Research

The Mechanism by which Irradiation Causes Genomic Instability

Cells have sophisticated mechanisms to respond to DNA damage caused by external stress, such as radiation. Genomic abnormalities in transcriptionally active regions can lead to diseases, including many types of cancer. Our laboratory focuses on elucidating how chromosome translocations occur in transcriptionally active regions. For example, it is well known that fusion genes generated by chromosome translocations at specific gene regions, such as BCR-ABL, produce oncogenic proteins. However, the mechanisms of how chromosome translocations occur in cells remain unclear.

　Chromosome translocations can be generated by three steps: 1) DNA double-strand breaks (DSBs) on two different chromosomes, 2) Proximity of those chromosomes, and 3) abnormal DSB repair. Our approaches to understanding these steps of chromosome translocation vary from molecular/cell biological methods to biochemical techniques. Moreover, we have been developing unique systems to analyze cancer genome databases.

　In our previous studies, we identified two novel transcription-associated DSB repair pathways taking care of transcriptionally active regions (Yasuhara et al. Cell 2018, Cell Reports 2022). Furthermore, relating to the proximity of chromosomes, we discovered nucleolar condensates formed by RNA-binding proteins upon cellular stresses involve transcriptionally active regions of the genome into nucleoli and promote the proximity among these regions. (Yasuhara et al. Molecular Cell 2022).

DSB Repair Mechanism Initiated by R-loop

R-loop is a special nucleic acid structure consisting of DNA-RNA hybrid and single-stranded DNA. It is obvious that R-loops accumulate at genomic regions where transcription is active because of the abundance of RNA. Interestingly, recent studies have revealed that R-loops have many important roles in the process of DSB repair.

　In general, DSBs at transcriptionally active regions must precisely be repaired given the importance of these parts of the genome. We assume that the presence of R-loops around DSBs serves as a mark for cells to initiate precise repair mechanisms. Indeed, we revealed that R-loops around a DSB initiate two accurate repair mechanisms: transcription-associated homologous recombination repair (TA-HRR) or transcription-associated end-joining (TA-EJ) (Figure 1). Without these repair systems, irradiated cells had genomic abnormalities at a higher frequency, suggesting that these repair mechanisms guard our genome against mutations. We are currently working to elucidate the detailed molecular mechanisms of these repair pathways to understand how our DSB repair systems suppress chromosome translocations.

Figure 1. Two transcription-associated DNA double-strand break repair pathways initiated by R-loops.

Liquid-liquid phase separation and chromosome translocation

Chromosome translocations that lead to disease always occur in specific regions of the genome. For years, the question of whether this is due to survival selection pressure or whether there is any bias that these regions are fragile to cause genomic aberrations has been unsolved. Our work has provided evidence for the latter possibility, i.e., there is a plausible reason for transcriptionally active regions to have a higher chance of chromosome translocations.
　Recently, a set of proteins that have unstructured domains self-assembling via liquid-liquid phase separation has been rigorously studied. We hypothesized that liquid-liquid phase separation could cause the proximity of chromosomes by involving the genome together with unstructured proteins. We found that certain RNA-binding proteins form condensates (Condensate Induced by Transcription Inhibition; CITI) in nucleoli upon transcription inhibition (Figure 2) and that CITIs bring chromosomes, especially the transcriptionally active regions, into nucleoli to increase the chance of chromosome translocations. We are now working on the questions of 1) what kind of stresses cause CITIs, and 2) what the physiological functions of CITIs are.

Figure 2. CITI formed via liquid-liquid phase separation upon transcriptional stresses.

Diseases caused by chromosome translocation

Chromosome translocations are a major cause of cancer. They are also a risk factor for some neuropsychiatric disorders, infertility, and fetal chromosome aberrations. We believe that understanding how genomic instability occurs and causes the diseases will give us a clue to develop new preventive and therapeutic strategies against these diseases. In an age of long-life expectancy, we hope to contribute to solving various problems, such as cancer and infertility in reproductive medicine, through our research which sheds light on the underlying fundamental mechanisms behind diseases.

Competitive research funds related to this research

• FY2023 - FY2025 Asian Young Scientist Fellowship
  “The fundamental mechanisms of genome protection” (Principal Investigator)
• FY2023 - FY2024 Grant-in-Aid for Transformative Research Areas (A)
  “Genome instability induced by spatial positions in the nucleus” (Principal Investigator)
• FY2022 - FY2025 AMED-PRIME
  “Age-related changes in germline cells that cause chromosome translocations” (Principal Investigator)
• FY2022 - FY2023 Grant-in-Aid for Transformative Research Areas (A)
  “A strategy to transform genomic organization and change the gene expression pattern via controlling nucleolar condensates” (Principal Investigator)
• FY2022 - FY2026 Takeda Science Foundation
  “Elucidation of the mechanism of tumorigenesis via comprehensive understanding of transcription-associated DNA double-strand break repair” (Principal Investigator)
• FY2022 - FY2023 Mochida Memorial Foundation for Medical and Pharmaceutical Research
  “The mechanism by which 14q32 translocations occur in lymphocytes” (Principal Investigator)
• FY2022 - FY2023 Sumitomo Foundation
  “R-loop over-processing during DNA double-strand break repair responses” (Principal Investigator)
• FY2022 - FY2023 SGH Foundation
  “The mechanism of acrocentric chromosome translocations in blood cancer” (Principal Investigator)
• FY2022 - FY2023 Kowa Life Science Foundation
  “How chromosome translocations occur in leukemia cells” (Principal Investigator)
• FY2021 - FY2025 Grant-in-Aid for Scientific Research (B)
  “Investigation of the new factors regulating transcription-associated DNA double-strand break repair” (Principal Investigator)
• FY2020 - FY2023 Fund for the Promotion of Joint International Research (Fostering Joint International Research (A)
  “The mechanism for gene fusion mediated by condensates” (Principal Investigator)
• FY2019 - FY2020 Yamada Science Foundation
  “How the R-loop structure is protected during DNA double-strand break repair” (Principal Investigator)
• FY2019 - FY2020 Takeda Science Foundation
  “Elucidation of the mechanism of tumorigenesis via comprehensive understanding of transcription-associated DNA double-strand break repair” (Principal Investigator)
• FY2018 - FY2020 JSPS Grant-in-Aid for Early-Career Scientists
  “Genome stability maintenance mechanism via R-loop structure inducing DNA damage signaling” (Principal Investigator)

