Biology subjects

Awazu, A.

Publications and source records attributed to Awazu, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Transcriptomic response to histone deacetylase inhibitors in cultured feline cells

ObjectiveIn recent years, the number of cats kept as companion animals has increased, leading to a growing demand for veterinary care. Although some histone deacetylase (HDAC) inhibitors are promising for the treatment of human cancers and neurological diseases, comprehensive systematic research on HDAC inhibitors in domestic cats remains insufficient. Therefore, this study aimed to investigate the effects of HDAC inhibitors on the transcriptome of feline cells. MethodsTwo types of cells derived from domestic cats, Crandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived), were treated with four HDAC inhibitors (panobinostat, trichostatin A, valproic acid, and vorinostat) for 24 h. Transcriptomic changes after treatment were examined using RNA sequencing. ResultsHDAC inhibitor treatment upregulated the expression of intercellular chemical interactions and signal transduction-related genes, similar to observations in human cells. Although HDAC inhibitors did not suppress the expression of cell cycle-related genes in CRFK cells, as observed in human cells, the inhibitors downregulated the expression of organogenesis-related genes. Consistent with observations in human cells, HDAC inhibitors suppressed the expression of cell cycle- and cancer-related genes in PG-4 cells. Importantly, valproic acid, which is thought to be more effective for neurological diseases than for cancer, suppressed the expression of more cancer-related genes in PG-4 cells than the other three HDAC inhibitors. Conclusion and relevanceOur findings revealed that the responses of cells derived from feline organs to various HDAC inhibitors varied considerably depending on the organ of origin and species. Since few studies, including human studies, have comprehensively compared transcriptomic responses to multiple HDAC inhibitor classes across multiple cell types, the results of this study provide a foundation for future research on the treatment and prevention of cancer and neurological diseases in domestic cats and other mammals.

cell biology↗

bioRxiv2025

Whole-genome sequencing of CRFK and PG-4 cells to infer the phenotype of the original donors

BackgroundCrandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived) have been in use and have been passaged for decades in laboratories worldwide; however, no detailed information on the genetic background of the donor individuals is available, particularly regarding phenotype characteristics such as coat and iris color. ResultsWe performed whole-genome sequencing of CRFK and PG-4 cells. We analyzed the resulting data to infer the phenotype of the individual from which the cells were derived, specifically for the coat color, coat length, coat pattern, and iris color. Our data suggested that CRFK cells originated from a cat with long black fur lacking stripes and with non-blue irises; PG-4 cells originated from a cat with long bicolored white and black fur without stripes, and with non-blue irises. Analysis of publicly available RNA-seq data confirmed that genes associated with coat phenotype and iris color are expressed in the skin and eyes, as well as in various other organs. ConclusionsVariants of the genes affecting coat phenotype and iris color may influence physiological functions throughout the body. These results shed light on the previously unknown genetic background of commonly used feline cultured cells and the phenotype of the donor individuals. These findings may facilitate more accurate interpretation of data obtained from cultured feline cells and provide guidelines for developing cell lines with domestic cat genotypes exhibiting diverse phenotypes through genome editing. This will help to provide clarity regarding deafness in cats with white fur and blue irises and elucidate the effects of melanocyte destruction caused by KIT gene mutations on the nervous system and the influence of other coat-related factors in organs other than the skin or functions independent of coat formation.

genomics↗

bioRxiv2025

Common tissue-specific expressions and regulatory mechanisms of c-KIT isoforms with and without GNNK and GNSK sequences across five mammals

c-KIT is a transmembrane receptor tyrosine kinase involved in various signaling pathways. Alternative pre-mRNA splicing of KIT results in isoforms that differ in the presence or absence of four amino acid sequences in the extracellular juxtamembrane region, such as, isoforms with and without the GNNK sequence (GNNK+ and GNNK-, respectively) in humans and mice, and those with and without GNSK (GNSK+ and GNSK-, respectively) in domestic dogs, cats, and sheep. These isoforms have been extensively studied as disease-associated (particularly tumors or cancer) splice variants with differing kinase activities. However, the expression patterns and regulatory factors of each isoform in various animal species without tumors or cancer remain poorly understood. Studying these aspects can provide the basis for understanding the associations between c-KIT isoforms and disease. Therefore, in the present study, a comprehensive expression analysis of c-KIT isoforms was conducted using tissue-wide transcriptome data from humans, mice, dogs, cats, and sheep. We found that the expression ratio of c-KIT isoforms differs across tissues, and such features are conserved across animal species: GNNK+ and GNSK+ isoforms have high expression ratios in the central nervous system, while GNNK- and GNSK- predominate in other tissues. Furthermore, NOVA2, RBFOX1, RBFOX3, and DYRK1A were suggested to be candidate factors regulating the selection of the alternative 5' splice donor site of KIT.

