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

项目摘要 我们最近鉴定了超过4,000个大的基因间非编码RNA基因（lincRNA），包括数百个 lincRNA与表观遗传调控因子相互作用并影响广谱细胞内的表观遗传调控因子， 过程和疾病状态。例如，我们发现p53直接调节一种lincRNA， 所需的适当定位的染色质因子介导的p53依赖性细胞凋亡的反应 DNA损伤。p53通路在对DNA损伤剂的反应中起核心作用，包括 最终导致细胞转化的环境危害。因此，lincRNA可能预示着一种新的 在理解暴露于环境危害，表观遗传调节和 人体健康在这里，我们提出了一个多方面的实验和计算方法来识别lincRNA 调节p53依赖性表观遗传调节的基因、它们的功能作用和它们的生化机制。 目的1）鉴定p53调控的染色质相关lincRNA和表观遗传景观， 暴露于DNA损伤剂。我们将原代人类成纤维细胞暴露于一系列DNA损伤 试剂是环境危害，并确定p53调节的转录组，表观基因组及其 交互.具体地说，我们将使用RNA测序、表观遗传状态和基因组学方法来鉴定p53调控的lincRNA。 使用染色质免疫沉淀测序和与染色质物理相关的lincRNA 因子使用RNA免疫沉淀测序技术。 目的2）检查与lincRNA调节相关的细胞和表观遗传表型。这里我们将 进行功能丧失筛选以鉴定干扰p53依赖性表观遗传和细胞遗传的lincRNA， 表型我们将使用细胞分选和比色分析来检查在调节细胞中的扰动。 lincRNA耗竭后的细胞周期和细胞凋亡。我们还将监测lincRNA调节的表观遗传 使用称为"ChIP-string"的高通量和具有成本效益的方法改变lincRNA。 目的3）阐明p53调控lincRNA的生化机制。在这里，我们将剖析 lincRNA干扰p53依赖性DNA损伤的表观遗传调控的生化机制 反应我们将进行RNA下拉测定以鉴定与每个lincRNA缺失相关的蛋白质， 定位以确定这些复合物的RNA-蛋白质相互作用的位点。我们还将鉴定 使用基于RNA的免疫沉淀的新方法直接与lincRNA相互作用的位点 比色法然后，这些数据集将用于许多计算分析，以找到共同的序列， lincRNA内的结构基序，可能驱动其表观遗传和细胞调控作用。

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.