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.

与分化细胞相比，ES细胞对DNA损伤诱导的细胞凋亡高度敏感。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 细胞的低突变负担，因为具有由 DNA 损伤引起的突变 DNA 的 ES 细胞会被细胞凋亡去除。我们假设 ES 细胞特异性因素导致了 ES 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) 期间，我们在识别这些 ES 细胞特异性因素方面取得了重大进展，这些因素可能调节 mES 细胞对 DNA 损伤的超敏性。我们之前已经绘制了 ES 细胞中的全局 p53 信号传导（Li M 等人，Molecular Cell，2012）。根据这项早期研究生成的数据集，我们已经确定了可能参与 ES 细胞 p53 信号传导的 ES 细胞富集因子。我们决定将重点放在一个名为 Apela 的转录本上进行进一步研究，因为 Apela 受到 p53 抑制并编码一种假定的分泌肽 Apela。 Apela 肽与其受体 Aplnr 结合，调节分化细胞中的细胞运动。我们发现 Apela 正调节小鼠 ES 细胞中 p53 调节的细胞凋亡，因为 Apela 的敲低会损害细胞中 p53 介导的细胞凋亡。令人惊讶的是，Apela 的编码能力对于其在 p53 介导的细胞凋亡中的作用来说是可有可无的。相反，Apela 结合并拮抗异源核核糖核蛋白 L (hnRNPL) 的功能。 hnRNPL 与 p53 相互作用并抑制 p53 激活。因此，我们在小鼠 ES 细胞中发现了 Apela RNA 介导的负反馈环，可调节细胞凋亡。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 损伤的超敏性提供了解释。我们对 Apela 的研究还为研究 ES 细胞中 p53 信号传导调节的两个方面奠定了基础。首先，在无应激条件下，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 活性的因素尚不清楚。在这里，我们确定 hnRNPL 是控制 ES 细胞中 p53 活性的抑制因子之一。有趣的是，hnRNPL 敲除胚胎在囊胚阶段死亡。我们计划测试 hnRNPL 是否是体内 p53 活性的抑制因子。 p53 信号传导调节的第二个方面涉及激活的 p53 增强子干扰的机制。我们之前发现p53通过干扰ES细胞特异性基因的增强子活性来抑制它们，例如Nanog、Oct4和Sox2。然而，增强子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 下调不会导致 ES 细胞分化。因此，Apela 可以作为研究增强子干扰的良好模型基因。我们计划利用 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。