Biology subjects

Sakata, Y.

Publications and source records attributed to Sakata, Y..

12 recordsLinked to original sources

bioRxiv2025

A Conserved Stress-Inducible Hub Enhancer Governs Natriuretic Peptide Expression and Undergoes Dynamic and Reversible Activation in Human Heart Failure

BackgroundNatriuretic peptides (NPs), encoded by the NPPA and NPPB genes, serve as both diagnostic biomarkers and cardioprotective hormones in heart failure. Their expression is tightly regulated by mechanical load, yet the upstream enhancer mechanisms translating hemodynamic stress into transcriptional activation remain incompletely understood. MethodsWe investigated the Nppa/Nppb super-enhancer (SE), which resides in a well-insulated topologically associating domain (TAD) and regulates stress-inducible expression of Nppa and Nppb. We applied CRISPR-based enhancer perturbation, chromatin accessibility and histone profiling, chromatin conformation assays, genetic mouse models, human iPSC-derived cardiomyocytes, and paired failing human hearts before and after left ventricular assist device (LVAD) unloading. ResultsThrough integrative epigenomic and genetic analyses, we identified a conserved element, CR9, as a dominant hub enhancer within this SE. In neonatal rat cardiomyocytes, CRISPR-based activation and inhibition established that CR9 was both necessary and sufficient for NP induction, whereas neighboring elements (CR7/CR8) displayed modest activity but synergized with CR9, establishing a hierarchical and cooperative SE architecture. In vivo, CR9 deletion in mice markedly suppressed Nppa/Nppb expression and diminished chromatin accessibility and histone acetylation across adjacent enhancers and promoters, underscoring its structural as well as transcriptional role. Reinsertion of CR9, even in reverse orientation, restored transcription, confirming its orientation-independent activity. CR9 function remained confined within TAD boundaries, supporting a locus-specific regulation. RNAscope revealed spatial proximity between CR9 enhancer RNA and Nppa/Nppb nascent transcripts. Developmental profiling revealed a switch in enhancer usage from CR6/CR7 in the fetal heart to CR9 in adulthood, indicating a transition from developmental to stress-inducible regulation. In human induced pluripotent stem cell-derived cardiomyocytes, deletion of CR9 nearly abolished NPPA/NPPB expression, establishing its essential role in a human cellular context. Most notably, in paired human failing hearts before and after LVAD unloading, chromatin accessibility at CR9 was dynamically reversed, providing the first direct evidence that enhancer states are plastic and therapeutically modifiable in the human myocardium. ConclusionsCR9 functions as a stress-inducible hub enhancer that coordinates NP transcription under pathological load. This enhancer exhibits reversible activation in human hearts, underscoring enhancer plasticity as a modifiable regulatory layer mediating stress-responsive transcriptional adaptation in the diseased myocardium. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIWe identified CR9 as a stress-inducible hub enhancer within the Nppa/Nppb super-enhancer that is both necessary and sufficient for natriuretic peptide induction, defining a hierarchical and cooperative regulatory architecture controlling Nppa and Nppb expression. C_LIO_LIWe uncovered a developmental switch in enhancer usage from CR6 in the fetal heart to CR9 in adulthood, revealing how stress-inducible enhancer activity emerges as the heart transitions from developmental to adaptive regulation. C_LIO_LIUsing paired failing human hearts before and after LVAD unloading, we provide the first direct evidence that enhancer states are dynamically reversible in the human myocardium. C_LI What are the clinical implications?O_LIThe reversibility of CR9 activity in human heart failure provides a molecular basis for how natriuretic peptide levels decrease when cardiac function improves with effective therapy, reflecting the underlying enhancer plasticity of the diseasesd myocardium. C_LIO_LIEnhancer plasticity is an emerging and targetable mechanism in heart failure, raising the possibility that stress-inducible enhancers like CR9 could be engineered to switch on cardioprotective genes when the heart is under stress, offering a new strategy for precision gene therapy. C_LI

molecular biology↗

bioRxiv2024

The Rubicon-WIPI axis regulates exosome biogenesis during aging

Cells release intraluminal vesicles (ILVs) in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNAi screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for ILV formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for the age-dependent changes in exosome quantity and quality.

cell biology↗

bioRxiv2024

Induction of the aggresome and insoluble tau aggregation using an optogenetic tool

