2021 Fiscal Year Annual Research Report
Molecular analysis and universality exploration of DNA damage-triggered reprogramming to stem cells
| Project/Area Number |
21F21084
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| Research Institution | Utsunomiya University |
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Principal Investigator |
玉田 洋介 宇都宮大学, 工学部, 准教授 (50579290)
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| Co-Investigator(Kenkyū-buntansha) |
GU NAN 宇都宮大学, 工学部, 外国人特別研究員
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| Project Period (FY) |
2021-04-28 – 2023-03-31
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| Keywords | Stem cell / Reprogramming / Physcomitrium patens / DNA damage / snRNA-sequencing |
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| Outline of Annual Research Achievements |
We found that transient DNA damage can induce the reprogramming of differentiated leaf cells into stem cells without wounding and exogenous phytohormones in the moss Physcomitrium patens. Only a part of leaf cells reprogrammed into stem cells after the DNA damage. Thus, single-cell transcriptome analysis is effective to identify key genes and the regulatory network involved in DNA damage-triggered reprogramming. Physcomitrium leaf cells cannot be isolated due to rigid cell walls, thus we have to perform single-nucleus RNA-sequencing to achieve single-cell transcriptome analysis. To this end, we established a nuclei isolation method suitable for DNA-damage reagent treated gametophores and then successfully built single-nucleus RNA sequencing libraries with more than 40,000 nuclei in total. To confirm whether the positive role of DNA damage in cellular reprogramming also exists in other organisms, we selected and started culturing several angiosperm plants.
DNA Topoisomerase 1 (TOP1) is an enzyme, which releases the topological stress of DNA strands during DNA duplication, transcription, and damage repair. TOP1 induces the single-strand break, releases the topological stress, and repairs the break. We found that TOP1 is necessary for the synchronous and effective nuclei contraction during the Physcomitrium spermatogenesis. This suggests that tight chromatin condensation during spermatogenesis also involves DNA single strand break and the release of the topological stress mediated by TOP1.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
No effective nucleus isolation method for Physcomitrium was available and we unexpectedly met severe problems during the nucleus isolation including the aggregation and elution of the nuclei. However, we finally solved the problems and successfully established an effective nucleus isolation method and prepared high-quality sequencing libraries with more than 40,000 nuclei in total. In addition, we found that TOP1 is involved in the synchronous and effective nuclei contraction during the spermatogenesis, suggesting the novel role of DNA damage and releasing the topological stress in tight chromatin condensation. Taken together, we decided the current status as "Overall progressing well".
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| Strategy for Future Research Activity |
We will perform the sequencing of single-nucleus sequencing libraries prepared using the Physcomitrium gametophores with or without the treatment of a DNA-damage reagent. Then, we will analyze the data and obtain the cell-fate trajectory during the reprogramming from differentiated leaf cells to stem cells. Reprogramming and non-reprogramming cells can be separated by the expression of STEMINs. We will identify the candidate key genes involved in DNA damage-triggered reprogramming. We will analyze the role of the candidate genes by producing the knock-out mutant of these genes with a highly efficient gene knock-out system based on CRISPR-Cas9 and the fluorescent protein gene knock-in lines. Through above research, we will understand the whole gene regulatory networks underlying DNA damage-triggered reprogramming.
We found that DNA damage-induced STEMIN1 expression depends on the DNA damage sensor kinase ATR. To examine whether active ATR can induce STEMIN1 expression without DNA damage, we will generate an inducible expression line of phosphor-mimic ATR and examine whether STEMIN1 expression and the reprogramming is induced by the ectopic expression of phosphor-mimic ATR.
We will investigate whether DNA damage also plays a positive role in the reprogramming in other organisms. We will establish a suitable reprogramming system in each organism for DNA damage-induced reprogramming and confirm the positive role of DNA damage in cellular reprogramming.
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| Remarks |
1, Nan Gu, Yosuke Tamada, A lecture under the Science Dialogue Program of JSPS at Utsunomiya Girls' High School (Nov. 1, 2021)
2, 玉田 洋介, 宇都宮大学学長表彰 優秀賞（研究）（2022年1月12日）
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