中国科学院昆明动物研究所机构知识库

小鼠胚胎干细胞来源于小鼠胚胎囊胚内细胞团，能在体外进行长期稳定培养，并具有发育多能性。因此，小鼠胚胎干细胞是胚胎发育研究的重要模型，且在再生医学中具有巨大应用前景。基因组的稳定性对于胚胎发育至关重要，基因组不稳定将直接导致胚胎发育的失败以及机体癌变等，同时，基因组的稳定是小鼠胚胎干细胞在再生医学中应用的前提。相对于分化细胞，小鼠胚胎干细胞有着特殊而强大的基因组稳定性维持机制，但是，目前对于小鼠胚胎干细胞的这种特性还知之甚少，还有待进一步深入研究。复制压力是造成基因组不稳定的最主要內源因素之一。相对于分化细胞，小鼠胚胎干细胞细胞周期较短，DNA复制频繁，G1期短暂且没有G1期检查点，因此，小鼠胚胎干细胞遭受着相对更大的复制压力。小鼠胚胎干细胞是如何应对复制压力来维持基因组稳定？掌握其中的机制，将有助于在体外长期大量扩增过程中维持小鼠胚胎干细胞基因组稳定性，从而促进小鼠胚胎干细胞的应用，也将有利于掌握相关疾病的病因，从而为疾病的诊断与治疗带来理论指导。通过对小鼠胚胎干细胞与分化细胞进行比较，我们发现在同等程度的复制压力诱导过程中，小鼠胚胎干细胞保护新合成DNA链稳定性能力、启动阻滞复制叉重启复制能力以及启动处于休眠状态复制叉进行DNA复制的能力均相对较强，即小鼠胚胎干细胞具有更强的复制压力应对能力。通过iPOND实验分离小鼠胚胎干细胞及分化细胞复制叉上的蛋白进行质谱分析，我们找到了小鼠胚胎干细胞复制叉上特异的蛋白复合体Filia-Floped，并且发现Filia与Floped缺失后，虽然小鼠胚胎干细胞保护新合成DNA链稳定性能力以及启动处于休眠状态复制叉的能力没有显著变化，但是其启动阻滞复制叉重启复制能力显著降低，进一步导致了小鼠胚胎干细胞基因组出现不稳定现象。在Filia的质谱数据中发现Filia的相互作用蛋白包括了解旋酶Blm以及泛素化E3连接酶Trim25，并且经实验验证发现Filia-Floped-Trim25-Blm以复合体形成存在，都能定位于复制叉。当Trim25与Blm敲降后，小鼠胚胎干细胞也出现了与Filia-Floped缺失一样的表型，即小鼠胚胎干细胞在复制压力诱导过程中启动阻滞复制叉能力显著降低，并且在长期培养过程中小鼠胚胎干细胞积累了较严重的DNA双链损伤。对Filia-Floped-Trim25-Blm复合体之间调控关系进行分析后发现，Filia-Floped相互协同共同调控Trim25与Blm之间的相互作用。Filia-Floped常态性定位于复制叉，当复制压力发生时，Filia-Floped在复制叉上聚集量被诱导增加且FiliaS151位点被ATR相关通路激活从而形成有功能的复合体，进一步促进Trim25对Blm进行K63位泛素化修饰，使得受阻复制叉上聚集更多的Blm来调控阻滞复制叉重启。与此同时，Filia-Floped复合体还能独立于对Trim25-Blm的调控能对ATR的激活起到一定作用，从而通过ATR对复制压力反应进行调控。在分化细胞中，Trim25也能调控Blm进行K63位泛素化修饰并定位于复制叉，从而调控阻滞复制叉重启。在小鼠胚胎干细胞中，由于Filia-Floped特异复合体的存在，使得Trim25与Blm更高效的富集于复制叉发挥作用；同时Filia-Floped复合体也使得小鼠胚胎干细胞中ATR的激活效率高于分化细胞。因此，小鼠胚胎干细胞特异复合体Filia-Floped使得小鼠胚胎干细胞具有更强的启动阻滞复制叉能力。 

Mouse embryonic stem cells (mESCs) come from the inner cell mass of a blastocyst. It can be cultured stably in vitro and is pluripotent in development, which make it become a typical model for development research and has great application value in regenerative medicine. Genomic stability has a great important role on embryonic development; genomic instability will lead to carcinogenesis and the failure of embryonic development. At the same time, the genomic stability of mESCs is the precondition of its application in regenerative medicine. Mouse embryonic stem cells have unique mechanisms to maintain genomic stability, which is rarely uncovered.Replication stress is one of the main endogenous factors leading to genomic instability. Compared to differentiated cells, mESCs have shorter G1 phase of cell cycle, lack G1 phase check point and have frequent DNA replication, which makes mESCs encounter greater replication stress. However, how do mESCs resolve the replication stress to keep genomic stability? Which still need us to explore to provide enough theory for the application of mESCs in regenerative medicine.Compared to differentiated cells, mESCs have greater ability to protect nascent DNA, to restart stalled replication forks and to fire new replication origins under the HU-induced replication stress. We can summarize that mESCs are superior to differentiated cells in coping with replication stress.We isolated proteins from replication fork through iPOND combined with mass spectrometry analysis in mESCs and differentiated cells. Fortunately, we detected mESCs –specific protein complex Filia-Floped binding to replication forks only in mESCs. The deficiency of Filia and Floped decreased the proportion of stalled replication forks restart, which caused the genomic instability of mESCs, but had no influence on the nascent DNA degradation and new replication origin firing. Mass spectrometry analysis identified the potential interaction partner of Filia， which included a helicase Blm and a E3 ubiquitination ligase Trim25. We demonstrated that Filia-Floped-Trim25-Blm located in the replication forks as a complex. The deficiency of Trim25 and Blm also decreased the proportion of stalled replication forks restart and increased the DNA doubled strands breaks of the long-term cultured mESCs.We analyzed the regulation relationships of Filia-Floped-Trim25-Blm complex, and the results displayed that Filia-Floped synergistically regulated the interaction between Trim25 and Blm. Filia-Floped normally locate in replication forks, which can be enhanced by the HU-induced replication stress, simultaneously, the serine 151 of Filia is phosphorylated through a ATR-dependent manner. So Filia-Floped complex can facilitate the K63 ubiquitination of Blm by trim25, which make Blm recruit on replication forks efficiently to regulate stalled replication forks restart. Meanwhile, Filia-Floped complex also regulate stalled replication forks restart through regulating the activation of ATR, a Trim25-Blm-independent manner.In differentiated cells, Trim25 also facilitate the recruitment of Blm on replication forks through regulating its K63 ubiquitination to promote stalled replication forks restart. In mESCs, the specific complex Filia-Floped boosts the interaction between Trim25 and Blm, and increase the recruitment of Trim25 and Blm on replication forks. Filia-Floped also makes the activation of ATR more efficient. So mESCs can restart stalled replication forks more efficiently through the specific complex Filia-Floped.