Biology subjects

Honda, M.

Publications and source records attributed to Honda, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanisms of fork stalling and reversal at pathogenic CANVAS (AAGGG)n repeats

Expansions of the pathogenic (AAGGG)n repeat within the RFC1 gene cause the hereditary disease CANVAS. We have shown that this repeat impedes DNA replication both in vitro and in vivo. Here, we investigated the mechanisms of replication fork stalling and reversal at (AAGGG)n repeats within an SV40-based episome in human cells. A comprehensive 2-dimensional electrophoretic analysis of replication intermediates revealed a strict orientation dependence of the replication block, evident only when the homopurine sequence was in the lagging-strand template. Fork accumulation was further increased in the presence of the G4-DNA stabilizer pyridostatin, implicating G4-DNA in the process. This stalling was followed by an accumulation of reversed forks and further analysis revealed roles for SMARCAL1 and RAD51 in this process. Biochemical studies showed that SMARCAL1, with assistance from RPA, efficiently remodels a fork carrying the G4-forming AAGGG repeat in the ssDNA gap, but not a substrate carrying the repeat in triplex conformation. We conclude that stalling results from G4-DNA formation by the repeat, which then triggers SMARCAL1-mediated fork reversal at the repeat. Given the mirror symmetry of the sequence, the nascent strands at the reversed forks likely adopt a triplex conformation, leading to a thorough accumulation of branched structures.

molecular biology↗

bioRxiv2026

Natriuretic Peptide Augmentation Attenuates Renin Cell Hyperactivation and Afferent Arteriolar Hypertrophy During Long-Term Renin-Angiotensin System Inhibition

BACKGROUNDChronic renin-angiotensin system (RAS) inhibition activates renin cells and induces afferent arteriolar hypertrophy, a maladaptive vascular response that may contribute to nephrosclerosis-like renal injury. Although genetic or cell ablation approaches have shown that renin cells are required for this remodeling, no pharmacological strategy to restrain hyperactivated renin cells while preserving RAS inhibition benefits has been established. Natriuretic peptides (NPs) counteract RAS; however, whether NP signaling modulates renin cell activation and afferent arteriolar remodeling remains unclear. METHODSWe examined direct effects of atrial natriuretic peptide (ANP) on As4.1 renin-producing cells using reverse transcription-quantitative PCR, ELISA, and RNA sequencing (RNA-seq). We established a mouse model of long-term RAS inhibition using valsartan, an angiotensin II receptor blocker (ARB), and compared it with sacubitril/valsartan, an angiotensin receptor-neprilysin inhibitor (ARNI). Valsartan was matched between the ARB and ARNI groups. Renin cell activation, afferent arteriolar remodeling, and renal injury were evaluated using biochemical assays, histology, immunostaining, single-nucleus RNA-seq, and region-specific photo-isolation chemistry RNA-seq of afferent arteriolar/juxtaglomerular regions. RESULTSANP suppressed Ren1 expression and renin secretion in As4.1 cells; this effect was attenuated by a natriuretic peptide receptor A antagonist. RNA-seq demonstrated that ANP induced receptor-dependent remodeling of renin cell gene programs. In mice, long-term ARB treatment induced renin cell hyperactivation, expansion of renin-positive juxtaglomerular regions, afferent arteriolar hypertrophy, renal dysfunction, tubular injury markers, and fibrosis. ARNI increased plasma ANP levels and attenuated these pathological changes, despite a comparable blood pressure reduction. Single-nucleus transcriptomics revealed attenuation of tubular injury-associated cellular states and altered renin cell-associated mesenchymal programs with ARNI. Region-specific transcriptomics further demonstrated distinct molecular states in afferent arteriolar/juxtaglomerular regions between ARB- and ARNI-treated kidneys. Integrated transcriptomic analysis suggested that NP signaling converges on vascular regulatory programs in hyperactivated renin-expressing cells. CONCLUSIONSNP signaling acts as a pharmacologically augmentable modulator of maladaptive renin cell activation. ARNI attenuates renin cell hyperactivation, afferent arteriolar hypertrophy, and renal injury during long-term RAS inhibition, suggesting that neprilysin inhibition may preserve RAS blockade benefits while limiting renin cell-driven renal vascular remodeling.

physiology↗

bioRxiv2025

The heterochronic activation of TGF-β signaling drives the diversity of the avian sterna