bioinformatics↗

bioRxiv2024

Multiple Alzheimer's disease progression pathways inferred from transcriptome data of the dorsolateral prefrontal cortex

Late-onset Alzheimers disease (AD) is a typical type of dementia for which therapeutic strategies have not yet been established. The database of the Rush Alzheimers Disease study by the ENCODE consortium contains transcriptome and various epigenome data. Although the Rush AD database may contain a satisfactory amount of data for women, the amount of data for men remains insufficient. Here, based on an analysis of publicly available data from female patients, this study found that AD pathology appears to be nonuniform; several different typical forms of cognitive deterioration were observed. First, cluster analysis was performed on individuals diagnosed with "No Cognitive Impairment (NCI)," "Mild Cognitive Impairment (MCI)," and "Alzheimers Disease (AD)" stages in clinical trials using gene expression. NCI was found to contain at least two substages, whereas MCI and AD contained four and six substages, respectively. The epigenome data, in particular genome-wide H3k4me3 distribution data, also supported the existence of multiple AD substages. However, APOE gene polymorphisms of individuals seemed to not correlate with disease stage. An inference of adjacency networks among substages, evaluated via partition-based graph abstraction using the gene expression profiles of individuals, suggested the possibility of multiple typical disease progression pathways from NCI to different AD substages through various MCI substages. These results will aid future research on the pathological features and mechanisms of AD and may induce changes in the strategic bases of conventional AD prevention and treatment.

bioinformatics↗

bioRxiv2024

Identification of anisotropy in chromosome dynamics by principal component analysis using integrated spatial genomics

Eukaryotic interphase chromosomes maintain a three-dimensional structure within the nucleus and undergo fluctuations. It has been reported that such dynamics are involved in transcription, replication, and DNA repair. However, the analysis of chromosomal dynamics has been limited to high-throughput chromosome conformation capture data, which records the contact frequencies between chromosomal regions and lack direct information about the dynamic. Herein, we investigated chromosome fluctuations as polymers based on experimental data from sequential fluorescence in situ hybridization (seqFISH)+ using a multiomics methodology. To describe the principal modes of chromosome fluctuations, we applied principal component analysis to the three-dimensional structure information of single chromosomes in 446 mouse embryonic stem cells (mESCs) obtained from seqFISH+ data analysis for spatial genomics and signals of nuclear factors (SNFs: histone marks, repeat DNAs, and nuclear compartments). We found that chromosome fluctuations exhibit both isotropic and anisotropic modes. The properties of anisotropy in chromosome fluctuation vary among chromosomes and appear to depend on the interaction between repeat DNAs on the chromosomes and nuclear compartments. Furthermore, our principal component analysis revealed anisotropic chromosome fluctuations before and after the mitotic phase, specifically when chromosomes adopt a spindle-like shape. This result suggests the potential involvement of anisotropic chromosomal fluctuations in the transition of nuclear organization during the cell cycle. Our results represent the first study to elucidate the dynamics of chromosomes as polymers based on real multiomics data.

bioinformatics↗

bioRxiv2023

Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided [~]600 Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5 % and 77.8 % respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcerrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of [~]100 Mb gaps, N50 = 143 kb, BUSCO completeness score = 86.1 %, and TMMR = 40.61. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using Hemicentrotus pulcherrimus.

genomics↗

bioRxiv2023

Skin coat color-dependent risks and risk factors of squamous cell carcinoma and deafness of domestic cats inferred via RNA-seq data

The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the 1st intron of KIT and its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism.

zoology↗

bioRxiv2023

Mathematical model of nuclear speckle morphology

Nuclear speckles are nuclear bodies consisting of populations of small and irregularly shaped droplet-like molecular condensates that contain various splicing factors. Recent experiments have shown the following morphological features of nuclear speckles: (I) Each molecular condensate contains SON and SRRM2 proteins, and MALAT1 non-coding RNA surrounds these condensates; (II) In the normal interphase of the cell cycle, these condensates are broadly distributed throughout the nucleus in multicellular organisms. In contrast, the fusion of condensates leads to the formation of strongly condensed spherical droplets when cell transcription is suppressed; (III) SON is dispersed spatially in MALAT1 knocked-down cells, whereas MALAT1 is dispersed in SON knocked-down cells by the collapse of nuclear speckles. However, the detailed interactions among molecules that reveal the mechanisms of this rich variety of morphologies remain unknown. In this study, a coarse-grained molecular dynamics model of the nuclear speckle was developed considering the dynamics of SON, SRRM2 or SRSF2, MALAT1, and pre-mRNA as representative components of condensates. The simulations reproduced the abovementioned morphological changes, by which the interaction strength among the representative components of the condensates was predicted.

