Isono, Mayu

写真a

Affiliation

Faculty of Pharmacy, Department of Pharmacy ( Shiba-Kyoritsu )

Position

Researcher (Non-tenured) / Project Researcher(Non-tenured)

Career 【 Display / hide 】

  • 2013
    -
    2017

    Gunma University

  • 2017
    -
    2018

    佐々木研究所

  • 2018
    -
    2019

    Nagasaki University

  • 2019
    -
    2022

    Nagoya University

  • 2022

    Gunma University

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Research Areas 【 Display / hide 】

  • Life Science / Molecular biology (放射線影響)

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Research Keywords 【 Display / hide 】

  • 53BP1

  • BRCA1

  • DNA修復

  • DSB修復

  • DSB修復経路選択性

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Papers 【 Display / hide 】

  • MeCP2 deficiency leads to the γH2AX nano foci expansion after ionizing radiation.

    Okumura H, Hayashi R, Unami D, Isono M, Yamauchi M, Otsuka K, Kato Y, Oike T, Uchihara Y, Shibata A

    DNA repair 145   103790 2025.01

    ISSN  15687864

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    DNA double-strand breaks (DSBs) trigger the recruitment of repair protein and promote signal transduction through posttranslational modifications such as phosphorylation. After DSB induction, ataxia telangiectasia mutated (ATM) phosphorylates H2AX on chromatin surrounds the mega-base pairs proximal to the DSBs. Advanced super-resolution microscopic technology has demonstrated the formation of γH2AX nano foci as a unit of nano domain comprised of multiple nucleosomes. The formation of γH2AX nano foci could be potentially affected by pre-existing chromatin structure prior to DSB induction; however, it remains unclear whether chromatin status around DSBs influences the formation of γH2AX nano foci. In this study, to investigate γH2AX nano foci formation in the context of chromatin relaxation, γH2AX nano foci were examined following the depletion of MeCP2, which is a factor promoting chromatin condensation. Remarkably, by using super-resolution imaging analysis, we found that the volume of γH2AX nano foci cluster in MeCP2-depleted cells was significantly greater than that in control cells, both 5 and 30 min after ionizing radiation (IR). Corresponding to the increased volume size, the number of γH2AX nano foci per cluster was greater than that in control cells, while the distance of each nano focus within foci clusters remained unchanged. These findings suggest that relaxed chromatin condition by MeCP2 depletion facilitates faster and more extensive γH2AX nano foci formation after IR. Collectively, our super-resolution analysis suggests that the chromatin status surrounding DSBs influences the expansion of γH2AX nano foci formation, thus, potentially influencing the DSB repair and signaling.

  • Exacerbated Inflammatory Gene Expression After Impaired G2/M-Checkpoint Arrest in Fibroblasts Derived From a Patient Exhibiting Severe Adverse Effects.

    Oike T, Okuda K, Haruna S, Shibata A, Hayashi R, Isono M, Tateno K, Kubo N, Uchiyama A, Motegi SI, Ohno T, Uchihara Y, Kato Y, Shibata A

    Advances in radiation oncology 9 ( 8 ) 101530 2024.08

    ISSN  2452-1094

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    Purpose: Recent radiation therapy (RT), such as intensity modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT. Methods and Materials: Dermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with 2 normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined. Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR. Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT.

  • Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation.

    Hayashi R, Okumura H, Isono M, Yamauchi M, Unami D, Lusi RT, Yamamoto M, Kato Y, Uchihara Y, Shibata A

    Journal of radiation research 65 ( 3 ) 263 - 271 2024.05

    ISSN  0449-3060

    　View Summary

    Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5′-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

  • Characterization of the signal transduction cascade for inflammatory gene expression in fibroblasts with ATM-ATR deficiencies after Ionizing radiation.

    Haruna S, Okuda K, Shibata A, Isono M, Tateno K, Sato H, Oike T, Uchihara Y, Kato Y, Shibata A

    Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology 194   110198 2024.05

    ISSN  0167-8140

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    Background and purpose: Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR. Materials and methods: Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor–based downstream gene expression mapping (TDEM) method developed in this study. Results: Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3–pathway between ATM and ATR deficiency. Conclusion: Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.

  • Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

    Yuka Nakazawa, Yuichiro Hara, Yasuyoshi Oka, Okiru Komine, Diana van den Heuvel, Chaowan Guo, Yasukazu Daigaku, Mayu Isono, Yuxi He, Mayuko Shimada, Kana Kato, Nan Jia, Satoru Hashimoto, Yuko Kotani, Yuka Miyoshi, Miyako Tanaka, Akira Sobue, Norisato Mitsutake, Takayoshi Suganami, Akio Masuda, Kinji Ohno, Shinichiro Nakada, Tomoji Mashimo, Koji Yamanaka, Martijn S Luijsterburg, Tomoo Ogi

    Cell 180 ( 6 ) 1228 - 1244 2020.03

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    Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

  • 放射線照射により惹起される炎症性サイトカイン存在下でのDNA修復能の検討

    2024.04
    -
    2028.03

    日本学術振興会, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Principal investigator

