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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结