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测定，以识别基因组中RNA的基因座：DNA杂交形成。我们还计划收集信息，以使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，这些数据显示了检测RNA的抗体：DNA杂种可用于在人类细胞系中免疫沉淀该基因座，从而使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基因座的特征， 息肉素偏斜，rDNA和端粒中的富集和基因座，如mtDNA复制的起源。我们从它们与完全沉默的基因的物理联系以及对从染色质免疫沉淀的蛋白质的质谱分析中对这些基因座的功能有一些早期见解。 我们的资金建议基于全基因组映射测定的进一步发展，包括使用其他核酸酶，亲和力试剂和化学诱变剂的一些正交方法。我们建议从人和小鼠中获取来自不同细胞类型的质谱数据的更详细的质谱数据，并对已识别的候选者进行严格的验证。最后，我们将进行生物信息学研究o看到RNA的序列：DNA杂种形成，扩展了当前的分析，这些分析显示了嘌呤的有趣模式：嘧啶偏斜，并将其形成与基因表达和DNA重复时的功能结果相关联。 基因组的许多调节机制都假设基本的双链DNA为默认值，但是如果在某个地方形成RNA：DNA杂种，它将改变我们对该基因座能力结合转录因子的能力，经过DNA甲基化或组织为核体的假设。至少我们将确定一个有可能混淆其中一些假设的变量。我们希望将洞察力提高到更高的水平，确定这些基因座的先天特性，这将使我们能够添加有关基因组功能的信息层。如果这个探索性项目成功，我们将有一个清晰的想法如何将研究扩展到将来更全面的项目。 PHS 398/2590（Rev. 11/07）延续格式页面 公共卫生相关性： 基因组本质上是复杂的，并且受我们刚刚开始理解的多种机制的调节。一个不足的区域是可能发挥不寻常的DNA结构的作用，这是相对难以研究的。如Watson和Crick所述，DNA通常存在于经典的双螺旋中，这两根DNA配对形成了双链DNA。作为一个领域，我们进行了许多实验，以研究基因的调节是基于以下假设：这是DNA如何在生物细胞中组织自身。但是，该规则有例外，其中一个是RNA的存在：基因组中的DNA杂交。 RNA通常仅在转录行为期间与DNA相关，然后它留下了母体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，某些基因座似乎没有放弃RNA，使其与DNA链紧密相关，形成了RNA：DNA杂交，并将其余未配对的DNA链留在单链构象中，即所谓的R-loop。 这些结构中的大多数不是在活细胞中鉴定出来的，而是在人工条件下hey在生物化学上重现的。我们的兴趣是查看我们是否可以识别它们在活着的人类细胞中形成的位置。我们表明，我们已经能够开发这种测定法，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来理解其功能。资金提案描述了我们计划如何进一步发展这些研究，但在筹资期间取得了重大进展。 拟议的项目不包括人类研究，因为在我们建立一个良好的系统之前，这些项目将为时过早，但是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更容易发生与年龄相关的氧化损伤，并且在衰老中非常有影响力的染色体的端粒已良好地成为RNA：DNA混合形成基因座。癌症是另一个感兴趣的领域 - 我们想看看这些基因座在易位中是否有作用，并充当某些化学治疗药物的特定基因组靶标。我们急于进入项目的阶段，这将使我们能够进行这些临床上可申请的研究，但要认识到有必要确保我们事先建立了一个精心设计和健壮的系统，从而促使当前的探索性赠款资金建议。