Search bioRxiv⌕ Search

Biology subjects

Hiraoka, Y.

Publications and source records attributed to Hiraoka, Y..

At least 19 recordsLinked to original sources

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↗

Nardilysin fine-tunes the mammalian circadian clock through selective modulation of PER2 function

Circadian rhythms in mammals are generated by negative feedback loops, in which CLOCK and BMAL1 bind to E-box to activate transcription of Period (Per) and Cryptochrome (Cry) and the E-box-dependent transactivation is inhibited by PER and CRY proteins. Although the core transcriptional feedback loop of the circadian clock has been well defined, how this machinery interfaces with broader nuclear regulatory systems remains incompletely understood. Here, we identify nardilysin (NRDC), a metalloendopeptidase previously implicated in nuclear transcriptional regulation and metabolic homeostasis, as an unexpected modulator of the circadian clock. NRDC deficiency led to elevated PER2 protein levels in the liver and enhanced PER2 dynamics in cell-autonomous circadian oscillators, and was accompanied by a significant shortening of behavioral rhythms in mice. Biochemical analyses demonstrated that NRDC selectively associates with PER2 and CRY2 and antagonizes PER2-mediated repression of CLOCK-BMAL1-dependent transcription. Genome-wide chromatin immunoprecipitation analyses reveal that NRDC is enriched at promoter-proximal E-box-containing regions, frequently co-localizing with CLOCK binding sites. Together, these findings uncover a previously unrecognized link between circadian timing and protease-based nuclear regulation, positioning NRDC as a critical modulator of PER2 function and circadian period determination.

molecular biology↗

Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

Understanding how molecular diversity across long-range neural circuits governs brain function remains a central challenge in neuroscience. Here, we developed a projection-specific spatial proteomics approach and revealed robust presynaptic molecular divergence across six projection-defined pathways of the medial prefrontal cortex (mPFC), including efferent projections to the basolateral amygdala (BLA), nucleus accumbens (NAc), thalamus (Thal), hypothalamus (HT) and cortex (CTX), as well as the afferent projection from the BLA to the mPFC. Among these pathways, we identify BLTP2 (KIAA0100), a previously uncharacterized transmembrane protein, as highly enriched in the projection from the mPFC to the BLA. BLTP2 localizes to excitatory presynaptic terminals and is enriched at synapses that are activated during memory formation. Loss of BLTP2 impairs synaptic structure and transmission, and reduces activity-dependent remodelling, resulting in selective deficits in contextual fear memory, anxiety-related behavior, and social behavior. Mechanistically, BLTP2 promotes presynaptic assembly by recruiting Neurexin 1. These findings reveal projection-specific presynaptic molecular diversity and provide mechanistic insights into circuit-level vulnerabilities in neuropsychiatric disorders.

neuroscience↗

Single-cell RNA sequencing reveals collagen interactions between detached osteoclasts and activated fibroblasts in granulation tissue surrounding sequestra in medication-related osteonecrosis of the jaw

ObjectivesMedication-related osteonecrosis of the jaw (MRONJ) is a rare but serious complication of anti-resorptive agents used for osteoporosis and bone metastasis. As the global population ages, the number of patients with MRONJ continues to rise. Resorptive osteoclasts firmly attached to bone surfaces cannot be detected using single-cell RNA-Seq (scRNA-Seq); however, we hypothesized that multinucleated giant and detached osteoclasts (DetOCs) can be detected by this method. This study aimed to characterize DetOCs with scRNA-seq. Materials and MethodsscRNA-Seq and immunohistochemistry staining was performed on granulation tissues surrounding sequestra from four patients with MRONJ and radicular cysts, a common odontogenic infectious disease with normal bone metabolism. ResultsscRNA-Seq and immunohistochemistry staining revealed that DetOCs were detected exclusively in the granulation tissues of MRONJ. These DetOCs expressed well-known osteoclast markers as well as COL27A1. Subcluster analysis revealed that SPP1+ TREM2+ macrophages differentiated into DetOCs and expressed several types of collagens. Immunostaining confirmed the expression of COL27A1 not only in DetOCs in MRONJ but also in osteoclasts in mandibular cancer. CellChat analysis revealed interactions mediated by collagen signaling pathways between DetOCs, myeloid cells, and activated fibroblasts. ConclusionsWe successfully characterized DetOCs and identified a distinctive microenvironment involving DetOCs and activated fibroblasts in the granulation tissues surrounding sequestra in MRONJ. Clinical RelevanceIdentification of DetOCs and their unique interaction with activated fibroblasts in MRONJ lesions provides novel insight into the pathophysiology of sequestrum formation, suggesting potential therapeutic targets to ameliorate MRONJ.

immunology↗

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↗

Functional and structural profiling of circulation via genetically encoded modular fluorescent probes.

