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

基本信息

批准号：
8529570
负责人：
John Greally
金额：
$ 20.14万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8529570
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 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 transcriptome sequencing

项目摘要

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

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 DNA 甲基化的能力的假设。组织为核小体。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