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

描述（由申请人提供）：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，其在广泛的细胞过程和疾病状态中与表观遗传调控因子相互作用并影响表观遗传调控因子。例如，我们发现p53直接调节lincRNA，lincRNA是染色质因子适当定位所需的，以介导响应于DNA损伤的p53依赖性细胞凋亡。p53通路在响应DNA损伤剂（包括最终可导致细胞转化的环境危害）中起核心作用。因此，lincRNA可能预示着一个新的范式，在了解暴露于环境危害，表观遗传调控和人类健康之间的联系。在这里，我们提出了一个多方面的实验和计算的方法来确定lincRNA调节p53依赖的表观遗传调控，其功能作用和生化机制。目的1）鉴定暴露于DNA损伤剂后由p53调控的染色质相关lincRNA和表观遗传景观。我们将原代人成纤维细胞暴露于一套DNA损伤试剂，这些试剂是环境危害，并确定p53调节的转录组，表观基因组及其相互作用。具体而言，我们将使用RNA测序鉴定p53调节的lincRNA，使用染色质免疫沉淀测序鉴定表观遗传状态，使用RNA免疫沉淀测序技术鉴定与染色质因子物理相关的lincRNA。目的2）检查与lincRNA调节相关的细胞和表观遗传表型。在这里，我们将进行功能缺失筛选以鉴定干扰p53依赖性表观遗传和细胞表型的lincRNA。我们将使用细胞分选和比色测定来检查lincRNA耗尽后细胞周期和细胞凋亡调节中的扰动。我们还将使用称为“ChIP-string”的高通量且具有成本效益的方法来监测lincRNA调节的表观遗传变化lincRNA。目的3）阐明p53调控lincRNA的生化机制。在这里，我们将剖析lincRNA的生化机制，干扰p53依赖的DNA损伤反应的表观遗传调控。我们将进行RNA下拉测定以鉴定与每个lincRNA相关的蛋白质，缺失作图以鉴定这些复合物的RNA-蛋白质相互作用的位点。我们还将使用基于RNA的免疫沉淀测定的新方法来鉴定与lincRNA直接相互作用的DNA位点。然后，这些数据集将用于许多计算分析，以找到lincRNA中可能驱动其表观遗传和细胞调控作用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：存在于每个细胞中的相同基因组如何呈现出不同的身份，从而协调不同的细胞状态，以及这些状态如何在疾病中失调。我们最近发现了一类新的大型基因间非编码RNA（lincRNA），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节。因此，我们寻求建立lincRNA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新的基于RNA的治疗方法。

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.