Tauopathy is a spectrum of diseases characterized by fibrillary tau aggregate formation in neurons and glial cells in the brain. Tau aggregation originates in the brainstem and entorhinal cortex and then spreads throughout the brain in Alzheimers disease (AD), which is the most prevalent type of tauopathy. Understanding the mechanism by which locally developed tau pathology propagates throughout the brain is crucial for comprehending AD pathogenesis. Therefore, a novel model of tau pathology that artificially induces tau aggregation in targeted cells at specific times is essential. This study describes a novel optogenetic module, OptoTau, which is a human tau with the P301L mutation fused with a photosensitive protein CRY2olig, inducing various forms of tau according to the temporal pattern of blue light illumination pattern. Continuous blue light illumination for 12 h to Neuro2a cells that stably express OptoTau (OptoTauKI cells) formed clusters along microtubules, many of which eventually accumulated in aggresomes. Conversely, methanol-resistant tau aggregation was formed when alternating light exposure and darkness in 30-min cycles for 8 sets per day were repeated over 8 days. Methanol-resistant tau was induced more rapidly by repeating 5-min illumination followed by 25-min darkness over 24 h. These results indicate that OptoTau induced various tau aggregation stages based on the temporal pattern of blue light exposure. Thus, this technique exhibits potential as a novel approach to developing specific tau aggregation in targeted cells at desired time points. SignificanceThis study developed an approach to manipulate tau aggregation in a blue light-dependent manner using cells that stably express OptoTau, which is an optogenetic tool based on the CRY2olig module. Tau accumulation in aggresomes or stable tau aggregation were selectively induced by blue light illumination conditions. These results are crucial as they provide a new technological basis for establishing a singular point of tau aggregation in specific targeted cells at a particular time.

biophysics↗

bioRxiv2024

Compromised actin dynamics underlie the orofacial cleft in Baraitser-Winter Cerebrofrontofacial Syndrome with a variant in ACTB

Craniofacial anomalies encompassing the orofacial cleft are associated with >30% of systemic congenital malformations. Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF) is a rare genetic disorder attributed to variants in the actin beta (ACTB) or actin gamma genes that are correlated with a range of craniofacial abnormalities, including cleft lip and/or palate. The underlying pathological mechanism of BWCFF remains elusive, and it is necessary to investigate the etiology of orofacial clefts in BWCFF. In this study, we identified a missense variant (c.1043C>T: p.S348L) in the ACTB gene from a patient with BWCFF and concomitant cleft lip and palate. Furthermore, we performed functional assessments of this variant using various disease models such as the MDCK cell line and Xenopus laevis. These models revealed a compromised capacity of mutated ACTB to localize at the epithelial junction, consequently affecting the behavior of epithelial cells. Additionally, we made the noteworthy discovery that mutated ACTB exhibited an impaired ability to bind PROFILIN1, a critical factor in actin polymerization. This defective ability may contribute to the molecular etiology of aberrant epithelial cell adhesion and migration, resulting in orofacial cleft formation in BWCFF. Author SummaryAdvances in clinical sequencing have rapidly elucidated the genetic basis of rare diseases affecting multiple organs. In this study, we investigate the role of a variant in the ACTB gene, which is fundamental to cell structure, in causing orofacial clefts in the Baraitser-Winter Cerebrofrontofacial Syndrome (BWCFF), known for its distinctive facial anomalies. Using epithelial cell lines and frog embryo models, we investigated the effects of the ACTB gene alteration on cellular functions. Our results indicate that this genetic change disrupts the normal adhesion and movement of epithelial cells, processes that are critical for facial development. This research underscores the importance of the ACTB gene in shaping the face and suggests new directions for future research, potentially informing the diagnosis and treatment of BWCFF and related conditions.

genetics↗

bioRxiv2022

Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

Gene therapy for hemophilia using adeno-associated virus (AAV) vectors allows long-term coagulation factor expression. We examined the potential of a novel engineered liver-tropic AAV3B-based vector AAV.GT5 for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, while in vivo transduction efficacy into the liver and the increase in coagulation factor IX (FIX) antigen following intravenous injection of these vectors were similar in PXB mice (chimeric mice with a humanized liver) and macaques. The discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the original AAV3B. The intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced vector transduction into the liver and reduced vector distribution to the kidney in pigs. In macaques, the intra-hepatic artery injection of AAV.GT5 yielded a comparable increase in FIX antigen with a one-third dosage of peripheral venous administration. Two of four macaques who received AAV.GT5 intravenously did not develop neutralizing antibodies (NAbs) against AAV.GT5, while AAV-Spark100 induced serotype-specific NAbs in all four macaques. The NAb produced after the administration was relatively specific to the serotype and less responsive to the other serotype. As a result, the administration of AAV.GT5 successfully boosted FIX expression in one animal previously given AAV-Spark100. Thus, AAV.GT5 has different biodistribution and immunogenic characteristics compared with AAV-Spark100, and the intra-hepatic vascular administration may lessen the vector dose and avoid vector distribution to other organs. Key PointsO_LIThe AAV.GT5 vector has a strong transduction efficacy in human hepatocytes but has a faster clearance after systemic administration. C_LIO_LIIntra-hepatic vascular administration of the AAV.GT5 vector is an effective liver transduction method for hemophilia gene therapy. C_LI

genetics↗

bioRxiv2022

Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells

The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we faithfully recapitulate human fetal adrenal cortex specification processes through stepwise induction of human induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenal progenitor-like states to ultimately generate fetal zone adrenal cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent {Delta}5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN and WNT signaling, and that steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of {Delta}5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro paving the way for cell-based therapies of adrenal insufficiency.

