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.

概括 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。转录受转录序列特异性结合的调节 DNA 因子 (TF)，通常位于启动子和增强子区域，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高度 TF 活性的信息特征，并且 eRNA 可能直接受 TF 结合调节。目标 1 旨在 确定单个 TF p53 的结合是否可以引起 eRNA 转录。我们首先将重点关注 单基因座以确定 p53 单独结合是否可以启动 eRNA 表达。 DRAM1 基因也是如此 作为上游约 20kb 的 p53 依赖性 eRNA，在用 p53 激活剂处理后迅速被诱导 药物，Nutlin-3。我将使用 CRISPR-Cas9 破坏 DRAM1 eRNA 内的 p53 结合位点，以便 防止 p53 刺激后结合。我们将使用 RT-qPCR 进行核运行来确定是否 p53 结合的丧失会导致 DRAM1 eRNA 和/或基因转录的丧失。接下来，我们将尝试 通过将 p53 反式激活结构域与 dCas9 结合并引导 构建突变的 p53 结合位点并测试 eRNA 转录是否恢复。这种创新的 该方法将明确证明单个 TF 的结合是否可以引起 eRNA 转录，以及是否 eRNA 转录与基因表达增加相关。 TF 内的错义突变很常见 在癌症中，但它们的功能影响往往难以预测。几种常见的 p53 错义突变 在癌症中观察到改变 p53s DNA 结合特异性和靶基因表达，并可能导致增加 相对于 p53 功能完全丧失而言，细胞增殖和预后不良。此类突变经常发生 p53 DNA 结合域内，导致 P53 反式激活能力改变和物理性质改变 p53 和其他转录因子之间的相互作用。确定 p53 突变体是否表现出改变的 p53 结合和/或 eRNA 表达谱，我们将使用 CRISPR-Cas9 生成表达单拷贝突变体的细胞系 HCT116 p53-/- 细胞中的 p53 和野生型形式，并在之前执行 p53 ChIP-seq 和 GRO-seq，并且一 Nutlin 激活 p53 后一小时。从这些分析中，我们将恢复直接的 p53 野生型和突变型 特定基因和 eRNA 目标。接下来，我们将执行增强器分析来测试其他 TF 是否 p53 突变体存在时被激活。最后，我们会问是否设计用于恢复野生型p53的药物 突变 p53 菌株中的功能能够恢复 p53 野生型 eRNA 和基因表达谱。