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

在2013财年，我们在胚胎干细胞中p53信号的研究取得了重大进展。1)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）。然而，目前尚不清楚胚胎干细胞中的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细胞和MEF细胞中的p53信号来解决这个问题。目前我们正在进一步研究胚胎干细胞中p53特异性调控的基因组，希望从抑制肿瘤的作用来剖析p53的发育作用。2)除了全基因组的研究，我们也在进行个体基因的补充研究。矛盾的是，p53不仅能杀死肿瘤细胞，还能促进肿瘤细胞的存活。p53的这种双重功能是在DNA损伤时由p53调控的单个基因介导的。在2013财年，我们重点研究了一个新的p53靶点Rap2b，发现它在DNA损伤后的促存活中起着重要作用。我们发现Rap2b是帮助肿瘤细胞在DNA损伤后存活的基因之一。Rap2b的抑制使肿瘤细胞对阿霉素（一种化疗药物）敏感。因此，对p53信号中单个基因的研究有可能发现新的癌症治疗靶点，并最大限度地发挥化疗的肿瘤杀伤作用。3)只有2%的基因组表达蛋白质编码转录物；其余部分积极表达非编码rna，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在胚胎干细胞中的作用在很大程度上尚未被探索。我们假设非编码rna部分介导了胚胎干细胞中p53的功能。在2013财年，我们发现了几个可能参与胚胎干细胞中p53信号传导的长链非编码rna （lncRs）。其中一些lncRs与染色质结合蛋白相互作用。我们目前正在研究这些lncRs在胚胎干细胞中的作用，重点是分化调控。本研究拓宽了我们对p53在ES细胞中的生物学功能的认识。4)人胚胎干细胞（hES）是小鼠胚胎干细胞的补充模型。在上一个财政年度，我们从康涅狄格大学干细胞研究所获得了两种人类胚胎干细胞系。这两种he细胞系是美国国立卫生研究院批准的he细胞系，可以使用联邦资金进行研究。在2013财年，我们与国家心肺血液研究所（NHLBI）的陈国凯博士的干细胞设施建立了合作，以产生p53敲除的人类胚胎干细胞。陈博士现在是NHLBI干细胞设施的负责人。我们还与Rafael Casellas博士（NIAMS， LCBG的助理研究员）合作，使用基因组编辑工具删除lncr。我们将使用这两种hES细胞系和p53敲除hES细胞系研究人类胚胎干细胞中的p53信号。然后，我们将比较小鼠胚胎干细胞和hES细胞中的p53信号，并得出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 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 is now the head of the stem cell facility at NHLBI. We are also collaborating with Dr. Rafael Casellas (NIAMS, an adjunct LCBG investigator) on using genome editing tools to delete the lncRs. 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.