cell biology↗

bioRxiv2022

Formation Of Small-World Network Containing Module Networks In Globally And Locally Coupled Map System With Changes In Global Connection With Time Delay Effects

In this study, we performed comprehensive morphological investigations of spontaneously formed network structures among elements in coupled map systems involving global connections that change depending on the synchronicity of states of elements and spatially local connections. The model formed various hierarchical networks, some of which were classified as small-world networks containing multiple module networks, similar to the neural network of mammalian brains. Moreover, such complex networks were formed in wider parameter regions when the global connection to an element from the other element was strengthened by the synchronization between the present and past states of the former and latter elements, respectively. This study suggests that the time delay effects for connection changed among elements and local interactions promoted the self-organization of small-world networks containing module networks, such as neural networks; neural networks contain them as spike-timing-dependent plasticity and inter-neuron interaction through glial cells.

biophysics↗

bioRxiv2022

Detailed profiling with MaChIAto reveals various genomic and epigenomic features affecting the efficacy of knock-out, short homology-based knock-in and Prime Editing

Highly efficient gene knock-out and knock-in have been achieved by harnessing CRISPR-Cas9 and its advanced technologies such as Prime Editor. In addition, various bioinformatics resources have become available to quantify and qualify the efficiency and accuracy of CRISPR edits, which significantly increased the user-friendliness of the general next-generation sequencing (NGS) analysis in the context of genome editing. However, there is no specialized and integrated software for investigating the preference in the genomic context involved in the efficiency and accuracy of genome editing using CRISPR-Cas9 and beyond. Here, we address this issue by establishing a novel analysis platform of NGS data for profiling the outcome of template-free knock- out and short homology-based editing, named MaChIAto (Microhomology- associated Chromosomal Integration/editing Analysis tools) (https://github.com/KazukiNakamae/MaChIAto). MaChIAto accommodates the classification and profiling of the NGS reads to uncover the tendency of the corresponding method of genome editing. In the profiling function, MaChIAto can summarize the mutation patterns along with the editing efficiency, and > 70 kinds of feature analysis, e.g., correlation analysis with thermodynamics and secondary structure parameters, are available. Additionally, the classifying function of MaChIAto is based on, but much stricter than, that of the existing tool, which is achieved by implementing a novel method of parameter adaptation utilizing Bayesian optimization. To demonstrate the functionality of MaChIAto, we analyzed the NGS data of knock- out, short homology-based knock-in, and Prime Editing. We confirmed that some features of (epi-)genomic context affected the efficiency and accuracy. These results show that MaChIAto is a helpful tool for understanding the best design for CRISPR edits. More importantly, it is the first tool for discovering features in the short homology-based knock-in outcomes. MaChIAto would help researchers profile editing data and generate prediction models for CRISPR edits, further contributing to revealing a "black box" process to produce a variety of CRISPR and Prime Editing outcomes.

bioinformatics↗

bioRxiv2022

Mathematical model of chromosomal dynamics during DNA double strand break repair in budding yeast

During the repair of double-strand breaks (DSBs) in DNA, active mobilizations for conformational changes in chromosomes have been widely observed in eukaryotes, from yeast to animal and plant cells. DSB-damaged loci in the yeast genome showed increased mobility and relocation to the nuclear periphery. However, the driving forces behind DSB-induced chromatin dynamics remain unclear. In this study, mathematical models of normal and DSB-damaged yeast chromosomes were developed to simulate their structural dynamics. The effects of histone degradation in the whole nucleus and the change in the physical properties of damaged loci due to the binding of SUMOylated repair proteins were considered in the model of DSB-induced chromosomes based on recent experimental results. The simulation results reproduced DSB-induced changes to structural and dynamical features by which the combination of whole nuclear histone degradation and the rigid structure formation of repair protein accumulations on damaged loci were suggested to be primary contributors to the process by which damaged loci are relocated to the nuclear periphery.

biophysics↗

bioRxiv2021

Epigenetic-structural changes in X chromosomes promote Xic pairing during early differentiation from mouse embryonic stem cells

