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，以确定基因组中RNA：DNA杂交形成的位点。我们还计划收集信息，这将使我们深入了解这些基因座的功能。该提议的基础是相当大量的初步数据，这些数据显示检测RNA的抗体：DNA杂交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，这些基因座的特征与体外和其他先前研究预测的特征一致， 多嘌呤偏斜、rDNA和端粒的富集以及类似mtDNA复制起点的位点。我们对这些基因座的功能有了一些早期的了解，从它们与完全沉默的基因的物理联系，以及从染色质免疫沉淀的蛋白质的质谱分析。 我们的资助建议是基于全基因组定位分析的进一步发展，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得更详细的质谱数据，并对所确定的候选人进行严格的验证。最后，我们将对我们看到的RNA：DNA杂交体形成的序列进行生物信息学研究，扩展我们目前的分析，这些分析显示了嘌呤：嘧啶偏斜的有趣模式，并将它们的形成与基因表达和DNA复制时机等功能结果相关联。 基因组的许多调节机制都假设潜在的双链DNA是默认的，但是如果RNA：DNA杂交体在某个位点形成，它将改变我们对该位点结合转录因子、进行DNA甲基化或组织成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，确定这些基因座的先天特性，这将使我们能够增加一层关于基因组如何运作的信息。如果这个探索性项目成功，我们将清楚地知道如何在未来将研究扩展到一个更全面的项目。PHS 398/2590（Rev.11/07） 公共卫生关系： 基因组本质上是复杂的，受到我们刚刚开始了解的许多机制的调节。一个未被探索的领域是不寻常的DNA结构可能发挥的作用，这是相对难以研究的。DNA通常以沃森和克里克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，我们已经进行了许多实验来研究基因是如何被调节的，这些实验是建立在DNA在活细胞中是如何组织自己的假设之上的。然而，这一规则也有例外，其中之一是基因组中存在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是，一些基因座似乎没有放弃RNA，使其与DNA链紧密结合，形成RNA：DNA杂交体，并使剩余的未配对DNA链处于单链构象，即所谓的R环。 这些结构中的大多数不是在活细胞中发现的，而是在人工条件下通过生物化学方法重新创建的。我们的兴趣是看看我们是否可以确定它们在活的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测定，开始，以确定这些序列结合的蛋白质，并了解其功能的计算生物学方法。拨款建议说明我们计划如何进一步发展这些研究，因为在拨款前阶段已取得一些重大进展。 拟议的项目不包括人类研究，因为在我们有一个完善的系统之前，这些研究还为时过早，但将分析系统应用于人类疾病研究将在一些领域。第一个是衰老-单链DNA更容易受到与年龄相关的氧化损伤，而染色体的端粒对衰老非常有影响，已经确定是RNA：DNA杂交形成位点。癌症是另一个感兴趣的领域-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，并作为某些化疗药物的特异性基因组靶点。我们急于进入项目阶段，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有一个精心设计和强大的系统，促使目前的探索性赠款资助提案。