Epigenetic Regulation by Large Non-Coding RNAs in the p53 Mediated DNA Damage Res

p53 介导的 DNA 损伤研究中大非编码 RNA 的表观遗传调控

• 批准号: 8335413
• 负责人: John Louis Rinn
• 金额: $ 40.41万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335413
• 关键词: Adverse effects; Apoptosis; Binding; Biochemical; Biological Assay; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Separation; Cell model; Cell physiology; Cells; Chromatin; Complex; Computer Analysis; DNA; DNA Damage; DNA Repair; Data Set; Disease; Doxorubicin; Environment; Environmental Hazards; Epigenetic Process; Exhibits; Exposure to; Fibroblasts; Functional RNA; Gene Expression Profile; Gene Targeting; Genes; Genome; Genomics; Goals; Health; Histones; Human; Human body; Immunoprecipitation; Lead; Link; Malignant Neoplasms; Maps; Measures; Mediating; Methods; Monitor; Mus; Mutation; Pathway interactions; Phenotype; Play; Protein p53; Proteins; RNA; RNA Sequences; RNA-Protein Interaction; Reagent; Regulation; Repression; Research; Role; Site; Technology; The Sun; Therapeutic; Transactivation; Ultraviolet Rays; base; chromatin immunoprecipitation; cost effective; design; gain of function; histone modification; innovation; insight; loss of function; metaplastic cell transformation; next generation; novel; novel strategies; research study; response

Project Summary We recently identified over 4,000 large intergenic non-coding RNA genes (lincRNAs) including hundreds of lincRNAs that interface with and influence epigenetic regulatory factors across a wide-spectrum of cellular processes and disease states. For example, we discovered that p53 directly regulates a lincRNA that is required for proper localization of chromatin factors to mediate p53 dependent cellular apoptosis in response to DNA damage. The p53 pathway plays a central role in response to DNA damaging agents including environmental hazards that can ultimately lead to cellular transformation. Thus, lincRNAs may herald a new paradigm in understanding the link between exposure to environmental hazards, epigenetic regulation and human health. Here we propose a multifaceted experimental and computational approach to identify lincRNAs that modulate p53-dependent epigenetic regulation, their functional roles and their biochemical mechanisms. Aim 1) To identify chromatin-associated lincRNAs and epigenetic landscapes regulated by p53 upon exposure to DNA-damaging agents. We will expose primary human fibroblasts to a suite of DNA damaging reagents that are environmental hazards and identify the p53 regulated transcriptome, epigenome and their interactions. Specifically, we will identify p53-regulated lincRNAs using RNA-sequencing, epigenetic states using chromatin immunoprecipitation sequencing and the lincRNAs physically associated with chromatin factors using RNA immunoprecipitation sequencing technologies. Aim 2) To examine cellular and epigenetic phenotypes associated with lincRNA modulation. Here we will perform a loss-of-function screen to identify lincRNAs that perturb p53 dependent epigenetic and cellular phenotypes. We will use and cell sorting and colorimetric assays to examine perturbations in the regulation of cell-cycle and cellular apoptosis upon lincRNA depletion. We will also monitor lincRNA regulated epigenetic changes lincRNAs using a high-throughput and cost-effective approach termed "ChIP-string". Aim 3) To elucidate the biochemical mechanisms of p53-regulated lincRNAs. Here we will dissect the biochemical mechanisms of lincRNAs that perturb epigenetic regulation of the p53 dependent DNA damage response. We will carry out RNA-pull down assays to identify proteins associated with each lincRNA, deletion mapping to identify the sites of RNA-protein interactions of these complexes. We will also identify the DNA sites that are directly interacting with lincRNAs using a new approach of RNA based immunoprecipitation assay. These datasets will then be used for numerous computational analyses to find common sequence and structural motifs within lincRNAs that may drive their epigenetic and cellular regulatory roles.

项目摘要 我们最近确定了超过4,000多个基因间非编码RNA基因（Lincrnas） 与广泛细胞相互接触并影响表观遗传调节因子的LincrNA 过程和疾病状态。例如，我们发现p53直接调节了lincrna 适当定位染色质因子需要介导p53依赖性细胞凋亡所必需的 DNA损伤。 p53途径在响应DNA损伤剂（包括）（包括）中起着核心作用 环境危害最终会导致细胞转化。因此，Lincrnas可能会预示着一个新的 了解对环境危害，表观遗传调节和 人类健康。在这里，我们提出了一种多面实验和计算方法来识别Lincrnas 调节p53依赖性表观遗传学调节，其功能作用及其生化机制。 目标1）鉴定由p53调节的染色质相关的LincrNA和表观遗传景观 暴露于DNA伤害剂。我们将将原代人成纤维细胞暴露于一套DNA损害 环境危害的试剂，并识别p53受调节的转录组，表观基因组及其 互动。具体而言，我们将使用RNA测序，表观遗传态确定p53调节的lincRNA 使用染色质免疫沉淀测序和与染色质物理相关的lincRNA 使用RNA免疫沉淀测序技术的因素。 目标2）检查与Lincrna调制相关的细胞和表观遗传表型。我们会在这里 执行功能丧失屏幕以识别依赖p53的表观遗传和细胞的lincrnas 表型。我们将使用和细胞分类和比色测定法检查调节的扰动 LincRNA耗竭后的细胞周期和细胞凋亡。我们还将监视Lincrna调节的表观遗传学 使用称为“碎屑”的高通量和成本效益的方法更改lincrnas。 目标3）阐明了p53调节的LincrNA的生化机制。在这里，我们将剖析 lincRNA的生化机制，这些lincRNA扰动p53依赖性DNA损伤的表观遗传调节 回复。我们将进行RNA-PULL下测定，以识别与每个Lincrna相关的蛋白质，缺失 映射以识别这些复合物的RNA蛋白相互作用的位点。我们还将识别DNA 使用基于RNA的免疫沉淀的新方法直接与LincRNA相互作用的站点 测定。然后，这些数据集将用于大量计算分析，以查找常见序列和 LincrNA中的结构基序可能会驱动其表观遗传和细胞调节作用。