Adaptive evolution has driven the diversification of vertebrate skeletal morphology, enabling a wide array of locomotor modes and lifestyles. In volant birds (carinates), the sternum develops a ventral keel that provides an attachment site for massive pectoral muscles essential for powered flight. In contrast, flightless ratites have lost both flight capability and the keel, resulting in a striking morphological contrast between their sterna. Here, we interrogated the cellular and molecular basis underlying this morphological divergence by using chicken embryos as a carinate model and emu embryos as a ratite model. Through a series of analyses including spatiotemporal transcriptomics and a spheroid culture system, we found that TGF-{beta} signaling, which promotes proliferation of ventral sternal chondroprogenitors, is activated in both species until the stage when the left and right sternal progenitors meet; however, in chicken this activation persists beyond this stage, driving ventral extension of the keel primordium, whereas in emu it shuts off early, culminating in the absence of a protruding keel. Together, our findings suggest that skeletal morphological changes associated with behavioral transitions can arise from heterochrony in developmental signaling, thereby deepening our understanding of the evolutionary logic shaping skeletal diversification.

developmental biology↗

bioRxiv2025

Passive and active demethylation in immune diseases with proliferation driving global hypomethylation

Global DNA hypomethylation is a hallmark of immune-mediated diseases, yet its regulatory significance remains unclear. Replication-associated loss of DNA methylation at solo-WCGW (W = A/T) CpGs has recently been proposed as a consequence of cell division. Here we systematically investigated genome-wide hypomethylation patterns across seven immune-mediated diseases. Most diseases exhibited global hypomethylation, particularly at solo-WCGW CpGs in transcriptionally repressed regions, potentially reflecting increased immune cell proliferation. By contrast, CpG sites whose methylation levels were associated with cytokine exposure or SNP genotypes were predominantly located in transcriptionally active regions. To investigate whether immunological events driving immune cell proliferation may also be imprinted in transcriptionally active regions, we searched for differentially methylated regions (DMRs) correlated with an index reflecting the extent of solo-WCGW hypomethylation. In narcolepsy, we identified a DMR within the T-cell receptor alpha (TRA) locus, and greater hypomethylation was associated with increased clonality of both TRA and TRB repertoires, with a similar pattern in an independent cohort. In multiple sclerosis, a DMR was also detected within the IGH locus encoding the B-cell receptor. Together, these findings suggest that the hypomethylation index captures the impact of disease-specific immune dynamics, while reflecting a shared epigenetic signature of immune cell proliferation across diseases.

genomics↗

bioRxiv2024

The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51 presynaptic filaments

Human RAD52 is a prime target for synthetical lethality approaches to treat cancers with deficiency in homologous recombination. Among multiple cellular roles of RAD52, its functions in homologous recombination repair and protection of stalled replication forks appear to substitute those of the tumor suppressor protein BRCA2. However, the mechanistic details of how RAD52 can substitute BRCA2 functions are only beginning to emerge. RAD52 forms an undecameric ring that is enveloped by eleven [~]200 residue-long disordered regions, making it a highly multivalent and branched protein complex that potentiates supramolecular assembly. Here, we show that RAD52 exhibits homotypic phase separation capacity, and its condensates recruit key players in homologous recombination such as single-stranded (ss)DNA, RPA, and the RAD51 recombinase. Moreover, we show that RAD52 phase separation is regulated by its interaction partners such as ssDNA and RPA. Using fluorescence microscopy, we show that RAD52 can induce the formation of RAD51-ssDNA fibrillar structures. To probe the fine structure of these fibrils, we utilized single-molecule super-resolution imaging via DNA-PAINT and atomic force microscopy and showed that RAD51 fibrils are bundles of individual RAD51 nucleoprotein filaments. We further show that RAD52 induces end-to-end tethering of RAD51 nucleoprotein filaments. Overall, we demonstrate unique macromolecular organizational features of RAD52 that may underlie its various functions in the cell.

biophysics↗

bioRxiv2024

Heterogeneous Appetite Patterns in Depression: Computational Modeling of Nutritional Interoception, Reward Processing, and Decision-Making

