多能干细胞高效调控DNA复制压力反应的关键lncRNA鉴定与功能分析

基因组稳定是多能干细胞（PSC）干性维持和再生医学应用的前提，研究PSC如何在快速自我更新中维持基因组稳定性具有重要意义。高效处理DNA复制压力是PSC维持基因组稳定的重要方式，但机制远未清楚。我们近期在PSC维持基因组稳定性研究中取得重要进展（Cell Stem Cell 2015; Cell Res 2018）。我们还发现，PSC中存在独特的lncRNAs，参与高效调控复制压力反应。本项目针对这一重要现象，拟系统筛选小鼠胚胎干细胞（ESC）中参与调控DNA复制压力反应的特异lncRNAs，深入研究关键lncRNAs在复制压力反应中的作用及分子机制，揭示关键lncRNA的功能模块，构建ESC高效应答DNA复制压力的调控网络。并构建小鼠模型，解析重要lncRNAs对个体发育的影响。本研究将揭示PSC基因组稳定维持的新机制，并为检测其遗传稳定性提供lncRNA分子标记。

Pluripotent stem cells (PSCs) have promising applications in cell-based regenerative medicine. But genomic instability hampers their applications. Understanding how PSCs maintain their genomic stability is therefore of paramount importance. We and others have shown that PSCs have increased capacity for resolving the DNA replication stress, thereby maintaining genomic stability. However, the underlying molecular mechanisms remain largely unknown. Long non-coding RNAs (lncRNAs) participate in a wide range of cellular activities and functions, by forming functional modules with other proteins, DNA and RNA molecules. We hypothesize that PSCs might utilize specific lncRNAs to deal with DNA replication stress. In this project, we aim to systematically identify the potential lncRNAs playing unique roles in replication stress response in mouse embryonic stem cells (ESCs). We plan to utilize iROND (isolate RNAs on nascent DNA) combined with RNA-seq to find out the lncRNA candidates, and to investigate their potential involvement in replication stress response and genomic stability through DNA fiber assay and neutral comet assay. Following the screening, we plan to pick up several key lncRNAs with the most significant phenotypes for deep functional and mechanistic studies. In the mechanistic studies, we hope to illustrate the key lncRNA interaction factors and understand how these factors mediate the functions of lncRNAs. We will also generate lncRNA knockout mice to examine their in vivo functions on embryonic development. Finally, we will integrate all known ESC-specific replication stress response regulators by network analysis to construct a regulatory network. These studies can deepen our understanding on how PSCs efficiently cope with DNA replication stress to maintain genomic stability. They can also provide us with new biomarkers for detecting the genomic instability in PSCs.