X chromosome inactivation center (Xic) pairing is robustly observed during the differentiation of embryonic stem (ES) cells from female mouse embryos, and this process is related to X chromosome inactivation, the circadian clock, intra-nucleus architecture, and metabolism. However, the mechanisms underlying the identification and approach of X chromosome pairs in the crowded nucleus are unclear. To elucidate the driving force of Xic pairing, we developed a coarse-grained molecular dynamics model of intranuclear chromosomes in ES cells and in cells 2 days after the onset of differentiation (2-days cells) by considering intrachromosome epigenetic-structural feature-dependent mechanics. The analysis of the experimental data showed X-chromosomes change to specifically softer than autosomes during the cell differentiation by the rearrangement of their distributions of open-close chromatin regions, and the simulations of these models exhibited such softening promoted the mutual approach of the Xic pair. These findings suggested that local intrachromosomal epigenetic features may contribute to the regulation of cell species-dependent differences in intranuclear architecture.

biophysics↗

bioRxiv2021

H+/K+ ion pump enhances cytoskeletal polarity to drive gastrulation in sea urchin embryo

Gastrulation is a universal process in the morphogenesis of many animal embryos. Although morphological and molecular events in gastrulation have been well studied, the mechanical driving forces and underlying regulatory mechanisms are not fully understood. Here, we investigated the gastrulation of embryos of a sea urchin, Hemicentrotus pulcherrimus, which involves the invagination of a single-layered vegetal plate into the blastocoel. We observed that omeprazole, a proton pump inhibitor capable of perturbing the left-right asymmetry of sea urchin embryo, induced "partial exogastrulation" where the secondary invagination proceeds outward. During early gastrulation, intracellular apical-basal polarity of F-actin distribution in vegetal half were higher than those in animal half, while omeprazole treatment disturbed the apical-basal polarity of F-actin distribution in vegetal half. Furthermore, gastrulation stopped and even partial exogastrulation did not occur when F-actin polymerization or degradation in whole embryo was partially inhibited via RhoA or YAP1 knockout. A mathematical model of the early gastrulation reproduced the shapes of both normal and exogastrulating embryos using cell-dependent cytoskeletal features based on F-actin. Additionally, such cell position-dependent intracellular F-actin distributions might be regulated by intracellular pH distributions. Therefore, apical-basal polarity of F-actin distribution disrupted by omeprazole may induce the partial exogastrulation via anomalous secondary invagination.

developmental biology↗

bioRxiv2021

Identification of transcription termination defects at DNA hypomethylated transcription termination sites in DNA methyltransferase 3a-deficient vertebrates.

CpG methylation in genomic DNA is well known as a repressive epigenetic marker in eukaryotic transcription, and DNA methylation of the promoter regions is correlated with silencing of gene expression. In contrast to the promoter regions, the function of DNA methylation during transcription termination remains to be elucidated. A recent study has revealed that mouse DNA methyltransferase 3a (Dnmt3a) mainly functions in de novo methylation in the promoter and gene body regions (including transcription termination sites (TTSs)) during development. To investigate the relationship between DNA methylation overlapping the TTSs and transcription termination, we employed two strategies: informatic analysis using already deposited datasets of Dnmt3a-/- mouse cells and the zebrafish model system. Bioinformatic analysis using methylome and transcriptome data showed that hypomethylated differentially methylated regions overlapping the TTSs were associated with increased read counts and chimeric transcripts downstream of TTSs in Dnmt3a-/- Agouti-related protein neurons, but not in Dnmt3a-/- embryonic stem cells and mouse embryonic fibroblasts. We experimentally detected increased read-through and chimeric transcripts downstream of hypomethylated TTSs in zebrafish maternal-zygotic dnmt3aa-/- mutant. This study is the first to identify transcription termination defects in DNA hypomethylated TTSs in Dnmt3a-/- vertebrates.

genomics↗

bioRxiv2020

Structural Dynamics of DNA Depending on Methylation Patterns

DNA methylation is associated with a number of biological phenomena, and plays crucial roles in epigenetic regulation of eukaryotic gene expression. It is also suggested that DNA methylation alters the mechanical properties of DNA molecules, which is likely to affect epigenetic regulation. However, it has not been systematically investigated how methylation changes the structural and dynamic features of DNA. In this research, to elucidate the effects of methylation on DNA mechanics, a fully atomic molecular dynamics simulation of double-stranded DNA with several methylation patterns was performed. Through the analysis of the relative positioning of the nucleotides (base-step variables), characteristic changes in terms of local flexibility were observed, which further affected the overall DNA geometry and stiffness. These findings may serve as a basis for a discussion on methylation-dependent DNA dynamics in physiological conditions.

biophysics↗

bioRxiv2019

Affinity of rhodopsin to raft enables the aligned oligomer formation from dimers: Coarse-grained molecular dynamics simulation of disk membranes