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    炎症は古くから知られる特徴的な放射線障害の一つである。近年、放射線照射後に多くの炎症性遺伝子の発現が高まること、炎症性サイトカイン存在下ではDNA二本鎖切断（DSB）が蓄積することが示された。そこで申請者は、既報のデータベースを用いて遺伝子発現解析を行った結果、炎症性サイトカイン存在下ではDSB修復の遺伝子発現が低下することを見出した。つまり、炎症性サイトカイン存在下では放射線誘発DSBに対するDNA修復能が低下し、遺伝子異常や生存率の低下が引き起こされる可能性がある。本研究では、放射線誘発炎症性微小環境因子が、その後のDSB修復能に影響を与えるのかを検証し、さらにその分子機構の解明に迫る。

  • RNAポリメラーゼIIのDNA二本鎖切断修復機構への関与の解明

    2020.04
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    2023.03

    日本学術振興会, Grants-in-Aid for Scientific Research, Grant-in-Aid for Early-Career Scientists, No Setting

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    転写領域におけるRNAポリメラーゼ(Pol II)の停滞やRNA:DNAハイブリッド構造の蓄積はゲノムの不安定性を誘発し、発がんや難治性神経変性疾患につながることを示唆する報告がある。ゲノムの不安定化はDNA損傷応答・修復機構に障害があると誘発される。また、Pol IIが内因性のDNA二本鎖切断(DSB)の誘発に寄与することは示されている。しかしながら、転写領域のDSB修復の分子機構の詳細は明らかにされていない。Pol IIのDSB修復経路への関与と役割について明らかにすることによって、上記で示すような病態解明の一助になると考えている。
    カンプトテシンによってDSBが誘発されている状況下で、Pol II抗体による免疫沈降法を用いたウェスタンブロットを行い、DSB修復関連タンパクの抗体を用いたスクリーニングから、いくつかのタンパクがPol IIと相互作用することを確認した。その中の相同組換え修復関連タンパクの一つ(名前は伏せる)に注目して実験を行い、SUMO認識型ユビキチンE3リガーゼ(名前は伏せる)の欠損によって、Pol IIとの相互作用が見られなくなることを確認した。
    今後は、Pol IIとDSB修復関連タンパクの相互作用やSUMO認識型ユビキチンリガーゼがDSB修復にどのような役割を持つのか詳細を検討したいと考えている。

  • Regulation of 53BP1 repositioning and foci formation during homologous recombination

    2017.04
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    2020.03

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, ISONO Mayu, Grant-in-Aid for Young Scientists (B), No Setting

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    DNA double-strand break (DSB) caused by ionizing radiation or anti-cancer drugs is a deleterious damage, which can lead to chromosomal instability. Cells have the two main repair pathways for DSB, non-homologous end-joining (NHEJ) and homologous recombination (HR). While NHEJ functions through cell cycle, HR ensues if NHEJ fails to repair DSB in S/G2 phase. 53BP1 is known as a factor promoting NHEJ via inhibiting HR. We show the activation of ATM and ATR is dispensable for the maintenance of 53BP1 foci at sites of DSBs generated by ionizing radiation at early time points (0.5 h). Interestingly, inhibition of ATR (but not ATM) disturbs the maintenance of 53BP1 foci at later time points (4 h), which is fully recovered when the inhibitor is omitted. Collectively, the results in this study suggest that localization of 53BP1 foci at DSB sites during HR is dependent on ATR activation.

  • Visualization of heavy-ion induced clustered DNA double strand break formation using super-resolution microscopy

    2015.04
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    2017.03

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Isono Mayu, SHIBATA Atsushi, Grant-in-Aid for Challenging Exploratory Research, No Setting

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    Heavy-ion radiation induces clustered DNA damage, which contains multiple type of DNA damage, e.g. DNA double strand break (DSB), single strand break and base damage. However so far, such clustered DNA damage has not been visualized in the cellular level. Here we aimed to visualize the clustered DNA damage, particularly about the formation of DSB, after heavy-ion radiation by using 3D super-resolution microscopy. To address the question whether heavy-ion irradiation generates clustered DSB formation or not, we monitored RPA foci as a DSB marker. The super-resolution analysis revealed that heavy-ion radiation induced multiple RPA foci in close proximity. Furthermore, our analysis demonstrated that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. In summary, our data suggests that these closely localized DSBs are considered to be a risk for chromosomal translocations, deletions and cell death after heavy-ion irradiation.

  • The measurement of cell death in human neural stem cells and glioblastoma cells

    2013.08
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    2015.03

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, ISONO Mayu, Grant-in-Aid for Research Activity Start-up, No Setting

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    We analyzed the level of IR-induced cell death in neural stem and glioblastoma cells. To measure the type of cell death, i.e. necrosis and apoptosis, after IR, we carried out Acridine Orange and Ethidim Bromide assay. We observed that neural stem cells show greater IR sensitivity than glioblastoma cells. Imporantly, neural stem cells preferentially undergo apoptosis after IR. Further, we observed greater p53 phosphorylation in neural stem cells compared with that in glioblastoma cells. In addition, we performed a preliminary study on the effect of bystander in these cells. We found less effect of bystander in terms of cell death in neural stem cells.

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