Biology subjects

Hayashi, M. T.

Publications and source records attributed to Hayashi, M. T..

6 recordsLinked to original sources

bioRxiv2026

Uncoupling of STING Accumulation and Activation upon Chromosome-Derived Extrachromosomal Circular DNA Formation

The innate immune system maintains cellular homeostasis by recognizing cytosolic nucleic acids. While the cGAS-STING pathway is well characterized in detecting non-self-DNA, its response to self-DNA originating from the host nucleus remains unclear. Extrachromosomal circular DNA (eccDNA), a form of self-DNA often enriched in cancer, has been implicated in immune modulation, yet its ability to activate cGAS-STING remains unresolved. Here, we developed a Cre-loxP-based eccDNA reporter system, where Cre-mediated eccDNA formation triggers expression of the fluorescent protein mCitrine, enabling time-resolved, single-cell analysis of chromosome-derived eccDNA dynamics and STING activation. Using an mRuby3-STING reporter, we found that low-dose plasmid transfection induced STING activation, as evidenced by modest STING accumulation and downstream cytokine induction. Cells carrying chromosome-derived cytosolic eccDNA also showed a modest, cGAS-dependent accumulation of STING. However, this accumulation was uncoupled from detectable STING-Ser366 phosphorylation or induction of downstream cytokines. These findings indicate that chromosome-derived eccDNA does not drive detectable STING signaling under the conditions tested, and that STING accumulation and its activation are mechanistically separable steps. This work provides a foundation for understanding the immunological roles of eccDNA in genome instability and cancer.

cell biology↗

bioRxiv2026

The BLM DHBN domain and structure-selective nucleases drive mitotic arrest-dependent telomere deprotection under WRN and TRF2 control

Telomeres shield chromosome ends from DNA damage response through T-loops, lariat DNA structures formed and stabilized by the shelterin protein TRF2. During prolonged mitotic arrest, telomeres lose this protection through a process termed mitotic arrest-dependent (MAD) telomere deprotection, which elicits telomere-specific DNA damage signaling in the absence of telomere shortening or chromosome end-to-end fusions. We previously demonstrated that the RecQ helicase BLM promotes MAD telomere deprotection, whereas the related helicase WRN suppresses it independently of its catalytic activities. However, the molecular interplay between BLM and WRN at mitotic telomeres and whether additional recombination-associated enzymes contribute to MAD telomere deprotection have remained unresolved. Here, we identify the Dimerization Helical Bundle in the N-terminal (DHBN) domain of BLM helicase as the critical determinant of MAD telomere deprotection and show that WRN selectively restrains this activity without interfering with BLM's canonical genome-protective functions. We further show that both MUS81 and GEN1 contribute to MAD telomere deprotection. Moreover, the exacerbation of MAD telomere deprotection observed upon TRF2 depletion is strongly attenuated by additional depletion of these enzymes, demonstrating that TRF2 normally protects T-loop junctions from their enzymatic activities. Collectively, our findings reveal how telomeres become selectively vulnerable during prolonged mitotic arrest and uncover a regulated enzymatic mechanism that repurposes recombination machinery at chromosome ends.

molecular biology↗

bioRxiv2024

A CPC-shelterin-BTR axis regulates mitotic telomere deprotection

Telomeres prevent ATM activation by sequestering chromosome termini within telomere loops (t-loops). Mitotic arrest promotes telomere linearity and a localized ATM-dependent telomere DNA damage response (DDR) through an unknown mechanism. Using unbiased interactomics, biochemical screening, molecular biology, and super-resolution imaging, we found that mitotic arrest-dependent (MAD) telomere deprotection requires the combined activities of the Chromosome passenger complex (CPC) on shelterin, and the BLM-TOP3A-RMI1/2 (BTR) complex on t-loops. During mitotic arrest, the CPC component Aurora Kinase B (AURKB) phosphorylated both the TRF1 hinge and TRF2 basic domains. The former enhanced CPC and TRF1 interaction through the CPC Survivin subunit, while the latter promoted telomere linearity, telomere DDR activation dependent upon BTR double Holliday junction dissolution activity, and mitotic death. We identify that the TRF2 basic domain functions in mitosis-specific telomere protection and reveal TRF1 regulation over a physiological ATM-dependent telomere DDR. The data demonstrate that MAD telomere deprotection is a sophisticated active mechanism that exposes telomere ends to signal mitotic stress.

molecular biology↗

bioRxiv2023

Micronucleus Is Not a Potent Inducer of cGAS-STING Pathway

Micronucleus (MN) has been associated with the innate immune response. The abrupt rupture of MN membranes results in the accumulation of cGAS, potentially activating STING and downstream interferon-responsive genes. However, direct evidence connecting MN and cGAS activation has been lacking. We have developed the FuVis2 reporter system, which enables the visualization of cell nucleus carrying a single sister chromatid fusion and, consequently, MN. Using this FuVis2 reporter equipped with cGAS and STING reporters, we rigorously assessed the potency of cGAS activation by MN in individual living cells. Our findings reveal that cGAS localization to membrane-ruptured MN during interphase is infrequent, with cGAS primarily capturing MN during mitosis and remaining bound to cytosolic chromatin. We found that cGAS accumulation during mitosis neither activates STING in the subsequent interphase nor triggers the interferon response. Gamma-ray irradiation activates STING independently of MN formation and cGAS localization to MN. These results suggest that cGAS accumulation in the cytosol is not a robust indicator of its activation and that MN is not the primary trigger of the cGAS/STING pathway.

cell biology↗

bioRxiv2019

A Single Defined Sister Chromatid Fusion Destabilizes Cell Cycle through Micronuclei Formation

Chromosome fusion is deleterious among oncogenic chromosome rearrangements, and has been proposed to cause multiple tumor-driving abnormalities. Conventional methodologies, however, lack the strictness of the experimental controls on the fusion such as the exact timing, the number and the types of fusion in a given cell. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. Fused chromosome developed numerical and structural abnormalities, including chromosome fragmentation, an indicative of eventual chromothripsis. Live cell imaging and hierarchical Bayesian modeling indicated that micronucleus (MN) is generated in the first few cell cycle, and that cells with MN tend to possess cell cycle abnormalities. These results demonstrate that, although most cells can tolerate a single fusion, even a single sister chromatid fusion destabilizes cell cycle through MN formation.

cell biology↗

bioRxiv2018

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death promotes genome instability in replication stressed cells through diverse mechanisms depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and genome instability.

molecular biology↗