The visual photopigment protein rhodopsin (Rh) is a typical G protein-coupled receptor (GPCR) that initiates the phototransduction cascade in retinal disk membrane of rod-photoreceptor cells. Rh molecule has a tendency to form dimer, and the dimer tends to form rows, which is suggested to heighten phototransduction efficiency in single-photon regime. In addition, the dimerization confers Rh an affinity for lipid raft, i.e. raftophilicity. However, the mechanism by which Rh-dimer raftophilicity contributes to the organization of the higher order structure remains unknown. In this study, we performed coarse-grained molecular dynamics simulations of a disk membrane model containing unsaturated lipids, saturated lipids with cholesterol, and Rh-dimers. We described the Rh-dimers by two-dimensional particle populations where the palmitoyl moieties of each Rh exhibits raftophilicity. We simulated the structuring of Rh in a disk for two types of Rh-dimer, i.e., the most and second most stable Rh dimers, which exposes the raftophilic regions at the dimerization-interface (H1/H8 dimer) and two edges away from the interface (H4/H5 dimer), respectively. Our simulations revealed that only the H1/H8 dimer could form a row structure. A small number of raftophilic lipids recruited to and intercalated in a narrow space between H1/H8 dimers stabilize the side-by-side interaction between dimers in a row. Our results implicate that the nano-sized lipid raft domains act as a "glue" to organize the long row structures of Rh-dimers.

biophysics↗

bioRxiv2019

Histone Tail Dynamics in Partially Disassembled Nucleosomes During Chromatin Remodeling

Nucleosomes are structural units of the chromosome consisting of DNA wrapped around histone proteins, and play important roles in compaction and regulation of the chromatin structure. While the structure and dynamics of canonical nucleosomes have been studied extensively, those of nucleosomes in intermediate states, that occur when their structure or positioning is modulated, have been less understood. In particular, the dynamic features of partially disassembled nucleosomes have not been discussed in previous studies. Using all-atom molecular dynamics simulations, in this study, we investigated the dynamics and stability of nucleosome structures lacking a histone-dimer. DNA in nucleosomes lacking a histone H2A/H2B dimer was drastically deformed due to loss of local interactions between DNA and histones. In contrast, conformation of DNA in nucleosomes lacking H3/H4 was similar to the canonical nucleosome, as the H2A C-terminal domain infiltrated the space originally occupied by the dissociated H3/H4 histones and stabilized DNA in close proximity. Our results suggest that, besides histone chaperones, the intrinsic dynamics of nucleosomes support the exchange of H2A/H2B, which is significantly more frequent than that of H3/H4.\n\nStatement of SignificanceEukaryotic chromosomes are composed of nucleosomes, in which the DNA wraps around the core histone proteins. To enable transcription and replication of DNA, or to modulate these functions by exchange of histones, nucleosomes should be at least partially disassembled, as evidenced by the observation of nucleosome structures lacking an H2A/H2B histone dimer by crystallography. The dynamic behavior of nucleosomes in such intermediate states may affect gene expression and repair, however it has not been completely elucidated so far. In this study, we adopted molecular dynamics simulations to analyze the conformational changes in partially disassembled nucleosomes. Enhanced structural fluctuations of DNA were observed in these nucleosomes, which may, as well as specific histone chaperones, support the exchange of H2A/H2B.

biophysics↗

bioRxiv2019

Insulator Activities of Nucleosome-Excluding DNA Sequences Without Bound Chromatin Looping Proteins

Chromosomes consist of various domains with different transcriptional activities separated by chromatin boundary sequences such as insulator sequences. Recent studies suggested that CTCF or other chromatin loop-forming protein binding sequences represented typical insulators. Alternatively, some long nucleosome-excluding DNA sequences were also reported to exhibit insulator activities in yeast and sea urchin chromosomes although specific binding of loop-forming proteins were not expected for them. However, the mechanism of the insulator activities of these sequences and the possibilities of similar insulators existing in other organisms remained unclear. In this study, we first constructed and performed simulations of a coarse-grained chromatin model containing nucleosome-rich and nucleosome-excluding DNA regions. We found that a long nucleosome-excluding region between two nucleosome-rich regions could markedly hinder the associations of two neighboring chromatin regions owing to the stronger long-term-averaged rigidity of the nucleosome-excluding region compared to that of nucleosome-rich regions. Subsequent analysis of the genome wide nucleosome positioning, protein binding, and DNA rigidity in human cells revealed that some nucleosome-excluding rigid DNA sequences without bound chromatin looping proteins could exhibit insulator activities, functioning as chromatin boundaries in various regions of human chromosomes.

genomics↗