Sustained labeling of fluids is crucial for their investigation in animal models. Here, we introduce a mouse line (Alb-mSc-ST), where blood and interstitial fluid are labeled with the red fluorescent protein mScarlet and SpyTag. The SpyTag-SpyCatcher technology is exploited to monitor circulating fluid properties by biosensors or detect blood-brain barrier disruption. This approach represents a valuable tool for studying vascular structure, permeability and microenvironment in body organs in vivo.

neuroscience↗

RTL4, a retrovirus-derived gene implicated in autism spectrum disorder, is a microglial gene that responds to noradrenaline in the postnatal brain

RTL4, a gene acquired from a retrovirus, is a causative gene in autism spectrum disorder. Its KO mice exhibit increased impulsivity, impaired short-term spatial memory, failure to adapt to novel environments, and delayed noradrenaline (NA) recovery in the frontal cortex. However, due to its very low expression in the brain, it remains unknown which brain cells express RTL4 and its dynamics in relation to NA. Here, using knock-in mice carrying endogenous Rtl4 fused to Venus, we demonstrated that RTL4 is a microglial gene with several important properties. The RTL4-Venus fusion protein was detected as a secreted protein in the midbrain, hypothalamus, hippocampus and amygdala in the postnatal brain. Its signal intensity was high during critical periods of neonatal adaptation to novel environments and was upregulated by various stimuli, such as handling the mice, mild environmental changes and administration of isoproterenol, an agonist of adrenergic beta receptors. It was decreased by anesthesia but was maintained by the administration of milnacipran, an NA reuptake inhibitor. These results suggest that RTL4 intensity depends on the arousal state via NA and is somehow related to the NA reuptake process. In vitro mixed glial culture experiments demonstrated that Rtl4 is a microglial gene and suggested that RTL4 secretion responds rapidly to isoproterenol. Taken together with previous results from Rtl4 KO mice, microglial RTL4 plays an important role in the NA response and it is likely involved in the development of the NAergic neuronal network in the brain.

molecular biology↗

Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes

The cell nucleus is a dynamic structure repeating disassembly and reformation during mitosis. Reformation of the nucleus is essential for cell proliferation, and therefore, the factors required for nuclear reformation are fundamental for eukaryotes. Although various factors have been identified in in vitro systems using frog egg extracts and in vivo imaging of somatic cells, little is known about the factors required for the formation of functional nuclear structures in living mouse eggs. To identify such factors, we used a reconstruction approach to construct an artificial nucleus around DNA in mouse eggs. T4 phage DNA (166 kbp) was microinjected into living mouse oocytes. Amounts of DNA injected and injection timing were examined to determine the conditions appropriate for the formation of functional nuclei. Microinjection of 100-500 ng/{micro}l DNA during metaphase through telophase of the second meiosis, but not the subsequent interphase, was important for the formation of artificial nucleus. This T4 DNA-derived artificial nucleus had the structure of nuclear lamina and nuclear pore complexes, and nuclear transport activity, similar to natural nuclei. These results suggest that exogenous DNA can form a functional nucleus in mouse oocytes, regardless of the sequence or the source of DNA.

cell biology↗

Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

DNA replication is tightly regulated to occur only once per cell cycle, as untimely re-initiation can lead to aneuploidy, which is associated with early senescence and cancer. The pre-replication complex (comprising Orc1-6, Cdc6, Cdt1, and MCM) is essential for the initiation of DNA replication, but the dynamics and function of Orc6 during the cell cycle remain elusive. Here, we demonstrate that Orc6 associates with chromatin during G1-phase and dissociates upon S-phase entry. The dissociation of Orc6 from chromatin is dependent on proteasome activity, and inhibition of the proteasome leads to the accumulation of chromatin-bound Orc6, which promotes abnormal MCM loading after S-phase entry without undergoing mitosis in human immortalized hTERT-RPE1 cells. Following release from proteasome inhibition, cells with elevated levels of chromatin-bound Orc6 and MCM proceed to the next replication phase as tetraploid cells. Our findings suggest that the proteasome-dependent dissociation of Orc6 after DNA replication is critical for preventing inappropriate MCM reloading and tetraploid formation.

