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

描述(由申请人提供)： 我们建议开发进一步的分析方法来确定在基因组中形成RNA：DNA杂交体的位置。我们还计划收集信息，让我们深入了解这些基因座的功能。这一提议的基础是相当大量的初步数据，这些数据表明抗体可以检测RNA：DNA杂交物可以用来免疫沉淀人类细胞系中的这些基因座，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， 多发性偏斜，富含rDNA和端粒，类似于线粒体DNA复制的起源。我们通过它们与完全沉默的基因的物理联系，以及对染色质免疫沉淀蛋白质的质谱分析，对这些基因座的功能有了一些早期的见解。我们的资金建议是基于全基因组图谱分析的进一步发展，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类型的细胞中获得更详细的质谱学数据，并对确定的候选细胞进行严格的验证。最后，我们将对我们看到RNA：DNA杂交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：嘧啶倾斜的有趣模式，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。基因组的许多调控机制都假设潜在的双链DNA是默认的，但如果RNA：DNA杂交体在某个基因座上形成，它将改变我们对该基因座结合转录因子、经历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颠覆其中一些假设的变量。我们希望将这些洞察力带到更高的水平，识别这些基因座的固有属性，这将使我们能够增加一层关于基因组功能的信息。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(11/07版)延续格式页面 公共卫生相关性： 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控。一个未被探索的领域是不寻常的DNA结构可能扮演的角色，这是一个相对较难研究的领域。正如沃森和克里克所描述的那样，DNA通常存在于经典的双螺旋中，两条DNA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控的，建立在DNA是如何在活细胞中自我组织的假设之上。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通常只在转录过程中与DNA结合，之后它离开亲本DNA分子，允许DNA返回到通常的双链构象。先前一个有趣的观察是，一些基因座似乎不会放弃RNA，使其与DNA链紧密相关，形成RNA：DNA杂交体，并将剩余的未配对的DNA链以单链构象保留，即所谓的R环。 大多数这些结构不是在活细胞中鉴定出来的，而是在人工条件下用生化方法重新创造出来的。我们感兴趣的是看看我们是否能确定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样的分析方法，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。拨款建议描述了我们计划如何进一步发展这些研究，因为我们在筹资期内取得了一些重大进展。 拟议的项目不包括人体研究，因为在我们建立一个良好的系统之前，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许多领域进行。首先是衰老--单链DNA更容易受到年龄相关的氧化损伤，而染色体的端粒在衰老过程中非常有影响力，被公认为是RNA：DNA杂交形成的位点。癌症是另一个令人感兴趣的领域--我们希望了解这些基因座是否如已提出的那样在易位中发挥作用，并作为某些化疗药物的特定基因组靶点。我们急于进入项目的阶段，这将使我们能够进行这些临床适用的研究，但我们认识到有必要确保我们事先有一个精心设计和强大的系统，促使目前的探索性拨款提案。

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.