Biology subjects

Kawaji, H.

Publications and source records attributed to Kawaji, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual Developmental Origins and Activity-dependent Specification of Mammalian Subplate Neurons

Subplate neurons (SpNs) are among the earliest-generated cortical neurons and are essential for neocortical circuit assembly. Despite this central role, they have long been considered a mammalian innovation, yet their evolutionary origin remains unresolved. Here, using comparative single-cell and spatial transcriptomics across amniotes (mice, chicks, and turtles), we identify two distinct developmental and evolutionary origins of SpNs: atypical SpNs (aSpNs), an Nr4a2-negative population conserved across amniotes and originating from the medial pallium, and mammalian-type SpNs (mSpNs), an Nr4a2-positive population preferentially expanded in mammals and arising from early-born cortical neurons. Cross-species analyses show that early-born pallial neurons in non-mammalian amniotes differentiate into thalamic input neurons, whereas this ancestral program is repurposed in mammals, with early-born neurons transiently adopting a subplate identity. We further show that this fate switch is controlled by Zbtb18 repression linked to thalamic input. Collectively, these findings establish a dual-origin model for SpNs and provide a unifying framework for understanding neocortical evolution. One-Sentence SummaryDevelopmental rewiring of an ancestral input-neuron program gave rise to the mammalian subplate.

evolutionary biology↗

bioRxiv2026

BRCA2 loss triggers a downward spiral of genomic instability via ROS-dependent metabolic collapse

BRCA2 plays a central role in maintaining genome integrity through homologous recombination and replication-fork protection, yet the compensatory networks sustaining BRCA2-deficient cells remain unclear. Here we show BRCA2 enforces a homeostatic mechanism aligning mitochondrial respiration with DNA repair capacity in both cancer and non-cancer contexts. Genome-wide CRISPR screening identified glutathione metabolism and base-excision repair as the key compensatory networks sustaining BRCA2-deficient cells by detoxifying mitochondria-derived reactive oxygen species. BRCA2 loss provokes an acute mitochondrial ROS surge, causing 8-oxoguanine accumulation and a systemic metabolic crisis marked by NAD+ and glutathione depletion. PARP inhibitor targets DNA replication vulnerabilities, increasing the cellular requirement for BRCA2. The resulting oxidative burden primes cells for TP53-dependent apoptosis in G1 during olaparib treatment, which extends cytotoxicity beyond canonical S-phase stress. These findings indicate BRCA2 prevents metabolic flux from outpacing repair capacity, providing a rationale for combining PARP inhibition with redox modulation to enhance efficacy and overcome resistance. HighlightsO_LIAcute BRCA2 loss induces ROS and mitochondrial dysfunction creating a metabolic scar C_LIO_LIOxidative lesions drive PARP hyperactivation and precipitate a cellular NAD crisis C_LIO_LIPARP inhibitors provoke TP53-dependent apoptosis in G1 beyond replication stress in S phase C_LIO_LIGlutathione deficiency exacerbates bone marrow failure under BRCA2 depletion C_LIO_LIBRCA2 tightly couples mitochondrial redox homeostasis to genomic maintenance C_LI

cell biology↗

bioRxiv2025

Anti-amyloid beta therapy resolves stroke recovery impairment caused by Alzheimer's disease

Stroke and dementia are common comorbidities and a growing concern causing disability in aging societies worldwide. Although anti-amyloid beta (anti-A{beta}) antibodies have recently been anticipated to relieve preclinical Alzheimers disease pathology, we discovered that post-stroke administration of anti-A{beta} antibodies restored neural repair for stroke recovery impeded by cerebral A{beta} accumulation. Neuronal recovery-associated gene expression for stroke recovery was considerably impaired even by slight A{beta} accumulation in murine and human brain. Slight A{beta} accumulation had less impact on neurons without stroke but caused a unique myeloid immunity after an ischemic stroke that enhanced the inflammatory cascades impeding neural repair for stroke recovery. Aducanumab administration after ischemic stroke prevented formation of this malignant myeloid immunity, resolving the impairment of post-stroke neural repair caused by cerebral A{beta} accumulation. Thus, our study has revealed the ability of anti-A{beta} therapies to restore functional recovery after a stroke with cerebral A{beta} accumulation.