cell biology↗

The mechanism of homologous chromosome recognition and pairing facilitated by chromosome-tethered protein-RNA condensates

Pairing of homologous chromosomes during meiosis is crucial for successful sexual reproduction. Previous studies have shown that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 locus and plays a key role in mediating robust pairing during meiosis. Several RNA-binding proteins accumulate at the sme2 and other lncRNA gene loci in conjunction with the lncRNAs transcribed from these loci. These lncRNA-protein complexes form condensates that exhibit phase separation properties on chromosomes and are necessary for robust pairing of homologous chromosomes. To further understand the mechanisms by which phase separation affects homologous chromosome pairing, we conducted an in vitro phase separation assay with the sme2 RNA-associated proteins (Smps) and RNAs. Our research has revealed that one of the Smps, Seb1, exhibits phase separation, which is enhanced by the addition of another Smp, Rhn1, and significantly increased by the addition of purified RNAs. Additionally, we have found that RNAs protect Smp condensates from treatment with 1,6-hexanediol. The Smp condensates containing different types of RNA display distinct FRAP profiles, and the Smp condensates containing the same type of RNA tend to fuse together more efficiently than those containing different types of RNA. Taken together, these results indicate that the RNA species in condensates determine their physical properties and suggest that regional RNA-Smp condensates with distinct properties ensure the pairing of homologous chromosomes.

molecular biology↗

Nuclear membrane protein Bqt4 maintains nuclear envelope integrity by recruiting phosphatidic acid

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

cell biology↗

scEGOT: Single-cell trajectory inference framework based on entropic Gaussian mixture optimal transport

Time-series scRNA-seq data have opened a door to elucidate cell differentiation, and in this context, the optimal transport theory has been attracting much attention. However, there remain critical issues in interpretability and computational cost. We present scEGOT, a comprehensive framework for single-cell trajectory inference, as a generative model with high interpretability and low computational cost. Applied to the human primordial germ cell-like cell (PGCLC) induction system, scEGOT identified the PGCLC progenitor population and bifurcation time of segregation. Our analysis shows TFAP2A is insufficient for identifying PGCLC progenitors, requiring NKX1-2. Additionally, MESP1 and GATA6 are also crucial for PGCLC/somatic cell segregation. These findings shed light on the mechanism that segregates PGCLC from somatic lineages. Notably, not limited to scRNA-seq, scEGOTs versatility can extend to general single-cell data like scATAC-seq, and hence has the potential to revolutionize our understanding of such datasets and, thereby also, developmental biology.

bioinformatics↗

Retrovirus-derived RTL9 plays an important role in innate antifungal immunity in the eutherian brain

Retrotransposon Gag-like (RTL) genes plays a variety of essential/important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (aka sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brains innate immunity against viruses and bacteria by their removal of double-stranded RNA and lipopolysaccharide, respectively. Here we demonstrate that RTL9 (aka SIRH10) also plays an important role, degrading fungal zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Interestingly, in Rtl9 knockout mice expressing RTL9{Delta}C protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost, demonstrating that RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system, RTL9, RTL5 and RTL6 responding to fungi, viruses and bacteria, respectively. Author SummaryWe have recently demonstrated that RTL5 and RTL6 are microglial genes that play important roles in the brains innate immunity against viruses and bacteria. In this report, we demonstrate that RTL9 is functional in innate antifungal immunity in the brain because Rtl9 KO mice lose zymosan degradation activity. Fungi are one of the most dangerous infectious pathogens, along with viruses, bacteria and protozoa. Phagocytic cells, such as microglia/macrophages, are essential for mounting a defense against fungal infection. As defects in these cells reduce host resistance to fungal infection, RTL9 is an antifungal therapy target as a newly identified member of innate antifungal immunity in eutherians.

evolutionary biology↗

A ubiquitin-proteasome pathway degrades the inner nuclear membrane protein Bqt4 to maintain nuclear membrane homeostasis

