Mechanisms of gene expression control in the p53 network

项目总结。 TP53是人类肿瘤中最常发生突变的抑癌基因。然而，大约一半的癌症 保留野生型的P53蛋白，它通常被其他机制减弱，例如 抑制因子MDM2的过表达。野生型p53的存在打开了一扇机会之窗 这种转录因子的治疗性再激活。这项提议的目标是阐明分子 以细胞类型特异的方式修改p53网络的机制，影响细胞命运选择和 肿瘤抑制下游的P53重新激活。最终目标是使有效的基于p53的靶向 治疗。利用全面的多组学实验和分析渠道，我们确定了三个主要的 以细胞类型特异性方式影响P53信号的调控机制：靶点染色质结构 基因座，次级‘放大’转录因子，以及特定细胞类型的翻译调节因子。我们会 通过制定以下具体目标，详细调查每个机制： 1.明确细胞转化对P53直接编程的影响。我们发现了那个细胞 直接P53转录程序中的类型特定变异与不同的模式相关 染色质结构，包括DNA甲基化的差异。我们现在将检验这一假设 直接P53程序在肿瘤演变过程中通过基于染色质的机制而减弱，该机制限制了 可用的直接P53靶点数量。我们将通过应用我们的多组学管道来研究直接 正常肠上皮和结直肠癌外植体中P53蛋白的表达。 2.明确次级转录因子在P53反应中的作用。我们发现了两部小说 直接的p53靶基因，转录因子ETV7和POU2F2，预计会引发间接的 P53具有完整的肿瘤抑制活性的细胞中的转录程序。我们将定义 ETV7和POU2F2在观察到的P53对全球转录组的影响以及它们的 在体外对依赖p53的细胞反应的贡献，以及在异种移植模型中的肿瘤抑制活性。 3.确定控制细胞对P53激活反应的翻译调控因子。翻译： 数以百计的mRNAs在P53下游以一种细胞类型特异的方式被激活或抑制。我们确认了 3‘非编码区基序和RNA结合蛋白(RBPs)被预测为介导细胞类型特异性翻译 控制力。我们将继续识别和表征功能RNA基序和相互作用的限制性商业惯例在 不同的上下文，并确定它们在体外对P53依赖的细胞反应的贡献以及对 P53在异种移植模型中诱导肿瘤消退。 完成后，这项建议将大大促进我们对内部监管机制的理解 P53网络，并有助于未来设计改进的基于P53的治疗策略。

PROJECT SUMMARY. TP53 is the most frequently mutated tumor suppressor gene in human cancer. However, about half of cancers retain a wild type version of the p53 protein, which is commonly attenuated by other mechanisms, such as overexpression of the repressor MDM2. The presence of wild type p53 opens a window of opportunity for therapeutic reactivation of this transcription factor. The goal of this proposal is to elucidate the molecular mechanisms that modify the p53 network in a cell type-specific manner, affecting cell fate choice and tumor suppression downstream of p53 reactivation. The ultimate goal is to enable effective p53-based targeted therapies. Using a comprehensive multi-omics experimental and analytical pipeline we identified three major regulatory mechanisms affecting p53 signaling in a cell type-specific fashion: chromatin architecture at target loci, secondary ‘amplifying’ transcription factors, and cell type-specific translational regulators. We will investigate each mechanism in detail by developing the following Specific Aims: 1. To define the impact of cellular transformation on the direct p53 program. We discovered that cell type-specific variations in the direct p53 transcriptional program are associated with distinct patterns of chromatin architecture, including differences in DNA methylation. We will now test the hypothesis that the direct p53 program is attenuated during tumor evolution via chromatin-based mechanisms that restrict the number of available direct p53 targets. We will do this by applying our multi-omics pipeline to study the direct p53 program in intestinal organoids derived from normal epithelium and colorectal carcinoma explants. 2. To define the role of secondary transcription factors in the p53 response. We identified two novel direct p53 target genes, the transcription factors ETV7 and POU2F2, which are predicted to elicit the indirect transcriptional program in cells where p53 has an intact tumor suppressive activity. We will define the role of ETV7 and POU2F2 in the observed amplification of p53 effects on the global transcriptome, as well as their contribution to p53-dependent cellular responses in vitro, and tumor suppressive activity in xenograft models. 3. To identify translational regulators controlling the cellular response to p53 activation. Translation of hundreds of mRNAs is activated or repressed downstream of p53 in a cell type-specific manner. We identified 3’UTR motifs and RNA-binding proteins (RBPs) that are predicted to mediate cell type-specific translational control. We will continue to identify and characterize functional RNA motifs and interacting RBPs acting in different contexts, and define their contribution to p53-dependent cellular responses in vitro and to the ability of p53 to induce tumor regression in xenograft models. Upon completion, this proposal will significantly advance our understanding of regulatory mechanisms within the p53 network, and contribute to the future design of improved p53-based therapeutic strategies.