Characterization of RNA-Chromatin Interactome by RNA-DNA Ligation and Sequencing

通过 RNA-DNA 连接和测序表征 RNA-染色质相互作用组

• 批准号: 8827738
• 负责人: YIJUN RUAN
• 金额: $ 71.24万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8827738
• 关键词: Address; Binding; Bioinformatics; Biological; Biological Assay; Biological Markers; Books; Cancer Patient; Cell Line; Cell Lineage; Cells; Chromatin; Chromatin Fiber; Chromatin Modeling; Chromosomes; Communities; Complementary DNA; Complex; DNA; DNA Ligation; DNA Sequence; Data; Data Analyses; Diagnostic; Dimensions; Disease; Elements; Endothelial Cells; Epigenetic Process; Event; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Genome; Genomics; Goals; HeLa S3; Health; Human; Human Cell Line; Immunofluorescence Immunologic; Individual; Investigation; Karyotype; Knowledge; Libraries; Ligation; Linker DNA; Location; MCF7 cell; Malignant Neoplasms; Malignant neoplasm of ovary; Maps; Mediating; Methods; Neoplasm Metastasis; Noise; Normal Cell; Nuclear; Oligonucleotides; Output; Patients; Proteins; Protocols documentation; Public Health; RNA; RNA Sequences; RNA analysis; RNA immunoprecipitation sequencing; RNA-Protein Interaction; Role; Sensitivity and Specificity; Sequence Analysis; Site; Sonication; Specificity; Stem cells; Technology; Time; Umbilical vein; Untranslated RNA; Validation; Work; base; biological systems; chromatin protein; comparative; crosslink; design; epigenomics; experimental analysis; genome-wide; human data; human embryonic stem cell; improved; in vivo; insight; interest; method development; neoplastic cell; new technology; novel; prototype; research study; therapeutic target; tool; transcriptome sequencing; tumor

DESCRIPTION (provided by applicant): Noncoding RNAs (ncRNAs) are now believed to be transcribed pervasively in the genome, and large numbers of ncRNAs have been identified. However, disproportionally, we still know very little about their functional roles. Many of the known ncRNA functions were inferred by perturbation experiments, which lack the details of what specific target an ncRNA interact with. Technologies like CLIP/RIP-Seq have provided tremendous insights of what kind of ncRNA the protein factors associated, and ChIRP-Seq have generated the chromatin loci for some ncRNAs to interact with, which have suggested that in particular long non-coding RNAs (lncRNAs) are involved in epigenomic regulation of gene expression and chromatin modeling. However, current methods are limited to examine ncRNA or interacting target one at a time. It is desirable to have an unbiased genome-wide strategy to identify the functional targets for all ncRNAs. We hypothesize that if an ncRNA had an epigenetic regulatory role in the nuclear space, it would have to either directly or indirectly interact with chromatin at certain locations in chromosomes, in which functions take place for modulating chromatin states and target gene activity. Hence, we propose to develop a new technology to globally map ncRNA-chromatin interactions through RNA-DNA ligation followed by paired-end-tag sequencing (R&D-PET). In brief, this method includes three main parts: 1) chromatin crosslinking to capture all molecular interaction events between RNA, DNA and proteins in vivo; 2) ligation of the tethered interactive RNA and the chromatin DNA fragment through specifically designed RNA linker and DNA linker oligos; 3) sequencing and mapping analysis of the RNA-DNA ligation products to localize ncRNAs' transcription sites and their chromatin target sites in the genome. We also realize that this RNA-DNA ligation approach can be applied to study RNA-protein interaction at specific chromatin locations. Thus a ChIP-based R&D-PET method could provide additional specificity of RNA-protein- chromatin interaction information. We have developed a prototype protocol for R&D-PET analysis, and have generated some promising preliminary data from human cells. In this proposal, we plan to further refine the R&D-PET method through systematic optimizations of key experimental conditions and improvement of bioinformatic analysis pipeline. We also plan to apply this method to comprehensively characterize the ncRNA- chromatin interactomes for a number of established human cell lines and stem cells derived from individual cancer patients. The successful development of this method will significantly increase our capability of investigating the immense complex world of RNA functions in regulating the output of the genome, and the successful completion of the proposed characterization of RNA-chromatin interactomes would provide a comprehensive chromatin address book for most of ncRNA species, which would add another dimension of genomic information to help understand how the genome functions in healthy and disease conditions.

描述（由申请人提供）： 现在认为非编码 RNA (ncRNA) 在基因组中普遍转录，并且已鉴定出大量 ncRNA。然而，不成比例的是，我们对它们的功能作用仍然知之甚少。许多已知的 ncRNA 功能都是通过微扰实验推断出来的，但缺乏 ncRNA 与特定靶点相互作用的细节。 CLIP/RIP-Seq 等技术为了解与蛋白质因子相关的 ncRNA 类型提供了深入的见解，ChIRP-Seq 生成了一些 ncRNA 相互作用的染色质位点，这表明长非编码 RNA (lncRNA) 特别参与基因表达和染色质建模的表观基因组调控。然而，目前的方法仅限于一次检查一个 ncRNA 或相互作用的靶标。希望有一个公正的全基因组策略来识别所有 ncRNA 的功能靶标。我们假设，如果 ncRNA 在核空间中具有表观遗传调节作用，那么它必须直接或间接与染色体中某些位置的染色质相互作用，在该位置上发挥调节染色质状态和靶基因活性的功能。因此，我们建议开发一种新技术，通过 RNA-DNA 连接和配对末端标签测序 (R&D-PET) 来全局绘制 ncRNA-染色质相互作用图谱。简而言之，该方法包括三个主要部分：1）染色质交联，捕获体内RNA、DNA和蛋白质之间的所有分子相互作用事件； 2) 通过专门设计的RNA接头和DNA接头寡核苷酸连接连接的相互作用RNA和染色质DNA片段； 3) 对RNA-DNA连接产物进行测序和作图分析，以定位基因组中ncRNA的转录位点及其染色质靶位点。我们还意识到这种 RNA-DNA 连接方法可用于研究特定染色质位置的 RNA-蛋白质相互作用。因此，基于 ChIP 的 R&D-PET 方法可以提供 RNA-蛋白质-染色质相互作用信息的额外特异性。我们开发了用于 R&D-PET 分析的原型方案，并从人体细胞中生成了一些有希望的初步数据。在本提案中，我们计划通过关键实验条件的系统优化和生物信息分析流程的改进，进一步细化R&D-PET方法。我们还计划应用这种方法来全面表征许多已建立的人类细胞系和源自个体癌症患者的干细胞的 ncRNA-染色质相互作用组。该方法的成功开发将显着提高我们研究RNA功能在调节基因组输出方面的巨大复杂世界的能力，并且成功完成所提出的RNA-染色质相互作用组表征将为大多数ncRNA物种提供全面的染色质地址簿，这将增加基因组信息的另一个维度，以帮助了解基因组在健康和疾病条件下如何发挥作用。