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

描述(申请人提供)：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lincRNAs，它们与广泛的细胞过程和疾病状态中的表观遗传调控因子相互作用并产生影响。例如，我们发现p53直接调节一种lincRNA，该lincRNA是适当定位染色质因子所必需的，以介导依赖于p53的细胞凋亡，以响应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可能预示着一种新的范式，可以理解暴露于环境危害、表观遗传调控和人类健康之间的联系。在这里，我们提出了一种多方面的实验和计算方法来识别调节p53依赖的表观遗传调控的lincRNAs，它们的功能作用和它们的生化机制。目的1)研究DNA损伤剂对染色质相关的lincRNAs和受p53调控的表观遗传景观的影响。我们将使原代人成纤维细胞暴露于一套对环境有害的DNA损毁试剂，并鉴定P53调节的转录组、表观基因组及其相互作用。具体地说，我们将使用RNA测序来识别P53调节的lincRNAs，使用染色质免疫沉淀测序来识别表观遗传状态，以及使用RNA免疫沉淀测序技术来识别与染色质因子物理相关的lincRNAs。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表型的lincRNAs。我们将使用和细胞分选和比色分析来检查lincRNA耗尽对细胞周期和细胞凋亡调节的扰动。我们还将使用一种称为“芯片串”的高通量和高成本效益的方法来监测lincRNA调节的表观遗传学变化。目的3)阐明p53调控的lincRNAs的生化机制。在这里，我们将剖析lincRNAs扰乱P53依赖的DNA损伤反应的表观遗传调节的生化机制。我们将进行RNA下拉试验，以确定与每个lincRNA相关的蛋白质，缺失图谱，以确定这些复合体的RNA-蛋白质相互作用的位置。我们还将使用一种基于RNA的免疫沉淀分析的新方法来确定直接与lincRNAs相互作用的DNA位点。然后，这些数据集将被用于大量的计算分析，以找到可能驱动其表观遗传学和细胞调控作用的lincRNAs中的共同序列和结构基序。 公共卫生相关性：我们的研究旨在了解人类健康中的一个普遍问题：每个细胞中存在的相同基因组是如何呈现出协调不同细胞状态的替代身份的，以及这些状态在疾病中是如何被错误调控的？我们最近发现了一类新的大的基因间非编码RNA(LincRNAs)，它们具有调节细胞身份的能力，并且在人类癌症和DNA损伤途径中被错误调控。因此，我们试图建立lincRNAs调节细胞身份及其在人类健康中的作用的基本原则和机制。这些创新的实验和计算方法的最终目标是开发基于RNA的新疗法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Our research aims to understand a universal problem in human health: How does the same genome present in every cell take on alternate identities that orchestrate distinctive cell states and how are these states misregulated in diseases? We recently discovered a novel class of large intergenic non-coding RNAs (lincRNAs) that have the ability to regulate cellular identity and are misregulated in human cancers and DNA damage pathways. Thus, we seek to establish the fundamental principles and mechanisms by which lincRNAs regulate cellular identity and their roles in human health. The ultimate goal of these innovative experimental and computational approaches is to develop novel RNA based therapeutics.