Molecular analysis and universality exploration of DNA damage-triggered reprogramming to stem cells

本研究旨在了解立碗藓分化叶细胞向干细胞DNA损伤触发重编程的分子机制。为此，我们通过单核RNA测序（snRNA-seq）进行单细胞转录组分析，以跟踪重编程过程中的转录变化。我们成功地获得了高质量的snRNA-seq数据。在核转录组聚类后，我们获得了一组在每个聚类中特异表达的基因。通过对这些特异表达基因的启动子报告基因系的荧光观察，将多个簇注释到特定组织。基于这些聚类注释结果，我们可以提取从叶细胞到新形成的原丝体干细胞的细胞核重编程轨迹。通过分析，我们可以确定DNA损伤触发重编程过程中的阶段特异性基因，以及重编程和非重编程叶细胞在后期的差异表达基因，这将提供一个潜在的驱动细胞重编程的关键基因列表。为了探索DNA损伤触发重编程在陆地植物中的普遍性，我们建立了多种被子植物的培养条件，并用Zeocin处理每种植物。在大多数情况下，DNA损伤试剂导致测试植物中的细胞死亡，这表明有效的DNA损伤修复对于成功的细胞重编程至关重要。

Our study aimed to understand the molecular mechanism of DNA damage-triggered reprogramming from differentiated leaf cells into stem cells in Physcomitrium patens. To this end, we conducted single cell transcriptome analysis through single nucleus RNA-sequencing (snRNA-seq) to track transcriptional changes during the reprogramming process. We successfully obtained high quality snRNA-seq data. After the clustering of nuclear transcriptomes, we obtained a set of genes which were specifically expressed in each cluster. With fluorescence observation of promoter reporter lines of these specifically expressed genes, multiple clusters were annotated to specific tissues. Based on these cluster annotation results, we can extract the reprogramming trajectory of nuclei from leaf cells to newly formed protonema stem cells. From this analysis, we can identify stage-specific genes during DNA damage-triggered reprogramming and differentially expressed genes between reprogramming and non-reprogramming leaf cells at the late stage, which will provide a potential list of key genes driving the cellular reprogramming.To explore the universality of DNA damage-triggered reprogramming in land plants, we established culture conditions for multiple angiosperms and treated each plant with Zeocin. In most cases, the DNA damage reagent caused cell death in the tested plants, suggesting that efficient DNA damage repair is critical for successful cellular reprogramming.