Quantifying the Genome-wide Distribution of Transcriptionally-engaged RNA Polymer

描述(由申请人提供)： 本方案的主要目标是开发一种通用的、高度敏感的方法，该方法可以在哺乳动物细胞和果蝇的整个基因组中定位转录参与的RNA聚合酶的位置、数量和方向。这种全球核连续和大规模并行测序方法(GRO-SEQ)的初步结果表明，这种方法有可能提供基因组转录状态的全球图景。此外，这些早期结果发现了许多已知启动子上游和邻近的新的反义短转录单位，并提示了迄今为止尚未考虑的调控模式。这项提案旨在严格测试并进一步发展Gro-seq方法，以提供前所未有的、近核苷酸的对基因组上RNA聚合酶位置的解析。目标1是对已经产生结果的第一代GRO-SEQ方法进行优化和批判性评估。这一程序的简单变体应该能够识别聚合酶的类型，即RNA聚合酶I、II或III。目标2是开发第二代超高分辨率的Gro-Seq版本，它以几个核苷酸的分辨率映射邻近RNA的3‘端，与所有转录参与的聚合酶的活性部位相关。Gro-Seq的另一个改编将以高灵敏度识别转录参与的RNA聚合酶II(POL II)的转录起始点。POL II相对于转录起始点的准确位置和状态将识别转录延伸面临速率限制步骤的序列。事实上，最近的研究证明，POL II在启动子下游立即暂停是许多后生动物基因调控的限速步骤。此外，POL II位置与全基因组转录因子图谱的相关性将识别参与这一调控的候选因子。AIM 3将开发GRO-SEQ方法，以便它们可以普遍应用于关键模型系统，包括具有代表性的人类细胞系、小鼠胚胎干细胞和果蝇胚胎。这最后一个目标还将测试全球检测转录快速变化以响应调控信号的能力。最后，虽然该提案的重点是开发强大的、普遍适用的转录参与的RNA聚合酶的分析，但测试这些细胞和生物模型所产生的数据也将为现有的人类ENCODE和模型生物modENCODE项目以及国际调节组联盟的小鼠干细胞努力提供关键的转录信息。由于GRO-SEQ可以在正常情况下以及在感染和疾病期间定义细胞中参与的RNA聚合酶，因此它为许多疾病的基础研究提供了一个有价值的工具，并可以在诊断研究中产生关键信号。项目简介这是一项建议，旨在开发一种新的方法，以前所未有的近核苷酸分辨率绘制整个基因组中转录参与的RNA聚合酶的位置、数量和方向。GRO-SEQ将提供一个通用工具，该工具将适用于ENCODE和modENCODE项目的工作，以分子方式定义高等真核生物中的DNA密码是如何被读取和调节的，以提供发展和维持多细胞生物体所需的基因表达模式。GRO-SEQ可以确定在正常情况下以及在感染和疾病期间细胞中所有参与的RNA聚合酶的水平和状态，为基础和诊断研究提供了一个强大的工具。

DESCRIPTION (provided by applicant): Project Summary/Abstract The broad goal of this proposal is to develop a general and highly-sensitive method that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases across the entire genomes of mammalian cells and Drosophila. Preliminary results from this Global nuclear Run-On and massively parallel Sequencing method (GRO-seq) demonstrate the potential of this approach to provide a global picture of the transcription status of genome. Additionally, these early results uncovered novel antisense short transcription units upstream and adjacent to many known promoters and suggest modes of regulation that heretofore have not been considered. This proposal seeks to rigorously test and further develop the GRO-seq method to provide unprecedented, near-nucleotide resolution of RNA polymerase positions over the genome. Aim 1 is to optimize and critically evaluate the first generation GRO-seq method, which is already producing results. Simple variations of this procedure should be capable of identifying the type of polymerase, that is, RNA polymerases I, II, or III. Aim 2 is to develop second generation, ultra-high resolution versions of GRO-seq that map with several-nucleotide resolution the 3' ends of nacent RNA, which are associated with active site of all transcriptionally- engaged polymerases. An additional adaptation of GRO-seq will identify transcription start sites of transcriptionally- engaged RNA polymerase II (Pol II) with high sensitivity. The precise position and status of Pol II relative to transcription start sites will identify sequences where transcription elongation is confronted with a rate limiting step. Indeed, recent studies are documenting that Pol II pausing immediately downstream of promoters is a rate-limiting step in the regulation of many metazoan genes. Furthermore, the correlation of Pol II position to genome-wide transcription factor maps will identify candidate factors involved in this regulation. Aim 3 will develop GRO-seq methods so they can be generally applied to key model systems including representative classes of human cell lines, mouse embryonic stem cells, and Drosophila embryos. This last aim will also test the ability to globally detect rapid changes in transcription in response to regulatory signals. Finally, although the focus of the proposal is to develop robust, generally-applicable assays of transcriptionally-engaged RNA polymerases, the data generated in testing these cell and organismal models will also provide critical transcription information to existing the human ENCODE and model organism modENCODE projects as well as to the mouse stem cell efforts of the International Regulome Consortium. Because GRO-seq can define engaged RNA polymerases in cells under normal conditions as well as during infection and disease, it provides a valuable tool for basic research of numerous diseases and can produce critical signatures in diagnostic studies. Project Narrative This is a proposal to develop a novel method that maps the position, amount, and orientation of transcriptionally-engaged RNA polymerases across the genome at unprecedented, near-nucleotide resolution. GRO-seq will provide a general tool that will be applicable to efforts of ENCODE and modENCODE projects to define in molecular terms how the DNA code in higher eukaryotes is read and regulated to provide the patterns of gene expression required to develop and maintain a multicellular organism. GRO-seq can define both the level and status of all engaged RNA polymerases in cells under normal conditions, and during infection and disease providing a powerful tool for basic and diagnostic studies.