Biology subjects

Asada, S.

Publications and source records attributed to Asada, S..

8 recordsLinked to original sources

bioRxiv2026

Midbody inheritance predicts re-entry into quiescence, but not lineage potential, in mouse hematopoietic stem cells

Hematopoietic stem cells (HSCs) give rise to all blood cell lineages and possess long-term self-renewal potential. HSCs undergo symmetric division for their expansion and asymmetric division to generate one HSCs and one progenitor cells which contribute to production of mature blood cells. The midbody is a structure which is formed in the center of the intercellular bridge during cytokinesis. However, the midbody is either asymmetrically inherited by one daughter cell or symmetrically released after cell division, whether these distinct patterns of midbody inheritance influence HSC fate remain poorly understood. In this study, we designed a fusion protein hmKO2 and MgcRacGAP which is a component of midbody. We then traced the midbody inheritance during cell division and the future cell fates of HSC daughters after division by time-lapse imaging. As a result, we found that the midbody release correlated with the delay of the time to the next division but not to the lineage potential of HSCs, indicating the possibility that midbody remnant plays some roles in cell cycle progression. HighlightHematopoietic stem cells exhibit a low frequency of midbody inheritance. Midbody inheritance does not affect the lineage potential of daughter cells. Midbody loss is associated with delayed entry into the next cell cycle.

cell biology↗

bioRxiv2026

DNA barcoding-based paired daughter cell analysis reveals division preferences of hematopoietic stem cells

Hematopoietic stem cells(HSCs) maintain their pools by stem-stem division and produce mature blood cells through stem-progenitor or progenitor-progenitor division. A paired daughter cell(PDC) assay combined with single cell transplantation is a powerful method to compare the lineage outputs of two HSC daughter cells. However, single-cell transplantation precludes large-scale analysis of daughter-cell pairs, as only one cell can be transplanted per recipient. Here, we developed a DNA barcoding-based PDC assay to overcome this limitation, enabling simultaneous analysis of 476 daughter pairs from individual HSC divisions and revealing that daughter-cell fates are coordinated and that HSC division patterns are biased toward stem-stem and progenitor-progenitor divisions rather than stem-progenitor divisions. These findings indicate that HSC fate outcomes are directed toward symmetric division outcomes. Integration of single-cell RNA sequencing with DNA barcoding revealed a continuum of HSC states--from balanced HSCs to myeloid-biased HSCs and ultimately to a low-output HSC subset--in which progressively reduced production of mature blood cells relative to stem cell expansion, a proxy for stem-stem division bias, exhibits distinct activities of transcription factors and signaling pathways. Overall, our analysis uncovers characteristic patterns of HSC division and links stem maintenance with distinct molecular features.

cell biology↗

bioRxiv2026

A RAD18 SAP domain PIP motif enables PCNA mono-ubiquitination and USP1-BRCA1 synthetic lethality

The proliferating cell nuclear antigen (PCNA) sliding clamp is mono-ubiquitinated by RAD6-RAD18 in response to DNA damage, initiating the DNA damage tolerance pathway of translesion synthesis. The molecular basis by which RAD18 engages PCNA has, however, remained incompletely defined. Mono-ubiquitinated PCNA is subsequently poly-ubiquitinated with K48-linked chains that target PCNA for degradation. Ubiquitin-specific protease 1 (USP1) reverses PCNA mono- and poly-ubiquitination; accordingly, inhibiting USP1 causes the accumulation of mono-ubiquitinated PCNA at replication forks and a reduction in total PCNA levels. USP1 inhibitors promote the accumulation of ssDNA gaps (ssGAPs) in newly replicated DNA and are synthetic lethality in BRCA1-deficient cells. Here, we combine computational and structural approaches to identify and characterize a PCNA-interacting peptide (PIP) motif in RAD18. This PIP motif is required for RAD18-dependent DNA damage-induced PCNA ubiquitination and PCNA turnover. Mutation of the RAD18-PCNA interface reduces ssGAP accumulation and USP1 inhibitor sensitivity in BRCA1-deficient cells. Furthermore, cells adapted to prolonged USP1 inhibition exhibit reduced RAD18 levels, suggesting that deregulation of RAD18 contributes to a biologically relevant drug resistance mechanism. This resistance could be overcome by inhibiting the Ataxia telangiectasia and Rad3-related (ATR) kinase. Together, these findings define a molecular interface required for RAD18-dependent PCNA mono-ubiquitination and identify it as a key determinant of USP1-BRCA1 synthetic lethality.

biochemistry↗

bioRxiv2025

Defective Microhomology-Mediated End-joining in SMARCB1-Deficient Tumors

Rhabdoid tumors (RTs) are highly aggressive cancers driven by biallelic mutation of SMARCB1, a core subunit of the BAF (SWI/SNF) complex. We found that SMARCB1-deficient tumors have a defect in the microhomology-mediated end-joining (MMEJ) pathway, and SMARCB1 is essential for maintaining the protein level of the core MMEJ protein Polymerase Q (POLQ). Mechanistically, SMARCB1 facilitates the nuclear export of the POLQ mRNA through its interaction with the nuclear pore complex. Interestingly, loss of MMEJ in RT cells leads to a compensatory activation of, and a hyper-dependence on, the Fanconi Anemia (FA)/BRCA pathway. Knockout or inhibition of this pathway selectively kills RT cells. Notably, RBM39 degraders, novel splicing modulators, effectively inhibit the FA/BRCA pathway and kill RT cells. SMARCB1 and other cBAF/pBAF components are important for maintenance of MMEJ activity and POLQ protein level, suggesting that BAF-deficient cancers more broadly may be treated by targeted inhibition of the FA/BRCA pathway. TeaserSMARCB1-deficient tumors lose POLQ protein and MMEJ repair, forcing a hyper-dependence on the FA/BRCA pathway.

