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

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