Accurate interoceptive processing in decision-making is essential to maintain homeostasis and overall health. Disruptions in this process have been associated with various psychiatric conditions, including depression. Recent studies have focused on nutrient homeostatic dysregulation in depression for effective subtype classification and treatment. Neurophysiological studies have associated changes in appetite in depression with altered activation of the mesolimbic dopamine system and interoceptive regions, such as the insular cortex, suggesting that disruptions in reward processing and interoception drive changes in nutrient homeostasis and appetite. This study aimed to explore the potential of computational psychiatry in addressing these issues. Using a homeostatic reinforcement learning model formalizing the link between internal states and behavioral control, we investigated the mechanisms by which altered interoception affects homeostatic behavior and reward system activity via simulation experiments. Simulations of altered interoception demonstrated behaviors similar to those of depression subtypes, such as appetite dysregulation. Specifically, reduced interoception led to decreased reward system activity and increased punishment, mirroring the neuroimaging study findings of decreased appetite in depression. Conversely, increased interoception was associated with heightened reward activity and impaired goal-directed behavior, reflecting an increased appetite. Furthermore, effects of interoception manipulation were compared with traditional reinforcement learning parameters (e.g., inverse temperature {beta} and delay discount{gamma} ), which represent cognitive-behavioral features of depression. The results suggest that disruptions in these parameters contribute to depressive symptoms by affecting the underlying homeostatic regulation. Overall, this study findings emphasize the importance of integrating interoception and homeostasis into decision-making frameworks to enhance subtype classification and facilitate the development of effective therapeutic strategies.

systems biology↗

bioRxiv2024

Multiomics analysis of narcolepsy T cells: global hypomethylation in solo-WCGW motif linked to T cell proliferation

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a loss of orexin-producing cells in the brain and involves autoimmune mechanisms, including the presence of autoreactive T cells. We performed a genome-wide DNA methylation analysis using CD4+ and CD8+ T cells of NT1 patients. Analysis of differentially methylated regions as well as multiomics analysis with genomic and transcriptomic data obtained from the same samples indicated that cell chemotaxis pathways are implicated as a cause in the pathogenesis of NT1. Additionally, we found global hypomethylation in both the T cells of NT1 cases (CD4+: P = 1.69E-67; CD8+: P = 4.83E-12). These NT1-associated hypomethylated sites were significantly more abundant in solo-WCGW (sequences without neighboring CpGs, where W is an A or T base; P = 9.87E-194). Solo-WCGW tends to lose DNA methylation over the course of cell divisions, suggesting enhanced T cell proliferation in NT1.

genomics↗

bioRxiv2023

A double-ring of human RAD52 remodels replication forks restricting fork reversal

Human RAD521,2 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation3-7. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL15. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1. One Sentence SummaryUsing cryo-EM, biochemical and single-molecule approaches we show that the structure of stalled DNA replication fork promotes a unique two-ring organization of human RAD52 protein which remodels the fork via DNA strand exchange.

biochemistry↗

bioRxiv2023

Atf3 controls transitioning in female mitochondrial cardiomyopathy as identified by single-cell transcriptomics

Oxidative phosphorylation defects results in mitochondrial diseases, with cardiac involvement markedly impacting prognosis. However, the mechanisms underlying the transition from compensation to dysfunction in response to metabolic deficiency remain unclear, impeding the development of effective treatments. Here, we employed single-nucleus RNA sequencing (snRNA-seq) on hearts from mitochondrial cardiomyopathy (MCM) mice with cardiac-specific Ndufs6 knockdown (FS6KD). Pseudotime trajectory analysis of cardiomyocytes from early stage of female FS6KD hearts revealed dynamic cellular state transitioning from compensation to severe compromise, coincided with transient upregulation of a critical transcription factor, activating transcription factor 3 (Atf3). Genetic ablation or adeno-associated virus-mediated Atf3 knockdown in FS6KD mice effectively delayed cardiomyopathy progression in a female-specific manner. Notably, human MCM snRNA-seq revealed a similar transition, including the dynamic expression of ATF3. In conclusion, our findings highlight a fate-determining role of Atf3 in female MCM progression, providing a promising therapeutic candidate for the currently intractable disease.

cell biology↗

bioRxiv2023

Contrasting roles of different mismatch repair proteins in basal-like breast cancer

