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.

与分化的细胞相比，ES细胞对DNA损伤诱导的凋亡性过敏。人们认为，对DNA损伤诱导的细胞凋亡的过敏性有助于ES细胞的低突变负担，因为通过凋亡通过DNA损伤引起的突变DNA的ES细胞被凋亡消除。我们假设ES细胞特异性因素有助于ES细胞特异性的超敏反应，从而导致DNA损伤诱导的凋亡。在2015年财政年度（FY）期间，我们取得了重大进展，以识别这些ES细胞特异性因素，这些因素可能可能调节MES细胞对DNA损伤的超敏反应。我们以前已经在ES细胞中绘制了全局p53信号传导（Li M等，分子细胞，2012年）。基于这项早期研究产生的数据集，我们已经确定了ES细胞中可能涉及的ES细胞增强因子。我们决定将重点放在一个名为APELA的成绩单上进行进一步研究，因为Apela被p53抑制并编码推定的分泌肽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，随着细胞中APELA损害的p53介导的细胞凋亡，APELA阳性调节小鼠ES细胞中p53调节的细胞凋亡。令人惊讶的是，Apela的编码能力因其在p53介导的细胞凋亡中的作用而易用。取而代之的是，Apela结合并拮抗异质性核核糖蛋白L（HNRNPL）的功能。 HNRNPL与p53相互作用并抑制p53激活。因此，我们发现了调节凋亡的小鼠ES细胞中APELA RNA介导的负反馈回路。鉴于APELA在ES细胞中特异性表达，我们的发现为ES细胞对DNA损伤的过敏性提供了解释。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建立了基础。首先，在没有压力的情况下，需要控制p53活性以使ES细胞增殖。然而，该因子（S）在未知条件下抑制p53活性是未知的。在这里，我们将HNRNPL确定为控制ES细胞中p53活性的这种抑制因素之一。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活性的抑制因素。 p53信号传导调节的第二个方面是对激活p53干扰增强子干扰的机制的关注。我们以前已经发现，p53通过干扰其增强剂活性来抑制ES细胞特异性基因，例如Nanog，Oct4和Sox2。但是，增强子干扰的机制尚不清楚。我们无法将Nanog，Oct4或Sox2用作模型基因来研究机制，因为这两个基因中的任何一个的延长下调都会导致ES细胞分化。但是，APELA下调不会导致ES细胞分化。因此，Apela是研究增强子干扰的良好模型基因。我们计划使用APELA和CRISPR技术研究ES细胞中p53定向增强子干扰的分子机制。