Study the role(s) of p53 in embryonic stem cells

在2013财年，我们在胚胎干细胞中p53信号通路的研究取得了重大进展。1)我们先前已经绘制了ES细胞中的全局p53信号传导（Li M等人，Molecular Cell，2012）。然而，目前尚不清楚ES细胞中的p53信号与小鼠胚胎成纤维细胞（MEF）中的p53信号有何不同。在2013财年，我们通过在全基因组范围内比较和对比ES细胞与MEF细胞中的p53信号传导来解决这个问题。我们现在正在进一步研究ES细胞中p53特异性调控的基因集，并希望从其肿瘤抑制作用中剖析p53的发育作用。2)除了全基因组研究外，我们还对单个基因进行补充研究。有趣的是，p53不仅杀死肿瘤细胞，而且促进肿瘤细胞的存活。p53的这种双重功能是由DNA损伤时p53调控的单个基因介导的。在2013财年，我们专注于一个新的p53靶点Rap 2b，发现它在DNA损伤后的促生存中起着重要作用。我们发现Rap 2b是帮助肿瘤细胞在DNA损伤后存活的基因之一。Rap 2b的抑制使肿瘤细胞对化疗剂阿霉素敏感。因此，对p53信号传导中的单个基因的研究具有发现癌症治疗的新治疗靶点和最大化化疗的肿瘤杀伤效果的潜力。3)只有2%的基因组表达蛋白质编码转录本;其余的积极表达非编码RNA，在各种生物过程中发挥重要作用。然而，这些非编码RNA在ES细胞中的作用在很大程度上尚未探索。我们推测非编码RNA部分介导了p53在ES细胞中的功能。在2013财年期间，我们已经鉴定了几种可能参与ES细胞中p53信号传导的长非编码RNA（lncR）。这些lncR中的一些与染色质结合蛋白相互作用。我们目前正在研究这些lncRs在ES细胞中的作用，重点是分化调控。本研究为进一步了解p53在ES细胞中的生物学功能奠定了基础。4)人ES（hES）细胞是小鼠ES细胞的补充模型。在上一个财政年度，我们从康涅狄格大学的干细胞设施获得了两个人类ES细胞系。这两种hES细胞系是NIH批准的hES细胞系，可以使用联邦资金进行研究。在2013财政年度，我们与国立心肺血液研究所（NHLBI）的陈国凯博士的干细胞设施建立了富有成效的合作，以产生p53基因敲除的人类ES细胞。陈博士曾在威斯康星州大学与hES细胞研究的先驱James Thompson博士一起接受培训，他现在是NHLBI干细胞设施的负责人。我们将使用这两种hES细胞系和p53敲除的hES细胞系研究人ES细胞中的p53信号传导。然后，我们将比较小鼠ES细胞和人ES细胞中的p53信号传导，并推导出由p53控制的保守转录网络。

During fiscal year (FY) of 2013, we have made significant progress of studying the p53 signaling in embryonic stem cells. 1) We have previously mapped a global p53 signaling in ES cells (Li M, et al., Molecular Cell, 2012). However, it is unknown how the p53 signaling in ES cells is different from that in mouse embryonic fibroblast (MEF) cells. In FY2013, we have addressed this question by comparing and contrasting the p53 signaling in ES cells with that in MEF cells on a genome-wide scale. We are now further investigating the gene sets that are specifically regulated by p53 in ES cells, and hoping to dissect the developmental role of p53 from its tumor suppressive role. 2) In addition to the genome-wide study, we are also performing complementary studies on individual genes. Paradoxically, p53 not only kills but also promotes the survival of tumor cells. This dual function of p53 is mediated by individual genes regulated by p53 upon DNA damage. In FY2013, we focused on a novel p53 target, Rap2b, and found that it plays an important role in the pro-survival after DNA damage. We found that Rap2b is one of the genes that help tumor cells survive after DNA damage. The inhibition of Rap2b sensitizes tumor cells to Adriamycin, a chemotherapy agent. Therefore, studies on individual genes in the p53 signaling have the potential of discovering novel therapeutic targets for cancer treatment and maximizing the tumor-killing effect of chemotherapy. 3) Only 2 percentage of the genome expresses protein-coding transcripts; the rest actively expresses non-coding RNAs, which play important roles in various biological processes. However, the roles of these non-coding RNAs in ES cells are largely unexplored. We hypothesize that noncoding RNAs partially mediate the function of p53 in ES cells. During FY2013, we have identified several long non-coding RNAs (lncRs) that may be involved in p53 signaling in ES cells. Some of these lncRs interact with chromatin-binding protein. We are currently studying the roles of these lncRs in ES cells with a focus on differentiation regulation. This study could broaden our understanding of the biological function of p53 in ES cells. 4) Human ES (hES) cells are a complementary model to mouse ES cells. In the previous fiscal year, we have obtained two human ES cell lines from the Stem Cell Facility at the University of Connecticut. These two hES cell lines are NIH-approved hES cell lines that can be studied using federal funding. In FY2013, we have established a fruitful collaboration with Dr. Guokai Chen's stem cell facility at the National Heart, Lung, and Blood Institute (NHLBI) to generate p53 knockout human ES cells. Dr. Chen was trained with Dr. James Thompson, the pioneer of hES cell study, at the University of Wisconsin, and he is now the head of the stem cell facility at NHLBI. We will study p53 signaling in human ES cells using these two hES cell lines and the p53 knockout hES cell lines. Then, we will compare the p53 signaling in mouse ES cells to that in hES cells and derive a conserved transcriptional network commanded by p53.