The mismatch repair (MMR) pathway is known as a tumor suppressive pathway and genes involved in MMR are commonly mutated in hereditary colorectal or other cancer types. However, the function of MMR genes/proteins in breast cancer progression and metastasis are largely undefined. We found that MSH2, but not MLH1, is highly enriched in basal-like breast cancer (BLBC) and that its protein expression is inversely correlated with overall survival time (OS). MSH2 expression is frequently elevated due to genomic amplification or gain-of-expression in BLBC, which results in increased MSH2 protein to pair with MSH6 (collectively referred to as MutS). Genetic deletion of MSH2 or MLH1 results in a contrasting phenotype in metastasis, with MSH2-deletion leading to reduced metastasis and MLH1-deletion to enhanced liver or lung metastasis. Mechanistically, MSH2 - but not MLH1 - binds to the promoter region of interferon receptor 1 (IFNAR1) and suppresses its expression in BLBC. Deletion of MSH2 initiates a chain of immune reactions via the upregulation of IFNAR1 expression and the activation of type 1 interferon signaling, which explains a highly immune active tumor microenvironment in tumors with MSH2-deficiency. Our study supports the contrasting functions of MSH2 and MLH1 in BLBC progression and metastasis due to the differential regulation of IFNAR1 expression, which challenges the paradigm of the MMR pathway as a universal tumor suppressive mechanism.

cancer biology↗

bioRxiv2023

Identification of Region-Specific Gene Isoforms in the Human Brain Using Long-Read Transcriptome Sequencing and Their Correlation with DNA Methylation

BackgroundSite specificity is known in neuropsychiatric disorders, and differences in gene expression patterns could potentially explain this mechanism. However, studies using long-read transcriptome sequencing to analyze gene expression in different regions of the human brain have been limited, and none have focused on the hypothalamus, which plays a crucial role in regulating autonomic functions. ResultsWe performed long-read RNA sequencing on 12 samples derived from three different brain regions of the same individuals; the cerebellum, hypothalamus, and temporal cortex. We found that, compared to other regions, many genes with higher expression levels in the cerebellum and temporal cortex were associated with neuronal pathways, whereas those with higher expression levels in the hypothalamus were primarily linked to immune pathways. In addition, we investigated genes with different major isoforms in each brain region, even with similar overall expression levels among regions, and identified several genes, such as GAS7, that express different major isoforms in different regions. Many of these genes are involved in "actin filament-based process" and "cell projection organization" pathways, suggesting that region-dependent isoforms may have distinct roles in dendritic spine and neuronal formation in each region. Furthermore, we investigated the involvement of DNA methylation in these isoforms and found that DNA methylation may be associated with isoforms that have different first exons. ConclusionsOur results provide potentially valuable findings for future research on brain disorders and shed light on the mechanisms underlying isoform diversity in the human brain.

genomics↗

bioRxiv2023

Human hnRNPA1 reorganizes telomere-bound Replication Protein A

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence SummaryAt the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

biophysics↗

bioRxiv2023

Single-molecule analysis of the improved variants of the G-quadruplex recognition protein G4P

As many as 700,000 unique sequences in the human genome are predicted to fold into G-quadruplexes (G4s), non-canonical structures formed by Hoogsteen guanine-guanine pairing within G-rich nucleic acids. G4s play both physiological and pathological roles in many vital cellular processes including DNA replication, DNA repair and RNA transcription. Several reagents have been developed to visualize G4s in vitro and in cells. Recently, Zhen et al. synthesized a small protein G4P based on the G4 recognition motif from RHAU (DHX36) helicase (RHAU specific motif, RSM). G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity towards G4s than the previously published BG4 antibody. To get insight into the G4P-G4 interaction kinetics and selectivity, we purified G4P and its expanded variants, and analyzed their G4 binding using single-molecule total internal reflection fluorescence microscopy and mass photometry. We found that G4P binds to various G4s with affinities defined mostly by the association rate. Doubling the number of the RSM units in the G4P increases the proteins affinity for telomeric G4s and its ability to interact with sequences folding into multiple G4s.

biophysics↗

bioRxiv2021

Narcolepsy with cataplexy is caused by epigenetic silencing of hypocretin neurons

Narcolepsy with cataplexy is a chronic sleep disorder characterized by hypocretin deficiency. The condition is believed to result from autoimmune destruction of hypocretin (HCRT) neurons, although direct evidence is lacking and mere Hcrt gene inactivation causes full-blown narcolepsy in mice. Here we show that the expression of another hypothalamic neuropeptide, QRFP, is lost in mouse models with HCRT cell-ablation, but tends to be even increased in Hcrt gene knockout mice, suggesting that QRFP expression can be used as a proxy for the presence or absence of HCRT neurons. Similar to Hcrt knockout mice, narcolepsy patients show intact hypothalamic QRFP expression, and cerebrospinal fluid levels of QRFP peptide are increased, suggesting their HCRT neurons are intact. We show that the human HCRT gene promoter is methylation-sensitive in vitro, and is hypermethylated in the hypothalamus of patients selectively at a putative PAX5:ETS1 binding site within the proximal HCRT promoter. Ets1-KO mice display downregulated Hcrt expression, while pax5-ets1 knockdown in zebrafish causes decreased hcrt expression, decreased activity and sleep fragmentation, similar to narcolepsy patients. Our results suggest that HCRT neurons are alive, but epigenetically silenced, in the hypothalamus of narcolepsy patients, opening the possibility to reverse or cure narcolepsy.