cancer biology↗

bioRxiv2025

HPV16 E6 oncoprotein promotes microhomology-mediated viral integration by upregulating POLQ expression

Integration of the high-risk Human Papillomavirus 16 (HPV16) genome into the host chromosome, frequently driven by microhomology-mediated end joining (MMEJ), is a critical step in the carcinogenesis of HPV-associated tumors. However, the mechanisms by which viral oncoproteins manipulate the error-prone MMEJ pathway remain poorly defined. Here, we demonstrate that the HPV16 E6 oncoprotein upregulates MMEJ to facilitate viral genome integration. This heightened MMEJ activity is driven by a marked increase in the protein levels of DNA Polymerase Theta (POLQ), a central enzyme of the MMEJ pathway. Mechanistically, we show that the elevation of POLQ levels in response to HPV16 E6 expression is dependent on the host E3 ubiquitin ligase UBE3A/E6AP but is independent of p53 degradation. E6 activates UBE3A to promote the ubiquitination and degradation of RAD23A, a shuttle protein required for delivering polyubiquitinated POLQ to the proteasome. Consequently, the loss of functional RAD23A phenocopies the effect of HPV16 E6, leading to POLQ stabilization and increased MMEJ activity. By elucidating the E6-UBE3A-RAD23A-POLQ axis, our findings reveal a novel mechanism through which HPV manipulates the host DNA repair machinery to promote its integration and oncogenic potential.

molecular biology↗

bioRxiv2025

Disruption of Microhomology-mediated End-joining in Ewing Sarcoma

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POL{Theta}, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POL{Theta} expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POL0 mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS. Highlights- Ewing sarcoma tumors have a deficiency in POL{theta} expression and a corresponding loss of MMEJ activity - EWSR1 is a splicing factor that interacts with other splicing factors such as FUBP1 and KHSRP/FUBP2 to accurately splice the POLQ mRNA. - The EWS-FLI1 fusion oncoprotein, or loss of EWSR1, causes a splicing defect, leading to exon25 skipping of the POLQ pre-mRNA and loss of POL{theta} expression - The MMEJ deficiency of EwS cells results in cellular sensitivity to inhibitors of Fanconi Anemia, Homologous Recombination or Non-Homologous End-Joining - Exon 25 skipping of POLQ mRNA is a predictive biomarker for HR inhibitors in human cancers

cancer biology↗

bioRxiv2024

CH-related Mutant ASXL1 Promotes Atherosclerosis in Mice via Dysregulated Innate Immunity

Certain somatic mutations confer a fitness advantage in hematopoietic stem and progenitor cells (HSPCs) over normal HSPCs, resulting in the clonal expansion of mutant blood cells1, otherwise known as clonal haematopoiesis (CH). CH is frequently observed among healthy elderly people and is closely associated with the risk of cardiovascular diseases (CVDs). The most frequently mutated genes of CH include DNMT3A, TET2, and ASXL12. Among them, even though ASXL1 mutations are clinically associated with the highest risk for developing CVDs, little is known whether and how the mutations contribute to CVDs. Here we show accelerated development of atherosclerosis and increased inflammatory monocytes in mice transplanted with the bone marrow cells (BMCs) from the mice expressing mutant ASXL1 (ASXL1-MT) selectively in hematopoietic cells. RNA sequencing analysis of the plaque-macrophages derived from BMCs expressing ASXL1-MT showed more inflammatory signatures than those from control BMCs. Mechanistically, wild-type ASXL1 inhibited innate immune signalling through direct interactions with IRAK1/TRAF6/TAK1 in the cytoplasm, while ASXL1-MT, which only interacted with TAK1, lost this regulatory function, leading to NF-{kappa}B activation. This mechanism is unique and distinct from those of CH with Tet2 or Dnmt3a mutations, where overactivation of the IL-1{beta}/NLRP3 inflammasome plays critical roles3-5. Intriguingly, IRAK1/4 inhibition decreased the number of inflammatory monocytes and attenuated the development of atherosclerosis driven by ASXL1-MT. The present work connects the mutations of an epigenetic factor, ASXL1, with inflammation and CVDs and gives an indication for the prevention of CVDs in CH.

cell biology↗

bioRxiv2024

MECOM promotes leukemia progression and inhibits mast cell differentiation through functional competition with GATA2

MECOM is a nuclear transcription factor essential for the proliferation of hematopoietic stem cells (HSCs) and myeloid leukemia cells. MECOM contains N- and C-terminal zinc finger domains (ZFDs) and binding motifs for the corepressor CtBP to regulate gene expression. Recent studies have shown that germline MECOM variants are associated with thrombocytopenia, radioulnar synostosis, and bone marrow failure, collectively termed MECOM-associated syndromes. Although the mutations are clustered in the C-terminal ZFD, how these mutations affect MECOM function has remained unclear. In addition, the individual genes and pathways regulated by MECOM are less well understood. In this study, we showed that the C-terminal ZFD is a major DNA-binding domain of MECOM and that the disease-associated mutations abolish the DNA-binding ability. We also found that MECOM functionally antagonizes GATA2 through the C-terminal ZFD-mediated DNA binding and CtBP interaction, thereby promoting myeloid leukemogenesis while inhibiting mast cell differentiation. Furthermore, we generated mutant MECOM knockin mice harboring a C-terminal ZFD mutation that recapitulate several features of MECOM-associated syndromes, including HSC and B-cell reduction. Our study demonstrates that C-terminal ZFD mutations are loss-of-function mutations with reduced DNA-binding ability, reveals the critical role of MECOM in inhibiting GATA2, and provides a novel mouse model for MECOM-associated syndromes.

molecular biology↗