The roles of p53 in embryonic stem cells

p53在胚胎干细胞中的作用

• 批准号: 9556476
• 负责人: Jing Huang
• 金额: $ 64.32万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556476
• 关键词: Apoptosis; Binding; Biological Models; CRISPR/Cas technology; Cell Differentiation process; Cells; ChIP-seq; Code; DNA; DNA Damage; DNA Sequence Alteration; Data Set; Development; Down-Regulation; Embryo; Enhancers; Feedback; Foundations; Gene Expression Profile; Generations; Genes; Genetic Transcription; Genome; Genome Stability; Glioblastoma; Goals; Hypersensitivity; Inherited; Inner Cell Mass; Knock-out; Malignant Neoplasms; Mammary Neoplasms; Maps; Mediating; Modeling; Molecular; Mus; Mutate; Mutation; Normal Cell; Nuclear; Organism; Outcome; Peptides; Play; Proliferating; Prostatic Neoplasms; Proteins; RNA; Regulation; Role; Signal Transduction; Stress; TP53 gene; Technology; Testing; Tissues; Transcript; Tumorigenicity; base; blastocyst; cancer genome; cell motility; cell type; clinical practice; daughter cell; embryonic stem cell; genetic signature; genome-wide; in vivo; insight; knock-down; mutational status; neoplastic cell; novel; outcome forecast; programs; receptor; response; stem; transcriptome sequencing; tumor; tumor progression; tumorigenesis; tumorigenic

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.

与已分化的细胞相比，ES细胞对DNA损伤诱导的凋亡更敏感。人们认为，这种对DNA损伤诱导的细胞凋亡的超敏性有助于ES细胞的低突变负荷，因为具有由DNA损伤引起的突变DNA的ES细胞通过细胞凋亡被去除。我们推测，ES细胞特异性因子有助于这种ES细胞特异性的DNA损伤诱导的细胞凋亡的超敏反应。在2015财政年度（FY），我们在鉴定这些ES细胞特异性因子方面取得了重大进展，这些因子可能会调节mES细胞对DNA损伤的超敏反应。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）。基于这项早期研究产生的数据集，我们已经确定了ES细胞富集因子，这些因子可能参与ES细胞中的p53信号传导。我们决定集中在一个转录称为Apela的进一步研究，因为Apela是由p53抑制，并编码一个假定的分泌肽，Apela。Apela肽与其受体Aplnr结合，以调节分化细胞中的细胞运动。我们发现Apela阳性调节小鼠ES细胞中p53调节的凋亡，因为Apela的敲低损害了细胞中p53介导的凋亡。令人惊讶的是，Apela的编码能力与其在p53介导的细胞凋亡中的作用无关。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活化。因此，我们在小鼠ES细胞中发现了一个Apela RNA介导的负反馈环，它调节细胞凋亡。鉴于Apela在ES细胞中特异性表达，我们的研究结果为ES细胞对DNA损伤的超敏反应提供了解释。我们对Apela的研究也为研究ES细胞中p53信号调节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ES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，我们确定hnRNPL作为这样的抑制因子，控制p53在ES细胞的活动之一。有趣的是，hnRNPL敲除胚胎在胚泡阶段死亡。我们计划测试hnRNPL是否是体内p53活性的抑制因子。p53信号调节的第二个方面涉及激活的p53对增强子干扰的潜在机制。我们以前发现，p53抑制ES细胞特异性基因，如Nanog，Oct4和Sox 2，通过干扰它们的增强子活性。然而，增强子干扰的机制尚不清楚。我们不能使用Nanog，Oct4或Sox 2作为我们的模型基因来研究机制，因为这些基因中的任何一个的长期下调都会导致ES细胞分化。然而，Apela下调不会引起ES细胞分化。因此，Apela是研究增强子干扰的一个很好的模型基因。我们计划使用Apela和CRISPR技术来研究ES细胞中p53定向增强子干扰的分子机制。