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High-throughput functional characterization of human enhancers

人类增强子的高通量功能表征

基本信息

批准号：
10241101
负责人：
JOHN T LIS
金额：
$ 75.05万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-18 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10241101
关键词：
Address Affect Architecture Base Pairing Binding Binding Sites Biological Assay CRISPR/Cas technology Cell Line Cells Characteristics Chromatin Chromatin Remodeling Factor Classification Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Computing Methodologies Cues DNA DNA Polymerase II DNA-Directed RNA Polymerase Defect Deoxyribonuclease I Development Disease Distal Drosophila genus Elements Enhancers Environment Failure Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Genome Genomic DNA Goals HCT116 Cells HSF1 Heat-Shock Response Histones Human Hypersensitivity K-562 K562 Cells Knock-in Lead Maintenance Mammalian Cell Mass Spectrum Analysis Measures Mechanics Messenger RNA Methods Modeling Molecular Mutate Mutation Nutritional Oncogenes Organism Pattern Personal Satisfaction Phenotype Positioning Attribute Process Production Proteins RNA RNA Sequences Regulation Regulator Genes Research Resolution Role Run-On Assays Structure System Testing To specify Transactivation Transcription Initiation Site Transcriptional Regulation Transgenic Organisms Undifferentiated Work aptamer base biological adaptation to stress cell type chromatin immunoprecipitation chromatin remodeling chromosome conformation capture comparative design embryo cell follow-up genome-wide high throughput screening histone modification in vivo insight mutant novel novel therapeutics nutrition promoter recruit response spatiotemporal transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT: The genomes of higher organisms and their expression are highly regulated in response to a variety of developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to developmental defects and disease states. The overarching goal of this research is to understand basic regulatory mechanisms of genes at the level of transcription, the stage where RNA polymerase II (Pol II) transcribes the genes into mRNA. This regulation is conducted by the concerted effort of DNA elements (promoters and enhancers) and many protein machineries such as Pol II, TFs, and chromatin/histone modifying complexes. Recent studies in Drosophila and mammalian cells have revealed that transcription is primarily regulated at two rate-limiting steps; 1) Recruitment of Pol II to promoter and Pol II pausing at 20-50bp downstream of transcription initiation site, and 2) Release of paused Pol II to productive elongation. A battery of complementary approaches (some novel) are designed to identify human TFs and chromatin remodelers that act at these steps and then rigorously address the mechanism by which they exert transcriptional regulation. by TFs and chromatin modifiers with the highest possible spatiotemporal resolution genome-wide in distinct cell lines, including K562, HCT116, HEK293, and undifferentiated and differentiated WTC-11. The wealth of existing information in these cell lines will be complemented with Run-On assays (RO-seq), and others when necessary, to identify TFs that act primarily on one or both of the rate-limiting regulated steps in transcription. These observational assays include multiple highly-sensitive RO-seq assays for mapping the position and amount of transcription across genes and enhancers genome-wide, ChIP-exo for detecting TF binding and occupancy, STARR-seq assays to measure transcription regulatory activity of enhancers and promoters, and a degron system to rapidly degrade TFs to assess their primary effects by these ‘-seq’ assays. Together, these systems will test our regulatory models and assess their generality. In Aim 1, using a novel aptamer-based pulldown and mass spectrometry approach, we will identify pioneering TFs that work through remodeling complexes such as PBAP (SWI/SNF) to open chromatin for subsequent TF and Pol II recruitment. In Aim 2, we seek to identify cell- type-specific TFs that act at major regulated steps of transcription, Steps 1, 2, or both. This will be achieved by a comparative analysis of cell line specific nascent transcription profiles. Aim 3 will test the role of TF candidates, which are associated with one or both of the regulatory steps in the transcription cycle, in living cells using a Degron approach and multiple ‘-seq’ assays. In Aim 4, we will assess the roles of TFs in the interplay of enhancers and promoters interactions using high-throughput episomal and chromosomal enhancer assays. The proposal will identify TFs that act at specific steps in transcription regulation and provide insights to the mechanics of their action.

项目总结/摘要：高等生物的基因组及其表达受到多种生物因子的高度调节，发育、环境和营养线索。如果不能进行适当的基因调控，发育缺陷和疾病状态。这项研究的首要目标是了解基本的基因在转录水平上的调控机制，RNA聚合酶II（Pol II）将基因转录成mRNA。这种调节是由DNA元件的协同努力进行的（启动子和增强子）和许多蛋白质机制，如Pol II，TF和染色质/组蛋白修饰，配合物最近在果蝇和哺乳动物细胞中的研究表明，转录主要是 Pol Ⅱ在启动子上的募集和Pol Ⅱ在20- 50 bp的停顿是两个限速步骤转录起始位点下游，和2）暂停的Pol II释放到生产性延伸。的电池互补的方法（有些是新的）被设计来鉴定人类TF和染色质重塑，在这些步骤中，然后严格解决它们发挥转录调节的机制。由TFs 和在不同细胞系中具有全基因组最高可能时空分辨率的染色质修饰剂，包括K562、HCT 116、HEK 293和未分化和分化的WTC-11。现有的财富这些细胞系中的信息将用Run-On测定法（RO-seq）补充，必要时用其他测定法补充，以鉴定主要作用于转录中的一个或两个限速调节步骤的TF。这些观察性测定包括多种高灵敏度的RO-seq测定，用于定位蛋白质的位置和量。跨基因和增强子的全基因组转录，用于检测TF结合和占用的ChIP-exo，用于测量增强子和启动子的转录调节活性的STARR-seq测定，以及降解决定子系统来快速降解TF，以通过这些“-seq”测定来评估它们的主要作用。总之，这些系统将测试我们的监管模式并评估其通用性。在目标1中，使用一种新的基于适体的下拉，质谱方法，我们将确定开拓性的TF，通过重塑复合物，如 PBAP（SWI/SNF）打开染色质，用于随后的TF和Pol II募集。在目标2中，我们试图识别细胞- 在转录的主要调节步骤，步骤1，2或两者中起作用的类型特异性TF。这将通过一个细胞系特异性新生转录谱的比较分析。目标3将测试TF候选人的作用，其与转录周期中的一个或两个调节步骤相关，降解决定子方法和多重“-seq”测定。在目标4中，我们将评估TF在以下因素相互作用中的作用：使用高通量附加体和染色体增强子测定进行增强子和启动子相互作用。的该提案将确定在转录调控的特定步骤中起作用的转录因子，并为转录调控提供见解。他们的行动机制。