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

项目摘要 我们最近发现了4000多个大的基因间非编码RNA基因(LincRNAs)，其中包括数百个 与多种细胞表观遗传调控因子相互作用并对其产生影响的lincRNA 进程和疾病状态。例如，我们发现p53直接调节一种lincRNA，即 正确定位染色质因子以介导依赖p53的细胞凋亡所需的反应 DNA损伤。P53途径在对DNA损伤剂的反应中起着核心作用，包括 最终可能导致细胞转化的环境危害。因此，lincRNAs可能预示着一个新的 理解暴露于环境危害、表观遗传调控和 人类健康。在这里，我们提出了一个多方面的实验和计算方法来识别lincRNAs 调控P53依赖的表观遗传调控，它们的功能作用及其生化机制。 目的1)鉴定染色质相关的lincRNAs和受p53调控的表观遗传景观 暴露在DNA损伤剂中。我们将使原代人类成纤维细胞暴露于一套DNA损伤 对环境有害并识别P53调节的转录组、表观基因组及其 互动。具体地说，我们将使用RNA测序、表观遗传状态来识别P53调节的lincRNAs 利用染色质免疫沉淀测序和与染色质物理相关的lincRNAs 使用RNA免疫沉淀测序技术的因子。 目的2)检测与lincRNA调控相关的细胞和表观遗传表型。在这里，我们将 进行功能丧失筛查以确定干扰p53依赖的表观遗传学和细胞内的lincRNAs 表型。我们将使用和细胞分选和比色分析来检查调节的扰动 LincRNA耗尽时的细胞周期和细胞凋亡。我们还将监测lincRNA调节的表观遗传学 使用一种称为“芯片串”的高吞吐量和高成本效益的方法来改变lincRNA。 目的3)阐明p53调控的lincRNAs的生化机制。在这里，我们将剖析 干扰p53依赖DNA损伤表观遗传调控的lincRNAs生化机制 回应。我们将进行RNA下拉试验以确定与每个lincRNA缺失相关的蛋白质 作图以确定这些复合体的RNA-蛋白质相互作用的位置。我们还将鉴定DNA 使用基于RNA的免疫沉淀的新方法直接与lincRNAs相互作用的位点 化验。然后这些数据集将被用于大量的计算分析，以找到共同的序列和 LincRNAs中可能驱动其表观遗传和细胞调控作用的结构基序。

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.