neuroscience↗

bioRxiv2021

Don't think, feel: basal ganglia-cortical connectivity underlies self-regulation of brain oscillations in humans

Brain-computer interfaces (BCIs) provide an artificial link by which the brain can directly interact with the environment. To achieve fine BCI control, participants must modulate the patterns of the cortical oscillations generated from the motor and somatosensory cortices. However, it remains unclear how humans regulate cortical oscillations, the controllability of which substantially varies across individuals. Here, we performed simultaneous electroencephalography (to assess BCI control) and functional magnetic resonance imaging (to measure brain activity) in healthy participants. Self-regulation of cortical oscillations induced activity in the basal ganglia-cortical network (BgCN) and the neurofeedback control network (NfCN). Successful self-regulation correlated with striatal activity in the BgCN, through which patterns of cortical oscillations were likely modulated. Moreover, BgCN and NfCN connectivity correlated with strong and weak self-regulation, respectively. The findings indicate that the BgCN is important for self-regulation, the understanding of which should help advance BCI technology.

neuroscience↗

bioRxiv2020

Three-dimensional convolutional autoencoder extracts features of structural brain images with a diagnostic label-free approach: Application to schizophrenia datasets

There has been increasing interest in performing psychiatric brain imaging studies using deep learning. However, most studies in this field disregard three-dimensional (3D) spatial information and targeted disease discrimination, without considering the genetic and clinical heterogeneity of psychiatric disorders. The purpose of this study was to investigate the efficacy of a 3D convolutional autoencoder (CAE) for extracting features related to psychiatric disorders without diagnostic labels. The network was trained using a Kyoto University dataset including 82 patients with schizophrenia (SZ) and 90 healthy subjects (HS), and was evaluated using Center for Biomedical Research Excellence (COBRE) datasets including 71 SZ patients and 71 HS. The proposed 3D-CAEs were successfully reconstructed into high-resolution 3D structural magnetic resonance imaging (MRI) scans with sufficiently low errors. In addition, the features extracted using 3D-CAE retained the relevant clinical information. We explored the appropriate hyper parameter range of 3D-CAE, and it was suggested that a model with eight convolution layers might be relevant to extract features for predicting the dose of medication and symptom severity in schizophrenia.

neuroscience↗

bioRxiv2020

Deep Feature Extraction for Resting-State Functional MRI by Self-Supervised Learning and Application to Schizophrenia Diagnosis

In this study, we propose a novel deep-learning technique for functional MRI analysis. We introduced an "identity feature" by a self-supervised learning schema, in which a neural network is trained solely based on the MRI-scans; furthermore, training does not require any explicit labels. The proposed method demonstrated that each temporal slice of resting state functional MRI contains enough information to identify the subject. The network learned a feature space in which the features were clustered per subject for the test data as well as for the training data; this is unlike the features extracted by conventional methods including region of interests pooling signals and principle component analysis. In addition, using a simple linear classifier for the identity features, we demonstrated that the extracted features could contribute to schizophrenia diagnosis. The classification accuracy of our identity features was higher than that of the conventional functional connectivity. Our results suggested that our proposed training scheme of the neural network captured brain functioning related to the diagnosis of psychiatric disorders as well as the identity of the subject. Our results together highlight the validity of our proposed technique as a design for self-supervised learning.

neuroscience↗

bioRxiv2020

High resolution spatial transcriptome analysis by photo-isolation chemistry

In multicellular organisms, individual cells are characterized by their gene expression profiles and the spatial interactions among cells enable the elaboration of complex functions. Expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we established a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of whole tissues. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. After photo-irradiation of limited areas, gene expression was detected from at least 10 cells in the tissue sections. PIC transcriptome analysis detected genes specifically expressed in small distinct areas of the mouse embryo. Thus, PIC enables transcriptome profiles to be determined from limited regions at a spatial resolution up to the diffraction limit.

genomics↗