How does p53 binding affect eRNA-dependent transcriptional regulation?

p53 结合如何影响 eRNA 依赖性转录调控？

• 批准号: 9590365
• 负责人: Timothy Read
• 金额: $ 2.6万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9590365
• 关键词: Affect; Antineoplastic Agents; Binding; Binding Sites; CRISPR/Cas technology; Cancer Diagnostics; Cell Line; Cell Proliferation; Cells; ChIP-seq; Computer Simulation; DNA; DNA Binding; DNA Binding Domain; Data; Development; Drug Design; Enhancers; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; HCT116 Cells; Hour; Individual; Knowledge; Lead; Location; Malignant Neoplasms; Measures; Mediating; Methods; Missense Mutation; Molecular Biology; Molecular Profiling; Mutate; Mutation; Nuclear; Oncogenic; Pharmaceutical Preparations; Phenotype; Preclinical Drug Evaluation; Predisposition; Running; Site; Specificity; Statistical Models; TP53 gene; Testing; Transactivation; Transcript; Transcriptional Regulation; Work; cell type; experimental study; global run on sequencing; human disease; improved; innovation; mutant; nutlin 3; outcome forecast; prevent; promoter; protein protein interaction; tool; transcription factor; treatment response; virtual

SUMMARY Differences in transcriptional regulation contribute tremendously to phenotypic diversity, including susceptibility to human disease. Transcription is regulated by the sequence-specific binding of transcription factors (TFs) to DNA, typically within promoter and enhancer regions, where TF binding often correlates with the expression of bidirectional transcripts. Work in our lab suggests that these transcripts represent a highly informative signature of TF activity, and that eRNAs may be directly regulated by TF binding. Aim 1 seeks to determine whether the binding of a single TF, p53, can cause eRNA transcription. We will initially focus on a single locus to determine whether p53 binding alone can initiate eRNA expression. The DRAM1 gene, as well as a p53-dependent eRNA about 20kb upstream, is rapidly induced upon treatment with the p53-activating drug, Nutlin-3. I will use CRISPR-Cas9 to disrupt the p53 binding site within the DRAM1 eRNA in order to prevent p53 from binding following p53 stimulation. We will use nuclear run on RT-qPCR to determine whether the loss of p53 binding results in the loss of DRAM1 eRNA and/or gene transcription. Next, we will attempt to rescue p53 binding at this locus by tethering the p53 transactivation domain to dCas9 and guiding the construct to the mutated p53 binding site and testing whether eRNA transcription is restored. This innovative approach will prove definitively whether the binding of a single TF can cause eRNA transcription, and whether eRNA transcription is correlated with increased gene expression. Missense mutations within TFs are common in cancer, but their functional impact is often difficult to predict. Several p53 missense mutations commonly observed in cancer alter p53s DNA binding specificity and target gene expression, and can lead to increased cellular proliferation and poor prognosis relative to complete loss of p53 function. Such mutations often occur within the p53 DNA binding domain, resulting in altered P53 transactivating capacity and modified physical interactions between p53 and other TFs. To determine whether p53 mutants display altered p53 binding and/or eRNA expression profiles, we will use CRISPR-Cas9 to generate cell lines expressing a single copy of mutant and wildtype forms of p53 in HCT116 p53-/- cells and perform p53 ChIP-seq and GRO-seq before, and one hour after p53 activation by nutlin. From these analyses, we will recover direct p53 wildtype- and mutant- specific gene and eRNA targets. Next, we will perform enhancer profiling to test whether other TFs are activated in the presence of p53 mutants. Finally, we will ask whether drugs designed to recover wildtype p53 function in mutant p53 strains are capable of restoring p53 wildtype eRNA and gene expression profiles.

总结 转录调控的差异极大地促进了表型多样性，包括 对人类疾病的易感性。转录受转录的序列特异性结合的调控 转录因子（TF）与DNA的结合，通常在启动子和增强子区域内，其中TF结合通常与 双向转录本的表达。我们实验室的工作表明这些转录本代表了 TF活性信息标记，且eRNA可直接受TF结合调节。目标1： 确定是否结合一个单一的TF，p53，可以导致eRNA转录。首先，我们将专注于A 单个位点，以确定p53单独结合是否可以启动eRNA表达。DRAM 1基因， 作为p53依赖性eRNA，上游约20 kb，在用p53激活的 药物Nutlin-3我将使用CRISPR-Cas9破坏DRAM 1 eRNA内的p53结合位点， 防止p53在p53刺激后结合。我们将在RT-qPCR上使用核运行来确定是否 p53结合的丧失导致DRAM 1 eRNA和/或基因转录的丧失。接下来，我们将尝试 通过将p53反式激活结构域拴系到dCas 9并引导p53反式激活结构域与dCas 9结合来拯救p53在该基因座处的结合。 构建体与突变的p53结合位点结合并测试eRNA转录是否恢复。这一创新 该方法将明确证明是否结合单个TF可以引起eRNA转录，以及是否 eRNA转录与基因表达增加相关。TF内的错义突变是常见的 但它们的功能影响通常难以预测。常见的几种p53错义突变 在癌症中观察到改变p53 s DNA结合特异性和靶基因表达，并可导致增加 细胞增殖和相对于p53功能完全丧失的不良预后。这种突变经常发生在 在p53 DNA结合结构域内，导致P53反式激活能力改变和物理性质改变 p53与其他转录因子的相互作用为了确定p53突变体是否显示改变的p53结合和/或 eRNA表达谱，我们将使用CRISPR-Cas9来产生表达单拷贝突变体eRNA的细胞系。 和野生型形式的p53在HCT 116 p53-/-细胞中的表达，并在此之前进行p53 ChIP-seq和GRO-seq， nutlin激活p53后24小时。从这些分析中，我们将回收直接p53野生型和突变型- 特异性基因和eRNA靶标。接下来，我们将进行增强子分析，以测试其他TF是否 在p53突变体的存在下活化。最后，我们将询问设计用于恢复野生型p53的药物是否 在突变型p53菌株中的功能能够恢复p53野生型eRNA和基因表达谱。