neuroscience↗

bioRxiv2024

Drug-induced cis-regulatory elements in human hepatocytes affect molecular phenotypes associated with adverse reactions

BackgroundGenomic variations contribute to the phenotypic diversity of individuals. A number of polymorphisms in protein-coding regions that alter drug efficacy or lead to adverse reactions have been characterized; however, noncoding regions that affect drug responses are largely overlooked, except for a limited number of well-studied enhancers. ResultsWe conducted a quantitative assessment of cis-regulatory elements (CREs) based on transcription initiation profiling of mRNAs and noncoding RNAs, including enhancer RNAs, by using CAGE (Cap Analysis of Gene Expression). Candidate CREs identified in a hepatocellular carcinoma HepG2 cell line with stable expression of drug-responsive transcription factor pregnane X receptor (PXR) were further narrowed down by integrating data of PXR-binding sites in human primary hepatocytes and genome-wide association studies. We found more than 100-fold enrichments of the candidates to genetically associated loci with circulating levels of bilirubin and vitamin D, which implicated a link to adverse reactions of PXR ligands. We uncovered novel enhancers of UGT1A1 and TSKU through CRISPR/Cas9 knockout experiments. We identified alleles altering regulatory activities of UGT1A1 and CYP24A1enhancers by using luciferase reporter assay. Furthermore, our siRNA experiments revealed an unexpected impact of TSKU on the expression of vitamin D-metabolizing enzymes. ConclusionsOur transcriptome-based assessment of CREs expanded the list of drug-inducible and PXR-mediated enhancers and super-enhancers. We identified regulatory alleles that alter drug-induced gene expressions, and discovered a novel molecular cascade associated with an adverse reaction. Our results contribute a precise understanding of the noncoding elements of the human genome underlying drug responses.

genomics↗

bioRxiv2024

Sustaining microglial reparative function enhances stroke recovery

Neurological symptoms after brain injury can remain as lifelong detrimental sequelae since most spontaneous brain recovery disappears within a few months after brain injury. Microglia play an essential role in recovery processes after brain injury; however, cellular and molecular mechanisms that diminish spontaneous brain functional recovery remain unknown. We discovered by cellular fate analysis that reparative myeloid cells remained in the post-stroke brain even after losing their reparative function. ZFP384 was identified as a pivotal transcriptional regulator that diminished recovery phase-associated gene expression in reparative myeloid cells, turning them into ruined cells which lost reparative functions. ZFP384 diminished the YY1-mediated chromatin interaction necessary for expressing recovery phase-associated genes. Antisense oligonucleotide against Zfp384 sustained the broad range of neural repair effects of myeloid cells and enhanced stroke recovery, even in the chronic phase of ischemic stroke recovery. Thus, therapeutics preventing the myeloid reparative immunity from reaching a ruined state sustains brain functional recovery.

pathology↗

bioRxiv2023

Homologous recombination contributes to the repair of acetaldehyde-induced DNA damages.

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g., in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reaction among biological substances including DNA, RNA, and proteins, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we examined acetaldehyde sensitivity of DNA damage-deficient cells established from human TK6 cell line. Among the mutants, mismatch repair mutants did not show a hypersensitivity to acetaldehyde, while cells deficient in base and nucleotide excision repair pathways increased its sensitivity. We found a delayed repair and hypersensitivity in homologous recombination (HR)-deficient cells but not in non-homologous end joining-deficient cells after acetaldehyde treatment. By analyzing the formation of acetaldehyde-induced RAD51 foci, which represent HR intermediates, HR-deficient cells, but not NHEJ, exhibits delayed repair of acetaldehyde-induced DNA damages, compared with wild-type. These results suggest that acetaldehyde causes complex DNA damages that requires various types of repair pathways. Interestingly, mutants deficient in TDP1 and TDP2, which are involved in the removal of protein adducts from DNA ends, exhibited hypersensitivity to acetaldehyde. the acetaldehyde sensitivity of the TDP1-/-/RAD54-/- double mutant was similar to that of each single mutant. This epistatic relationship between TDP1 and RAD54 suggests that that the removal of protein-DNA adducts generated by acetaldehyde needs to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of genotoxic and mutagenic effects of acetaldehyde.

