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: 8673685
• 负责人: YIJUN RUAN
• 金额: $ 72.23万
• 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/8673685
• 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; Functional RNA; Gene Expression Regulation; Gene Targeting; Genetic Transcription; Genome; Genomics; Goals; HeLa S3; 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-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; public health relevance; research study; therapeutic target; tool; 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)： 非编码RNA(NcRNAs)现在被认为是在基因组中广泛转录的，并且已经发现了大量的ncRNAs。然而，不成比例地，我们仍然对它们的功能角色知之甚少。许多已知的ncRNA功能是通过扰动实验推断的，这些实验缺乏关于ncRNA与什么特定靶点相互作用的细节。像CLIP/RIP-Seq这样的技术已经为蛋白质因子相关的ncRNA提供了巨大的洞察力，而Chirp-Seq已经产生了一些ncRNA与染色质相互作用的染色质位点，这表明特别是长的非编码RNA(LncRNAs)参与了基因表达的表观基因组调控和染色质建模。然而，目前的方法仅限于一次一个地检测ncRNA或相互作用的靶标。人们希望有一种公正的全基因组策略来确定所有ncRNA的功能靶点。我们假设，如果ncRNA在核空间具有表观遗传调节作用，它将不得不直接或间接地与染色体上某些位置的染色质相互作用，在这些位置上发生着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的技术，通过RNA-DNA连接和配对末端标签测序(R&D-PET)来全球定位ncRNA-染色质的相互作用。简而言之，该方法包括三个主要部分：1)染色质交联，捕捉体内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连接子和DNA连接子寡核苷酸连接连接的相互作用的RNA和染色质DNA片段；3)对RNA-DNA连接产物进行测序和定位，以定位基因组中ncRNAs的转录位点和染色质靶点。我们还认识到，这种RNA-DNA连接方法可以用于研究特定染色质位置的RNA-蛋白质相互作用。因此，基于芯片的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。我们已经开发了一个用于R&D-PET分析的原型协议，并从人类细胞中产生了一些有希望的初步数据。在这项建议中，我们计划通过对关键实验条件的系统优化和生物信息分析流水线的改进，进一步完善研发-PET方法。我们还计划应用这种方法来全面表征一些已建立的人类细胞系和来自个别癌症患者的干细胞的ncRNA-染色质相互作用。这种方法的成功开发将显著提高我们研究调节基因组输出的巨大复杂的RNA功能的能力，而RNA-染色质相互作用的成功完成将为大多数ncRNA物种提供一个全面的染色质地址簿，这将增加另一个基因组信息维度，以帮助了解基因组在健康和疾病条件下的功能。