Biology subjects

Takashima, S.

Publications and source records attributed to Takashima, S..

9 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↗

bioRxiv2025

HiFAA: a high-confidence framework for transcription factor footprinting with ATAC-seq

Accurate inference of transcription factor (TF) binding from chromatin accessibility remains a major challenge, as conventional methods often miss true binding events, yield unstable motif rankings, or fail to resolve dynamic changes. Here we present high-confidence transcription factor footprint analysis using ATAC-seq (HiFAA), a framework that integrates topologically associating domain-restricted analysis, single-base-resolution footprint construction, and footprint ranking based on footprint bottom height. HiFAA markedly improves sensitivity while maintaining high specificity, enabling detection of binding events overlooked by existing analyses. It also ranks truly bound TFs with higher motif scores, enhancing predictive accuracy, and maintains consistently high geometric mean (G-mean) values, providing a stable balance between sensitivity and specificity. Furthermore, its high-resolution footprints capture subtle structural variations, allowing precise detection of developmental stage-specific TF-binding. By uniting sensitivity, specificity, and dynamic resolution, HiFAA establishes a robust paradigm for reliable TF footprinting from ATAC-seq data.

bioinformatics↗

bioRxiv2024

Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

Epithelial cells undergo epithelial-mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal-epithelial transition; MET). We established an experimental system that reproduces a three-compartment epithelial structure comprising the original epithelium, its EMT state, and its MET state. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-{micro}m or larger micropores. The 3.0-{micro}m-pored membrane induced an epithelial structure with three distinct states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the three-compartment epithelial structure. Live imaging revealed that KCs moved in an oscillatory manner, with actin-rich filopodia-like structures extending into and out of the 3.0-{micro}m micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry into and exit from the 3.0-{micro}m micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in an oscillatory manner, which might contribute to the formation of a three-compartment epithelial structure that recapitulates key aspects of wound healing.

cell biology↗

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

Single cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identified CD271+ progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271+ progenitors demonstrated robust in vivo angiogenic capacity, over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271+ progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271+ progenitors was strikingly reduced in insulin resistant donors. Our study highlights the identification of AT-CD271+ progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy. HIGHLIGHTSO_LIAdipose tissue stromal cells have a distinct angiogenic gene profile among human cell sources. C_LIO_LICD271+ progenitors in adipose tissue have a prominent angiogenic gene profile. C_LIO_LICD271+ progenitors show superior therapeutic capacities for limb ischemia. C_LIO_LICD271+ progenitors are reduced and functionally impaired in insulin resistant donors. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/527726v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@1b5e1aorg.highwire.dtl.DTLVardef@bf2c34org.highwire.dtl.DTLVardef@ef0e68org.highwire.dtl.DTLVardef@9aee1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Myosin phosphatase target subunit 1 governs integrity of the embryonic gut epithelium to circumvent atresia development in medaka, Oryzias latipes

Congenital intestinal atresia (IA) is a birth defect characterised by the absence or closure of part of the intestine. Although genetic factors are implicated, mechanistic understanding has been hindered by the lack of suitable animal models. Here, we describe a medaka (Oryzias latipes) mutant, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, that develops IA during embryogenesis. Positional cloning identified a nonsense mutation in mypt1, encoding myosin phosphatase target subunit 1. Mutant embryos exhibited ectopic accumulation of F-actin and phosphorylated myosin regulatory light chain (Mrlc) in the intestinal epithelium, consistent with disrupted actomyosin regulation. These cytoskeletal abnormalities were accompanied by epithelial disorganisation without notable alterations in cell proliferation, motility, or apoptosis. Inhibition of myh11a, encoding smooth muscle (SM) myosin heavy chain, ameliorated the IA phenotype but Blebbistatin treatment completely rescued the defect, suggesting a non-contractile role prior to SM maturation. Together, these findings demonstrate that mypt1 loss disrupts intestinal morphogenesis through actomyosin dysregulation. Given the recent clinical identification of IA associated with MYPT1 mutations, this medaka model offers a valuable platform to investigate the developmental and molecular basis of MYPT1-associated IA in human.

developmental biology↗

bioRxiv2021

Zebrafish model of human Zellweger syndrome reveals organ specific accumulation of distinct fatty acid species and widespread gene expression changes

In Zellweger syndrome (ZS), lack of peroxisome function causes physiological and developmental abnormalities in many organs such as the brain, liver, muscles, and kidneys, but little is known about the exact pathogenic mechanism. By disrupting the zebrafish pex2 gene, we established a disease model for ZS and found that it exhibits a pathological condition and metabolic failures similar to that of human patients. By comprehensive analysis of fatty acid profile, we found organ specific accumulation and reduction of distinct fatty acid species such as an accumulation of ultra-very-long-chain polyunsturated fatty acids (ultra-VLCPUFAs) in the brain of pex2 mutant fish. Transcriptome analysis using microarray also revealed mutant-specific gene expression changes that might lead to the symptom, which include reduction of crystallin, troponin, parvalbumin, and fatty acid metabolic genes. Our data indicated that the loss of peroxisome results in widespread metabolic and gene expression changes beyond the causative peroxisomal function. These results suggest the genetic and metabolic basis of the pathology of this devastating human disease.

developmental biology↗

bioRxiv2020

Subepidermal blisters are healed by wedge-shaped keratinocytes of hair follicle origin

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that tissue development can be sacrificed for the sake of wound regeneration and suggest that tissue repair does not coincide with the reactivation of developmental programs in all regenerative contexts. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

cell biology↗

bioRxiv2019

A method to determine the double bond position in unsaturated fatty acids by solvent plasmatization using liquid chromatography-mass spectrometry

Fatty acids (FAs) are the central components of life: they constitute biological membranes in the form of lipid, act as signaling molecules, and are used as energy sources. FAs are classified according to their chain lengths and the number and position of carbon-carbon double bond, and their physiological character is largely defined by these structural properties. Determination of the precise structural properties is crucial for characterizing FAs, but pinpointing the exact position of carbon-carbon double bond in FA molecules is challenging. Herein, a new analytical method is reported for determining the double bond position of mono- and poly-unsaturated FAs using liquid chromatography-mass spectrometry (LC-MS) coupled with solvent plasmatization. With the aid of plasma on ESI capirally, epoxidation or peroxidation of carbon-carbon double bond in FAs is facilitated. Subsequently, molecular fragmentation occurs at or beside the epoxidized or peroxidized double bond via collision-induced dissociation (CID), and the position of the double bond is elucidated. In this method, FAs are separated by LC, modified by plasma, fragmented via CID, and detected using a time-of-flight mass spectrometer in a seamless manner such that the FA composition in a mixture can be determined. Our method enables thorough characterization of FA species with distinguishing multiple isomers, and therefore can uncover the true diversity of FAs for their application in food, health, and medical sciences.

biochemistry↗