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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/文摘: