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

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，并且已经鉴定了大量的ncRNA。然而，从理论上讲，我们对它们的功能作用仍然知之甚少。许多已知的ncRNA功能都是通过扰动实验推断出来的，这些实验缺乏ncRNA与什么样的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类型提供了巨大的见解，ChIRP-Seq已经为一些ncRNA产生了与之相互作用的染色质位点，这表明特别是长非编码RNA（lncRNA）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检查一个ncRNA或相互作用靶标。期望具有无偏倚的全基因组策略来鉴定所有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，则它必须直接或间接地与染色体中某些位置的染色质相互作用，其中发生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，然后进行配对末端标签测序（R&D-PET），在全球范围内绘制ncRNA-染色质相互作用。简而言之，该方法包括三个主要部分：1）染色质交联以捕获体内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接头寡核苷酸连接拴系的相互作用RNA和染色质DNA片段; 3）RNA-DNA连接产物的测序和作图分析，以定位基因组中ncRNA的转录位点及其染色质靶位点。我们还认识到，这种RNA-DNA连接方法可以应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。我们已经开发了一种用于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。在该提案中，我们计划通过系统优化关键实验条件和改进生物信息学分析管道来进一步完善R&D-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著提高我们研究在调节基因组输出中的RNA功能的巨大复杂世界的能力，并且所提出的RNA-染色质相互作用组的表征的成功完成将为大多数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的功能。

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.