List of publications on radiation research

1. Yasuhara, T., Xing, YH., Bauer, NC., Lee, LK., Dong, R., Soberman, RJ., Rivera, MN., and Zou, L. Defective RNAPII elongation mislocalizes active chromatin to nucleoli and promotes gene fusion. Molecular Cell 82:2738-2753 2022.
2. Uchihara, Y., Permata, TBM., Sato, H., Kawabata-Iwakawa, R., Katada, S., Gu, W., Kakoti, S., Yamauchi, M., Kato, R., Gondhowiardjo, S., Hosen, N., Yasuhara, T., & Shibata, A. DNA damage promotes HLA class I presentation by stimulating a pioneer round of translation associated antigen production. Molecular Cell 82:2557-2570 2022.
3. Yasuhara, T.*,^#, Kato, R.^#, Yamauchi, M., Uchihara, Y., Zou, L., Miyagawa, K.*, and Shibata, A.* RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair. Cell Reports 38:110335 2022. (*co-corresponding, ^#co-first)
4. Krishnan, B., Yasuhara, T., Rumde, PH., Stanzione, M., Lu, C., Lee, H., Lawrence, MS., Zou, L., Nieman, LT., Sanidas, I., and Dyson, NJ.Active RB causes visible changes in nuclear organization. Journal of Cell Biology 221:e202102144 2022.
5. Kot, P., Yasuhara, T., Shibata, A., Hirakawa, M., Abe, Y., Yamauchi, M., and Matsuda, N. Mechanism of chromosome rearrangement arising from single-strand breaks. Biochemical and Biophysical Research Communications 572:191-196 2021.
6. Permata, T. B. M., Sato, H., Gu, W., Kakoti, S., Uchihara, Y., Yoshimatsu, Y., Sato, I., Kato, R., Yamauchi, M., Suzuki, K., Oike, T., Tsushima, Y., Gondhowiardjo, S., Ohno, T., Yasuhara, T., & Shibata, A. High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells. Journal of Radiation Research 62:773-781 2021.
7. Genois, MM., Gagné,JP., Yasuhara, T., Jackson, J., Saxena, S., Langelier, MF., Ahel, I., Bedford, MT., Pascal, JM., Vindigni, A., Poirier, GG, and Zou, L. CARM1 Regulates Replication Fork Speed and Stress Response by Stimulating PARP1. Molecular Cell 81: 784-800 2021.
8. Nakajima, N., Yamauchi, M., Kakoti, S., Cuihua, L., Kato, R., Permata, MBT, Iijima, M., Yajima, H., Yasuhara, T., Yamada, S., Hasegawa, S., and Shibata, A. RNF8 promotes high linear energy transfer carbon-ion-induced DNA double-stranded break repair in serum-starved human cells. DNA Repair 91-92:102872 2020.
9. Kakoti, S., Yamauchi, M., Gu, W., Kato, R., Yasuhara, T., Hagiwara, Y., Laskar, S., Oike, T., Sato, H., Held, KD., Nakano, T., and Shibata, A. p53 deficiency augments nucleolar instability after ionizing irradiation. Oncology Reports 42: 2293-2302 2019.
10. Permata, MBT, Hagiwara, Y., Sato, H., Yasuhara, T., Oike, T., Gondhowiardjo, S., Held, KD., Nakano, T., and Shibata, A. Base excision repair regulates PD-L1 expression in cancer cells. Oncogene 38:4452-4466 2019.
11. Yasuhara, T.*^,#, Kato, R.^#, Hagiwara, Y., Shiotani, B., Yamauchi, M., Nakada, S., Shibata, A., and Miyagawa, K. Human Rad52 promotes XPG-mediated R-loop processing to initiate transcription-associated homologous recombination repair. Cell 175:558-570 2018. (*Lead contact, ^#co-first)
12. Sato, H., Niimi, A., Yasuhara, T., Isono, M., Permata, MBT, Sekine, R., Oike, T., Nuryadi, E., Kakoti, S., Yoshimoto, Y., Held, KD, Suzuki, Y., Kono, K., Miyagawa, K., Nakano, T., and Shibata, A. DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells. Nature Communications 8:1751 2017.
13. Yasuhara, T., Suzuki, T., Katsura, M. and Miyagawa, K. Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength. Nature Communications 5:5426 2014.