Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

新生转录本测序揭示的转录控制机制

基本信息

批准号：
10584193
负责人：
Lee Stirling Churchman
金额：
$ 59.22万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584193
关键词：
Adenine Binding Binding Proteins Biochemical Cell Line Cell Nucleus Cells Chromatin Chromatin Fiber Complex Couples Cytoplasm DNA Data Data Set Detection Diagnosis Disease Down-Regulation Elements Enhancers Event Fiber Gene Expression Gene Expression Regulation Genes Genetic Transcription Genomics Heat-Shock Response Human Genome Introns Kinetics Knowledge Label Location Malignant Neoplasms Maps Measures Messenger RNA Metabolic Methods Methylation Methyltransferase Mission Modeling Molecular Monitor Nuclear Nuclear Export Nucleoplasm Nucleosomes Nucleotides Play Polymerase Positioning Attribute Process Production Promoter Regions Public Health RNA RNA Editing RNA Polymerase II RNA Processing RNA Splicing RNA chemical synthesis RNA purification RNA-Binding Proteins Regulator Genes Research Residencies Resolution Role Shapes Site Techniques Technology Testing Third Generation Sequencing Time Transcript Transcription Elongation Transcription Initiation Transcriptional Regulation U1 Small Nuclear Ribonucleoprotein United States National Institutes of Health Work base base editor biological adaptation to stress blind design epigenomics functional genomics genome-wide genomic data histone modification human disease mRNA Precursor nano new technology novel strategies nucleobase premature promoter recruit technology development termination factor tool transcription factor transcriptome transcriptome sequencing

项目摘要

Transcription initiation is highly regulated by genomic elements in promoters and enhancers that bind transcription factors and recruit RNA polymerase II (Pol II). After Pol II escapes from the promoter, many regulatory steps occur during transcription elongation that control Pol II progression, RNA processing, and finally release of the RNA from chromatin for nuclear export. These steps are tightly controlled by genomic and epigenomic features, such as histone modifications, nucleosome positions, and cis-elements that bind transcription factors and RNA-binding proteins (RBPs). Current functional genomic technologies tend to provide a static, averaged view of genomic function that obscure the transient roles that many genomic elements play in shaping the transcriptome. To identify and characterize all functional genomic elements, we need to develop approaches that quantitatively monitor the many layers of gene expression regulation, including transient interactions and short-lived events. In the proposed research, we will create a suite of genome-wide tools to map Pol II across single chromatin fibers, track the association of RBPs, and follow RNAs from transcription initiation to nuclear export. These techniques will highlight unappreciated connections between genomic elements, molecular events on chromatin, and post-initiation regulatory events. Our rationale is that by creating tools to quantify short-lived steps in mRNA production, unappreciated functional roles for genomic elements can be revealed. To demonstrate how these technologies reveal unappreciated gene regulatory mechanisms, we will apply them to the heat shock response (HSR), a stress response that has served as a model to study the many regulatory layers in gene expression. In Aim 1 we will develop a strategy that reveals Pol II locations across single long chromatin fibers. Fiber-seq is a method that identifies regions of accessible DNA across long (up to ~20 kb) chromatin fibers one molecule at a time. We will extend this approach to identify Pol II footprints, analyze the distributions of Pol II complexes across gene bodies, and characterize the interactions between Pol II complexes and nucleosomes. In Aim 2 we will measure the timing of binding of proteins across long pre-mRNA molecules. RBP binding will be marked by RNA editing and long- read direct RNA sequencing of nascent RNA (nano-COP) will enable us to resolve transient binding events that impact pre-mRNA processing and nuclear export. These events are transient, as many RBPs bind to introns and are released from RNA in the cytoplasm immediately after nuclear export. In Aim 3, we will create an approach to measure kinetics of RNA production, chromatin release, and nuclear export that couples 4sU metabolic labeling of newly synthesized RNAs with biochemical purification of RNAs from chromatin, nucleoplasm, and cytoplasm. The data from this technique will allow us to estimate the rates of release of RNA from chromatin, nuclear export of RNA, and cytoplasmic degradation of every RNA. Using public datasets from ENCODE and elsewhere, we will identify and test hypotheses for the determinants of RNA lifetimes.

转录起始受到启动子和增强子中结合的基因组元件的高度调控转录因子和招募 RNA 聚合酶 II (Pol II)。 Pol II 逃离启动子后，许多转录延伸过程中发生的调控步骤控制着 Pol II 进展、RNA 加工和最后从染色质中释放RNA用于核输出。这些步骤受到基因组和基因组的严格控制表观基因组特征，例如组蛋白修饰、核小体位置和结合的顺式元件转录因子和 RNA 结合蛋白 (RBP)。当前的功能基因组技术倾向于提供基因组功能的静态、平均视图，掩盖了许多基因组的短暂作用元素在转录组的形成中发挥作用。为了识别和表征所有功能基因组元件，我们需要开发定量监测基因表达调控多层的方法，包括短暂的相互作用和短暂的事件。在拟议的研究中，我们将创建一套用于跨单个染色质纤维绘制 Pol II 的全基因组工具，跟踪 RBP 的关联，并遵循 RNA从转录起始到核输出。这些技巧将突出不被重视的联系基因组元件、染色质分子事件和启动后调控事件之间的关系。我们的理由是通过创建工具来量化 mRNA 生产中短暂的步骤，可以揭示基因组元件。展示这些技术如何揭示未被重视的基因调节机制，我们将它们应用于热休克反应（HSR），这是一种应激反应，作为研究基因表达中许多调控层的模型。在目标 1 中，我们将制定一项战略揭示了单个长染色质纤维上的 Pol II 位置。 Fiber-seq 是一种识别区域的方法一次一个分子可跨越长（长达约 20 kb）染色质纤维的 DNA。我们将延长此方法来识别 Pol II 足迹，分析 Pol II 复合物在基因体中的分布，以及表征 Pol II 复合物和核小体之间的相互作用。在目标 2 中，我们将测量时间跨长前 mRNA 分子的蛋白质结合。 RBP 结合将通过 RNA 编辑和长链标记读取新生 RNA 的直接 RNA 测序 (nano-COP) 将使我们能够解决瞬时结合事件影响前 mRNA 加工和核输出。这些事件是暂时的，因为许多 RBP 绑定到内含子，并在核输出后立即从细胞质中的 RNA 中释放。在目标 3 中，我们将创建一种测量与 4sU 耦合的 RNA 产生、染色质释放和核输出动力学的方法通过从染色质中生化纯化 RNA，对新合成的 RNA 进行代谢标记，核质和细胞质。这项技术的数据将使我们能够估计 RNA 的释放速率来自染色质、RNA 的核输出以及每种 RNA 的细胞质降解。使用来自的公共数据集 ENCODE 和其他地方，我们将识别和测试 RNA 寿命决定因素的假设。