molecular biology↗

bioRxiv2023

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

AbstractThe Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

genomics↗

bioRxiv2023

Spatial transcriptome of developmental mouse brain reveals temporal dynamics of gene expressions and heterogeneity of the claustrum

During the development of the mammalian cerebral cortex, numerous neurons are arranged in a six-layer structure with an inside-out fashion to form the neocortex and wire neural circuits. This process includes cell proliferation, differentiation, migration, and maturation, supported by precise genetic regulation. To understand this sequence of processes at the cellular and molecular levels, it is necessary to characterize the fundamental anatomical structures by gene expression. However, markers established in the adult brain sometimes behave differently in the fetal brain, actively changing during development. Spatial transcriptomes yield genome-wide gene expression profiles from each spot patterned on tissue sections, capturing RNA molecules from fresh-frozen sections and enabling sequencing analysis while preserving spatial information. However, a deeper understanding of this data requires computational estimation, including integration with single-cell transcriptome data and aggregation of spots on the single-cell cluster level. The application of such analysis to biomarker discovery has only begun recently, and its application to the developing fetal brain is largely unexplored. In this study, we performed a spatial transcriptome analysis of the developing mouse brain to investigate the spatiotemporal regulation of gene expression during development. Using these data, we conducted an integrated study with publicly available mouse data sets, the adult brains spatial transcriptome, and the fetal brains single-cell transcriptome. Our data-driven analysis identified novel molecular markers of the choroid plexus, piriform cortex, thalamus, and claustrum. In addition, we revealed that the internal structure of the embryonic claustrum is composed of heterogeneous cell populations.

developmental biology↗

bioRxiv2022

mRNA-based generation of marmoset PGCLCs capable of differentiation into gonocyte-like cells

Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLC. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development. HighlightsO_LIAn mRNA transfection-based PGCLC induction method is developed C_LIO_LIMarmoset PGCLCs are efficiently induced from iPSCs C_LIO_LIMarmoset PGCLCs differentiate into gonocyte-like cells in mouse kidneys C_LIO_LIDevelopmentally regulated expression and demethylation are recapitulated C_LI eTOC blurbWatanabe et al. efficiently induced marmoset primordial germ cell-like cells (PGCLCs) using an mRNA transfection-based approach. PGCLCs further develop into gonocyte-like cells in the xenoreconstituted testes constructed under mouse kidney capsules. This system faithfully reproduced in vivo developmental processes (e.g., stage-specific expression of developmentally regulated genes and DNA demethylation).

developmental biology↗

bioRxiv2020

Chromosomal-scale De novo Genome Assemblies of Cynomolgus Macaque and Common Marmoset

Cynomolgus macaque (Macaca fascicularis) and common marmoset (Callithrix jacchus) have been widely used in human biomedical research. Their genomes were sequenced and assembled initially using short-read sequences, with the advent of massively parallel sequencing. However, the resulting contig sequences tended to remain fragmentary, and long-standing primate genome assemblies used the human genome as a reference for ordering and orienting the assembled fragments into chromosomes. Here we performed de novo genome assembly of these two species without any human genome-based bias observed in the genome assemblies released earlier. Firstly we assembled PacBio long reads, and the resultant contigs were scaffolded with Hi-C data. The scaffolded sequences obtained were further refined based on assembly results of alternate de novo assemblies and Hi-C contact maps by resolving identified inconsistencies. The final assemblies achieved N50 lengths of 149 Mb and 137 Mb for cynomolgus macaque and common marmoset, respectively, and the numbers of scaffolds longer than 10Mb are equal to their chromosome numbers. The high fidelity of our assembly is ascertained by concordance to the BAC-end read pairs observed for common marmoset, as well as a high resemblance of their karyotypic organization. Our assembly of cynomolgus macaque outperformed all the available assemblies of this species in terms of contiguity. The chromosome-scale genome assemblies produced in this study are valuable resources for non-human primate models and provide an important baseline in human biomedical research.

