bioRxiv2020

Effects of species traits and ecosystem characteristics on species detection by eDNA metabarcoding in lake fish communities

Although environmental DNA (eDNA) metabarcoding is acknowledged to be an exceptionally useful and powerful tool for monitoring surveys, it has limited applicability, particularly for nationwide surveys. To evaluate the performance of eDNA metabarcoding in broad-scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the consequences for community analysis. We conducted eDNA metabarcoding on fish communities in 18 Japanese lakes on a country-wide scale. By comparing species records, we found that certain species traits, including body size, body shape, saltwater tolerance, and habitat preferences, influenced eDNA detection. We also found that the proportion of species detected decreased significantly with an increase in lake surface area, owing to an ecosystem-size effect on species detection. We conclude that species traits, including habitat preferences and body size, and ecosystem size should be taken into consideration when assessing the performance of eDNA metabarcoding in broad-scale monitoring.

ecology↗