Aberrant accumulation of inner nuclear membrane (INM) proteins is associated with deformed nuclear morphology and mammalian diseases. However, the mechanisms underlying the maintenance of INM homeostasis remain poorly understood. In this study, we explored the degradation mechanisms of the INM protein, Bqt4, in the fission yeast Schizosaccharomyces pombe. We have previously shown that Bqt4 interacts with the transmembrane protein Bqt3 at the INM and is degraded in the absence of Bqt3. Here, we revealed that excess Bqt4, unassociated with Bqt3, was targeted for degradation by the ubiquitin-proteasome system localized in the nucleus and Bqt3 antagonized this process. The degradation process involved the Doa10 E3 ligase complex at the INM. Bqt4 is a tail-anchored protein and extraction from the membrane by the Cdc48 complex is required for its degradation. The C-terminal transmembrane domain of Bqt4 is necessary and sufficient for proteasome-dependent protein degradation. Accumulation of Bqt4 at the INM impaired cell viability with nuclear envelope deformation, suggesting that quantity control of Bqt4 plays an important role in nuclear membrane homeostasis. Summary statement Appropriate levels of the inner nuclear membrane protein Bqt4 are maintained by ubiquitin-proteasome-mediated degradation. Aberrant accumulation of Bqt4 disturbs nuclear membrane homeostasis.

cell biology↗

Resolution of the curse of dimensionality in single-cell RNA sequencing data analysis

Single-cell RNA sequencing (scRNA-seq) can determine gene expression in numerous individual cells simultaneously, promoting progress in the biomedical sciences. However, scRNA-seq data are high-dimensional with substantial technical noise, including dropouts. During analysis of scRNA-seq data, such noise engenders a statistical problem known as the curse of dimensionality (COD). Based on high-dimensional statistics, we herein formulate a noise reduction method, RECODE (resolution of the curse of dimensionality), for high-dimensional data with random sampling noise. We show that RECODE consistently eliminates COD in relevant scRNA-seq data with unique molecular identifiers. RECODE does not involve dimension reduction and recovers expression values for all genes, including lowly expressed genes, realizing precise delineation of cell-fate transitions and identification of rare cells with all gene information. Compared to other representative imputation methods, RECODE employs different principles and exhibits superior overall performance in cell-clustering and single-cell level analysis. The RECODE algorithm is parameter-free, data-driven, deterministic, and high-speed, and notably, its applicability can be predicted based on the variance normalization performance. We propose RECODE as a general strategy for preprocessing noisy high-dimensional data.

bioinformatics↗

Retrovirus-derived acquired genes, RTL5 and RTL6, are novel constituents of the innate immune system in the eutherian brain.

Retrotransposon Gag-like 5 (RTL5, also known as sushi-ichi-related retrotransposon homolog 8 (SIRH8)) and RTL6 (aka SIRH3) are eutherian-specific genes presumably derived from a retrovirus and phylogenetically related to each other. RTL5 encodes a strongly acidic protein while RTL6 encodes an extremely basic protein, and the former is well conserved and the latter extremely well conserved among the eutherians, indicating their unique and critically important roles as acquired genes. Here we report that RTL5 and RTL6 are microglial genes playing roles in the front line of brain innate immune responses against distinct pathogens. Venus and mCherry knock-in mice exhibited expression of RTL5-mCherry and RTL6-Venus fusion proteins in microglia and as extracellular granules in the central nervus system (CNS), and displayed a rapid response to pathogens such as lipopolysaccharide (LPS), double-stranded (ds) RNA analog and non-methylated CpG DNA. These proteins trapped pathogens in microglia in a variety of RTL-pathogen complexes depending on the pathogens. These results demonstrate that RTL5 and RTL6 exert functional effects against different hazardous substances cooperatively and/or independently to protect the developing and/or mature brain. This provides the first evidence that retrovirus-derived genes play a role in the innate immune system of the eutherian brain.

evolutionary biology↗

Rec8 cohesin-mediated loop-axis chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

cell biology↗

The inner nuclear membrane protein Lem2 coordinates RNA degradation at the nuclear periphery

Transcriptionally silent chromatin often localizes to the nuclear periphery. However, whether the nuclear envelope (NE) is a site for post-transcriptional gene repression is unknown. Here we demonstrate that S. pombe Lem2, an NE protein, regulates nuclear exosome-mediated RNA degradation. Lem2 deletion causes accumulation of non-coding RNAs and meiotic transcripts. Indeed, an engineered exosome substrate RNA shows Lem2-dependent localization to the nuclear periphery. Lem2 does not directly bind RNA, but instead physically interacts with the exosome-targeting MTREC complex and promotes RNA recruitment. The Lem2-assisted pathway acts independently of nuclear bodies where exosome factors assemble, revealing that multiple spatially distinct degradation pathways exist. The Lem2 pathway is environmentally responsive: nutrient availability modulates Lem2 regulation of meiotic transcripts. Our data indicate that Lem2 recruits exosome co-factors to the nuclear periphery to coordinate RNA surveillance and regulates transcripts during the mitosis-to-meiosis switch.

molecular biology↗