genomics↗

bioRxiv2020

Assessment of mapping strategies for determining the 5'-end of mRNAs and long-noncoding RNAs with short read sequences

BackgroundGenome mapping is an essential step in data processing for transcriptome analysis, and many previous studies have evaluated various methods and strategies for mapping RNA-seq data. Cap Analysis of Gene Expression (CAGE) is a sequencing-based protocol particularly designed to capture the 5{square}-ends of transcripts for quantitatively measuring the expression levels of transcription start sites genome-wide. Because CAGE analysis can also predict the activities of promoters and enhancers, this protocol has been an essential tool in studies of transcriptional regulation. Typically, the same mapping software is used to align both RNA-seq data and CAGE reads to a reference genome, but which mapping software and options are most appropriate for mapping the 5{square}-end sequence reads obtained through CAGE has not previously been evaluated systematically. ResultsHere we assessed various strategies for aligning CAGE reads, particularly [~]50-bp sequences, with the human genome by using the HISAT2, LAST, and STAR programs both with and without a reference transcriptome. One of the major inconsistencies among the tested strategies involves alignments to pseudogenes and parent genes: some of the strategies prioritized alignments with pseudogenes even when the read could be aligned with coding genes with fewer mismatches. Another inconsistency concerned the detection of exon-exon junctions. These preferences depended on the program applied and whether a reference transcriptome was included. Overall, the choice of strategy yielded different mapping results for approximately 2% of all promoters. ConclusionsAlthough the various alignment strategies produced very similar results overall, we noted several important and measurable differences. In particular, using the reference transcriptome in STAR yielded alignments with the fewest mismatches. In addition, the inconsistencies among the strategies were especially noticeable regarding alignments to pseudogenes and novel splice junctions. Our results indicate that the choice of alignment strategy is important because it might affect the biological interpretation of the data.

bioinformatics↗

bioRxiv2019

DUX4 regulates oocyte to embryo transition in human

During the human oocyte-to-embryo transition, the fertilized oocyte undergoes final maturation and the embryo genome is gradually activated during the first three cell divisions. How this transition is coordinated in humans is largely unknown. We show that the double homeodomain transcription factor DUX4 contributes to this transition. DUX4 knockdown in human zygotes caused insufficient transcriptome reprogramming as observed three days after fertilization. Induced DUX4 expression in human embryonic stem cells activated transcription of thousands of newly identified bi-directional transcripts, including putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 protein interacted with transcriptional modifiers that are known to couple enhancers and promoters. Taken together, our results reveal that DUX4 is a pioneer regulating oocyte-to-embryo transition in human through activation of intergenic genome, especially enhancers, and hence setting the stage for early human embryo development.

developmental biology↗

bioRxiv2019

Dynamics and Topology of Human Transcribed Cis-regulatory Elements

Promoters and enhancers are key cis-regulatory elements, but how they orchestrate to generate cell-type-specific transcriptomes remains elusive. We developed a simple and robust approach to globally determine 5-ends of nascent RNAs (NET-CAGE) in diverse cells and tissues, thereby sensitively detecting unstable transcripts including enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, uncovering the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without influence of RNA turnover, and identified enhancer-promoter pairs which were simultaneously activated upon cellular stimulation. By integrating NET-CAGE data with chromatin interaction maps, we revealed that cis-regulatory elements are topologically connected according to their cell-type specificity. We discovered new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our collection of NET-CAGE data from human and mouse significantly expanded the FANTOM5 catalogue of transcribed enhancers, with broad applicability to biomedical research. (148 words)

genomics↗

bioRxiv2019

CDK1 dependent phosphorylation of hTERT contributes to cancer progression

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses revealed that phosphorylation of hTERT at threonine 249 occurs more frequently in advanced cancers. Using CRISPR/Cas9 genome editing, we introduced substitution mutations at threonine 249 in the endogenous hTERT locus and found that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase activity. Cap Analysis of Gene Expression (CAGE) demonstrated that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomerase independent manner.

