The roles of p53 in embryonic stem cells

与分化细胞相比，胚胎干细胞对DNA损伤诱导的凋亡异常敏感。据认为，这种对DNA损伤诱导的凋亡的超敏感性有助于胚胎干细胞的低突变负荷，因为DNA损伤引起的DNA突变的胚胎干细胞被细胞凋亡清除。我们假设胚胎干细胞特异性因子导致了胚胎干细胞对DNA损伤诱导的细胞凋亡的特异性超敏反应。在2015财年（FY），我们在鉴定这些胚胎干细胞特异性因子方面取得了重大进展，这些因子可能调节胚胎干细胞对DNA损伤的超敏感性。我们之前已经在胚胎干细胞中绘制了一个全局p53信号（Li M等，Molecular Cell, 2012）。根据这项早期研究产生的数据集，我们已经确定了胚胎干细胞富集因子，这些因子可能参与胚胎干细胞中的p53信号传导。我们决定将重点放在一种名为Apela的转录本上进行进一步研究，因为Apela被p53抑制，并编码一种推定的分泌肽Apela。在分化细胞中，Apela肽与其受体applnr结合，调节细胞运动。我们发现Apela对小鼠胚胎干细胞中p53调控的细胞凋亡具有正向调节作用，因为Apela的敲低会损害p53介导的细胞凋亡。令人惊讶的是，在p53介导的细胞凋亡中，Apela的编码能力是不可或缺的。相反，Apela结合并拮抗异质核核糖核蛋白L （hnRNPL）的功能。hnRNPL与p53相互作用并抑制p53的激活。因此，我们在小鼠胚胎干细胞中发现了Apela rna介导的调控细胞凋亡的负反馈回路。鉴于Apela在胚胎干细胞中特异性表达，我们的研究结果为胚胎干细胞对DNA损伤的超敏感性提供了解释。我们对Apela的研究也为研究ES细胞中p53信号调控的两个方面奠定了基础。首先，在非应激条件下，需要控制p53活性以允许胚胎干细胞增殖。然而，在非应激条件下抑制p53活性的因子是未知的。在这里，我们发现hnRNPL是控制胚胎干细胞中p53活性的抑制因子之一。有趣的是，敲除hnRNPL的胚胎在囊胚期死亡。我们计划在体内测试hnRNPL是否是p53活性的抑制因子。p53信号调控的第二个方面涉及激活p53的增强子干扰的机制。我们之前发现p53通过干扰Nanog、Oct4和Sox2等ES细胞特异性基因的增强子活性来抑制它们。然而，增强剂干扰的机制尚不清楚。我们无法使用Nanog、Oct4或Sox2作为模型基因来研究其机制，因为这些基因中的任何一个的长期下调都会导致胚胎干细胞分化。然而，Apela下调并不会导致胚胎干细胞分化。因此，Apela是研究增强子干扰的良好模式基因。我们计划利用Apela和CRISPR技术研究p53定向增强子干扰ES细胞的分子机制。

Compared to differentiated cells, ES cells are hypersensitive to DNA damage-induced apoptosis. It is thought that this hypersensitivity to DNA damage-induced apoptosis contributes to the low mutation burden of ES cells because ES cells with mutated DNA caused by DNA damage are removed by apoptosis. We have hypothesized that ES cell-specific factors contribute to this ES cell-specific hypersensitivity to DNA damage-induced apoptosis. During fiscal year (FY) of 2015, we have made significant progress of identifying these ES cell-specific factors that could potentially regulate the hypersensitivity of mES cells to DNA damage. We have previously mapped a global p53 signaling in ES cells (Li M, et al., Molecular Cell, 2012). Based on the datasets generated by this earlier study, we have identified ES cell-enriched factors that may be involved in p53 signaling in ES cells. We have decided to focus on one transcript called Apela for further study because Apela is repressed by p53 and encodes a putative secretory peptide, Apela. Apela peptide binds to its receptor, Aplnr, to regulate cell movement in differentiated cells. We found that Apela positive regulates p53-regulated apoptosis in mouse ES cells as knockdown of Apela compromised p53-mediated apoptosis in the cells. Surprisingly, the coding ability of Apela is dispensable for its role in p53-mediated apoptosis. Instead, Apela binds and antagonizes the function of heterogeneous nuclear ribonuclear protein L (hnRNPL). hnRNPL interacts with p53 and inhibits p53 activation. Therefore, we have discovered an Apela RNA-mediated negative feedback loop in mouse ES cells that regulates apoptosis. Given that Apela is specifically expressed in ES cells, our findings provide an explanation to the hypersensitivity of ES cells to DNA damage. Our study on Apela also establishes foundations for investigating two aspects of the regulation of p53 signaling in ES cells. First, under unstressed condition, p53 activity needs to be controlled to allow ES cells to proliferate. However, the factor(s) inhibits p53 activity under unstressed condition is(are) unknown. Here, we identified hnRNPL as one of such inhibitory factors that control p53 activities in ES cells. Interestingly, hnRNPL knockout embryos die at the blastocyst stage. We plan to test whether hnRNPL is the inhibitory factor for p53 activity in vivo. A second aspect of the regulation of p53 signaling concerns about the mechanisms underlying the enhancer interference by activated p53. We have previously found that p53 represses ES cell-specific genes, such as Nanog, Oct4, and Sox2, by interfering with their enhancer activities. However, the mechanisms of enhancer interference are unclear. We were not able to use Nanog, Oct4, or Sox2 as our model genes to study the mechanisms because prolonged down-regulation of either of these genes will lead to ES cell differentiation. Apela down-regulation, however, does not cause ES cell differentiation. Therefore, Apela serves as a good model gene to study enhancer interference. We plan to use Apela and CRISPR technology to investigate the molecular mechanism of p53-directed enhancer interference in ES cells.