Mapping and Functional Analysis of RNA:DNA Hybrid-Forming Loci

RNA:DNA 杂交形成位点的定位和功能分析

• 批准号: 8316684
• 负责人: John Greally
• 金额: $ 25.05万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8316684
• 关键词: Affinity; Age; Antibodies; Area; Back; Bacteriophages; Binding; Binding Proteins; Bioinformatics; Biological; Biological Assay; Cells; Characteristics; Chemicals; Chromatin; Chromosomes; Communities; Complement; Complex; Cytosine; DNA; DNA Methylation; DNA Replication Timing; DNA Structure; DNA-Protein Interaction; Data; Detection; Development; Exploratory/Developmental Grant; Foundations; Funding; Funding Mechanisms; Future; G-Quartets; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Grant; Human; Human Cell Line; Hybrids; Immunoglobulins; Immunoprecipitation; In Vitro; Influentials; Left; Life; Literature; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mitochondrial DNA; Molecular Conformation; Mus; Mutagens; Nucleic Acids; Nucleosomes; Oligonucleotides; Outcome; Parents; Pattern; Pharmaceutical Preparations; Play; Principal Investigator; Property; Protein Analysis; Protein Binding; Proteins; Protocols documentation; Publications; Purines; Pyrimidine; RNA; RNA analysis; Reagent; Recombinant DNA; Regulation; Replication Origin; Research; Ribonucleases; Role; SS DNA BP; Shapes; Signal Transduction; Single-Stranded DNA; Site; Specificity; Staging; Structure; System; Testing; Transcript; Western Blotting; age related; base; candidate validation; cell type; design; ds-DNA; epigenomics; functional outcomes; genome-wide; human data; human disease; in vivo; information gathering; insight; interest; novel; nuclease; nucleic acid structure; oxidative damage; premature; purine; research study; telomere; transcription factor

DESCRIPTION (provided by applicant): We propose to develop further assays to identify the loci at which RNA:DNA hybrids form in the genome. We also plan to gather information that will give us insights into the function of these loci. The foundation for this proposal is a reasonably substantial amount of preliminary data that show an antibody detecting RNA:DNA hybrids can be used to immunoprecipitate such loci in a human cell line, allowing sequencing to map these loci back to the genome. We find that these loci have characteristics consistent with those predicted by in vitro and other prior studies, with polypurine skewing, enrichment in rDNA and telomeres and loci like the mtDNA origin of replication. We have some early insights into the function of these loci from their physical association with genes that are completely silenced, and from mass spectroscopy analysis of proteins immunoprecipitated from chromatin. Our funding proposal is based on the further development of the genome-wide mapping assay, including some orthogonal approaches using other nucleases, affinity reagents and chemical mutagens. We propose to get much more detailed mass spectroscopy data from different cell types from human and mouse, and perform rigourous validation of the candidates identified. Finally, we will perform bioinformatic studies o the sequences at which we see the RNA:DNA hybrids forming, extending our current analyses which show intriguing patterns of purine:pyrimidine skewing, and correlate their formation with functional outcomes like gene expression and DNA replication timing. A lot of the regulatory mechanisms for the genome assume underlying double stranded DNA as the default, but if RNA:DNA hybrids are formed at a certain locus it would change our assumptions about the ability of that locus to bind transcription factors, undergo DNA methylation or organize as nucleosomes. At the very least we will be identifying a variable that has the potential to confound some of these assumptions. We hope to take the insights to a higher level, identifying innate properties of these loci that will allow us to add a layer of information about how the genome functions. If this exploratory project is successful, we will have a clear idea how to expand the study to a more comprehensive project in the future. PHS 398/2590 (Rev. 11/07) Continuation Format Page PUBLIC HEALTH RELEVANCE: The genome is inherently complex and is regulated by numerous mechanisms that we are just beginning to understand. One underexplored area is the role that unusual DNA structures may play, something that has been relatively difficult to study. DNA usually exists in the classical double helix as described by Watson and Crick, two strands of DNA pairing to form double-stranded DNA. As a field, we have performed many experiments to study how genes are regulated built upon the assumption that this is how DNA organizes itself in living cells. There are, however, exceptions to this rule, one being the presence of RNA:DNA hybrids in the genome. RNA is usually associated with DNA only during the act of transcription, following which it leaves the parent DNA molecule and allows the DNA to return to its usual double-stranded conformation. An interesting prior observation is that some loci do not appear to relinquish the RNA, leaving it tightly associated with the DNA strand, forming an RNA:DNA hybrid and leaving the remaining unpaired DNA strand in a single-stranded conformation, a so-called R-loop. Most of these structures were identified not in living cells but in artificial conditions in which hey were recreated biochemically. Our interest was to see whether we could identify where they form in living human cells. We show that we have been able to develop such an assay, to begin to identify the proteins that bind to these sequences, and to understand their function by computational biological approaches. The funding proposal describes how we plan to develop these studies further, having made some significant progress in the pre-funding period. The proposed project does not include human studies, as these would be premature until we have a system well-established, but the application of the analytical system to human disease research will be in a number of areas. The first is ageing - single-stranded DNA is more prone to age-related oxidative damage, and the telomeres of chromosomes, which are very influential in ageing, are well-established to be RNA:DNA hybrid-forming loci. Cancer is another area of interest - we would like to see whether these loci have roles in translocations, as has been proposed, and act as specific genomic targets of certain chemotherapeutic drugs. We are anxious to proceed to the stage of the project that will allow us to perform these clinically-applicable studies, but recognize the need to make sure we have a carefully designed and robust system in place beforehand, prompting the current exploratory grant funding proposal.