cancer biology↗

bioRxiv2019

AGO1 in association with NEAT1 lncRNA contributes to nuclear and 3D chromatin architecture in human cells

Aside from their roles in the cytoplasm, RNA-interference components have been reported to localize also in the nucleus of human cells. In particular, AGO1 associates with active chromatin and appears to influence global gene expression. However, the mechanistic aspects remain elusive. Here, we identify AGO1 as a paraspeckle component that in combination with the NEAT1 lncRNA maintains 3D genome architecture. We demonstrate that AGO1 interacts with NEAT1 lncRNA and its depletion affects NEAT1 expression and the formation of paraspeckles. By Hi-C analysis in AGO1 knockdown cells, we observed global changes in chromatin organization, including TADs configuration, and A/B compartment mixing. Consistently, distinct groups of genes located within the differential interacting loci showed altered expression upon AGO1 depletion. NEAT1 knockout cells displayed similar changes in TADs and higher-order A/B compartmentalization. We propose that AGO1 in association with NEAT1 lncRNA can act as a scaffold that bridges chromatin and nuclear bodies to regulate genome organization and gene expression in human cells.

genomics↗

bioRxiv2018

dirHub: a trackHub configurator with directory structure projection

SummaryTrack Data Hub is a mechanism enabling us to visualize genomics data as tracks along genome coordinates and share them over the Internet, relying on a web server hosting data files and genome browsers offering graphical representations. It requires an accessible configuration file specifying all graphical parameters and track hierarchy, in addition to the data files. Here dirHub is developed to assist generation of the configuration file by projection of a file directory structure, which makes it possible to set up trackHub visualization mostly by file operations.\n\nAvailability and implementationIt is implemented in ruby and the source code is available at https://github.com/hkawaji/dirHub/. It is tested on the UCSC Genome Browser and the Hub Track Database Definition (v2).

bioinformatics↗

bioRxiv2018

Selective activation of alternative MYC core promoters by Wnt-responsive enhancers

In Metazoans, transcription of most genes is driven through the use of multiple alternative promoters. Although the precise spatio-temporal regulation of alternative promoters is important for proper gene expression, the mechanism that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types, and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in the colon carcinoma cell line HCT-116, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using both transgenic reporter assays and in the context of the endogenous Myc locus upon Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by distinct core promoter elements present in the two MYC promoters. Taken together, our results provide new insights into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more diverse combinatorial regulation of transcription initiation.

genetics↗

bioRxiv2018

Integrative analysis of transcription factor occupancy at enhancers and disease risk loci in noncoding genomic regions

Noncoding regions of the human genome possess enhancer activity and harbor risk loci for heritable diseases. Whereas the binding profiles of multiple transcription factors (TFs) have been investigated, integrative analysis with the large body of public data available so as to provide an overview of the function of such noncoding regions has remained a challenge. Here we have fully integrated public ChIP-seq and DNase-seq data (n ~ 70,000), including those for 743 human transcription factors (TFs) with 97 million binding sites, and have devised a data- mining platform --designated ChIP-Atlas--to identify significant TF-genome, TF-gene, and TF-TF interactions. Using this platform, we found that TFs enriched at macrophage or T-cell enhancers also accumulated around risk loci for autoimmune diseases, whereas those enriched at hepatocyte or macrophage enhancers were preferentially detected at loci associated with HDL-cholesterol levels. Of note, we identified \"hotspots\" around such risk loci that accumulated multiple TFs and are therefore candidates for causal variants. Integrative analysis of public chromatin-profiling data is thus able to identify TFs and tissues associated with heritable disorders.

genomics↗