developmental biology↗

bioRxiv2022

Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and other parameters. Here, we developed an assay system to dynamically measure second harmonic generation (SHG) polarization in pulsating cardiomyocyte and proved that the SHG anisotropy derived from myosin filaments in disease-model cardiomyocytes depended on their crossbridge status, providing an evaluation method for myosin force generation. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost force-generating ability upon myocardial differentiation, causing acquired dysfunction. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, along with the establishment of the methodology, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on living cardiomyocytes.

biophysics↗

bioRxiv2022

The developmental origin and the specification of the adrenal cortex in humans and cynomolgus monkeys

Development of the adrenal cortex, a vital endocrine organ, originates in the adrenogonadal primordium, a common progenitor for both the adrenocortical and gonadal lineages in rodents. In contrast, we find that in humans and cynomolgus monkeys, the adrenocortical lineage originates in a temporally and spatially distinct fashion from the gonadal lineage, arising earlier and more anteriorly within the coelomic epithelium. The adrenal primordium arises from adrenogenic coelomic epithelium via an epithelial-to- mesenchymal-like transition, which then progresses into the steroidogenic fetal zone via both direct and indirect routes. Notably, we find that adrenocortical and gonadal lineages exhibit distinct HOX codes, suggesting distinct anterior-posterior regionalization. Together, our assessment of the early divergence of these lineages provides a molecular framework for understanding human adrenal and gonadal disorders. One Sentence SummarySpecification of the adrenal cortex occurs in adrenogenic coelomic epithelium independent of gonadogenesis in humans and cynomolgus monkeys

developmental biology↗

bioRxiv2020

Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells

Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we established a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells (hPGCLCs) differentiated from human induced pluripotent stem cells were further induced into M-prospermatogonia-like cells (MLCs) and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing was used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibited gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enabled us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.

developmental biology↗

bioRxiv2020

Activation of SnRK2 by the Raf-like kinase ARK represents a common mechanism of ABA response in embryophytes

The Raf-like protein kinase ARK previously identified in the moss Physcomitrella patens acts as an upstream regulator of subgroup III SnRK2, the key regulator of abscisic acid (ABA) and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2 including the role of ABA receptor-associated group A PP2C (PP2C-A) are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in wild type. The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking PP2C-A was similar to that in wild type, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. These results indicate that ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototypal mechanism for ABA and abiotic stress-responses in embryophytes. One sentence summaryPhysiological characterization of various moss mutants revealed a common mechanism for phytohormone responses under water deficit in all land plants.

plant biology↗

bioRxiv2019

Population-specific and transethnic genome-wide analyses reveal distinct and shared genetic risks of coronary artery disease

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study (GWAS) of 168,228 Japanese (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,782 CAD cases and 3,148 controls. We detected 9 novel disease-susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a transethnic meta-analysis and discovered 37 additional novel loci. Using the result of the meta-analysis, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European GWAS. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improves clinical characterization of CAD genetics and suggests the utility of transethnic meta-analysis for PRS derivation in non-European populations.

genetics↗

bioRxiv2019

Transethnic meta-analysis of genome-wide association studies identifies three new loci and characterizes population-specific differences for coronary artery disease

BackgroundGenome-wide association studies (GWAS) provided many biological insights into coronary artery disease (CAD), but these studies were mainly performed in Europeans. GWAS in diverse populations have the potential to advance our understanding of CAD.\n\nMethods and ResultsWe conducted two GWAS for CAD in the Japanese population, which included 12,494 cases and 28,879 controls, and 2,808 cases and 7,261 controls, respectively. Then, we performed transethnic meta-analysis using the results of the CARDIoGRAMplusC4D 1000 Genomes meta-analysis with UK Biobank. We identified 3 new loci on chromosome 1q21 (CTSS), 10q26 (WDR11-FGFR2), and 11q22 (RDX-FDX1). Quantitative trait locus analyses suggested the association of CTSS and RDX-FDX1 with atherosclerotic immune cells. Tissue/cell type enrichment analysis showed the involvement of arteries, adrenal glands and fat tissues in the development of CAD. Finally, we performed tissue/cell type enrichment analysis using East Asian-frequent and European-frequent variants according to the risk allele frequencies, and identified significant enrichment of adrenal glands in the East Asian-frequent group while the enrichment of arteries and fat tissues was found in the European-frequent group. These findings indicate biological differences in CAD susceptibility between Japanese and Europeans.\n\nConclusionsWe identified 3 new loci for CAD and highlighted the genetic differences between the Japanese and European populations. Moreover, our transethnic analyses showed both shared and unique genetic architectures between the Japanese and Europeans. While most of the underlying genetic bases for CAD are shared, further analyses in diverse populations will be needed to elucidate variations fully.

genetics↗