描述（由申请人提供）： 我们建议开发进一步的检测方法来鉴定基因组中 RNA:DNA 杂交体形成的位点。我们还计划收集信息，让我们深入了解这些基因座的功能。该提案的基础是相当大量的初步数据，这些数据表明检测 RNA:DNA 杂交体的抗体可用于免疫沉淀人类细胞系中的此类基因座，从而允许测序将这些基因座映射回基因组。我们发现这些基因座具有与体外和其他先前研究预测的特征一致的特征， 多嘌呤倾斜、rDNA 和端粒以及复制起点等位点的富集。通过这些位点与完全沉默的基因的物理关联，以及对染色质免疫沉淀蛋白质的质谱分析，我们对这些位点的功能有了一些初步的了解。 我们的资助提案基于全基因组作图分析的进一步发展，包括使用其他核酸酶、亲和试剂和化学诱变剂的一些正交方法。我们建议从人类和小鼠的不同细胞类型中获取更详细的质谱数据，并对确定的候选者进行严格的验证。最后，我们将对形成 RNA:DNA 杂交体的序列进行生物信息学研究，扩展我们目前显示有趣的嘌呤:嘧啶倾斜模式的分析，并将它们的形成与基因表达和 DNA 复制时间等功能结果相关联。 基因组的许多调控机制都默认以双链 DNA 为基础，但如果在某个基因座形成 RNA:DNA 杂交体，就会改变我们对该基因座结合转录因子、进行 DNA 甲基化或组织为核小体的能力的假设。至少我们将确定一个有可能混淆其中一些假设的变量。我们希望将这些见解提升到更高的水平，识别这些基因座的先天特性，这将使我们能够添加一层有关基因组如何发挥作用的信息。如果这个探索性项目成功，我们将清楚地知道未来如何将研究扩展到更全面的项目。 PHS 398/2590（修订版 11/07）继续格式页 公共卫生相关性： 基因组本质上是复杂的，并受到我们刚刚开始了解的多种机制的调节。一个尚未得到充分探索的领域是不寻常的 DNA 结构可能发挥的作用，这是一个相对难以研究的领域。 DNA通常以沃森和克里克所描述的经典双螺旋形式存在，两条DNA链配对形成双链DNA。作为一个领域，我们进行了许多实验来研究基因是如何受到调节的，我们假设DNA就是在活细胞中组织自身的。然而，这一规则也有例外，其中之一就是基因组中存在 RNA:DNA 杂交体。 RNA 通常仅在转录过程中与 DNA 结合，随后它离开亲本 DNA 分子并允许 DNA 返回其通常的双链构象。一个有趣的先前观察结果是，一些位点似乎没有放弃 RNA，而是使其与 DNA 链紧密相连，形成 RNA:DNA 杂合体，并将剩余的未配对 DNA 链留在单链构象中，即所谓的 R 环。 大多数这些结构不是在活细胞中鉴定的，而是在人工条件下通过生化方法重建的。我们的兴趣是看看我们是否能够确定它们在活人类细胞中形成的位置。我们表明，我们已经能够开发这样的检测方法，开始识别与这些序列结合的蛋白质，并通过计算生物学方法了解它们的功能。资助提案描述了我们计划如何进一步开展这些研究，并在前期资助期间取得了一些重大进展。 拟议的项目不包括人类研究，因为在我们建立完善的系统之前，这些研究还为时过早，但分析系统在人类疾病研究中的应用将在许多领域。第一个是衰老——单链DNA更容易受到与年龄相关的氧化损伤，而染色体端粒对衰老影响很大，已被证实是RNA:DNA杂交形成位点。癌症是另一个令人感兴趣的领域——我们想看看这些基因座是否在易位中发挥作用，正如已经提出的那样，并充当某些化疗药物的特定基因组靶标。我们渴望进入项目阶段，使我们能够进行这些临床适用的研究，但认识到需要确保我们事先拥有一个精心设计和强大的系统，从而促成了当前的探索性拨款资助提案。