Imaging chromosome dynamics and measuring its impact on transcriptional activity

染色体动态成像并测量其对转录活性的影响

• 批准号: 9003587
• 负责人: Thomas Gregor
• 金额: $ 37万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003587
• 关键词: Animals; Architecture; Biological Assay; Biology; Cell Nucleus; Cells; Cellular biology; Chromatin; Chromosomes; Color; Complex; Computational algorithm; Computer Analysis; Custom; DNA; Data; Defect; Detection; Development; Disease; Drosophila genus; Embryo; Embryonic Development; Engineering; Enhancers; Exhibits; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Engineering; Genetic Enhancer Element; Genetic Transcription; Genome; Genome Mappings; Goals; Homeostasis; Homologous Gene; Image; Imagery; Individual; Investigation; Label; Lateral; Libraries; Life; Light; Malignant Neoplasms; Mammalian Cell; Mammals; Measurement; Measures; Methods; Microscope; Microscopy; Modeling; Multipotent Stem Cells; Mus; Noise; Nuclear; Nucleic Acid Regulatory Sequences; Optics; Process; RNA; Reading; Regulator Genes; Research; Resolution; Role; Sampling; Signal Transduction; Stem cells; System; Technology; Testing; Time; Tissues; Titrations; Transcriptional Regulation; base; cellular imaging; fly; functional gain; genetic information; genome editing; imaging modality; in vivo; interest; new technology; novel; programs; promoter; prototype; public health relevance; research study; stem; tissue fixing; tool; user-friendly

 DESCRIPTION (provided by applicant): One of the most fundamental problems in modern biology is to understand dynamic gene activity in time and space in the context of native chromosomes in living cells. The goal of the proposed study is to measure the levels of transcription produced by defined long-range chromosomal interactions in living cells. Traditional live imaging methods lack the spatial resolution to accurately determine the dynamics of gene activity, while bulk assays using fixed material strongly limit investigation of temporal dynamics. Here we propose to overcome these limitations by developing new methods of microscopy and computational analysis. Most of the studies will exploit the unique advantages of the early Drosophila embryo for the development of quantitative live cell imaging methods. Previous studies have identified hundreds of such interactions, and we will sample several of these to provide a "titration" of varying distances, from tens to hundreds of kilobases, as seen in mammalian systems. There are two specific aims: 1. Develop high-resolution imaging methods and associated computational algorithms for the visualization and quantification of dynamic enhancer-promoter interactions at select endogenous loci in living embryos. 2. Label regulatory regions and associated transcription units of individual genetic loci exhibiting long-range interactions, including trans-homolog associations during transvection at Hox loci, to measure in vivo the effect of chromosome topology on transcriptional activity. We plan to extend this approach to include the visualization of several hundred fluorescent DNA foci in a library of genetically engineered fly lines to establish a general overview of the dynamics of an entire chromosome in a living embryo and its impact on transcription. The successful realization of the proposed studies will greatly augment our current capacity to superimpose whole-genome maps based on fixed tissues onto the dynamic chromosomes of living cells. The resulting technologies will be immediately applied to the visualization of chromosome dynamics in mammalian tissues, particularly multipotent progenitor cells such as mouse hepatoblasts.

 描述（由申请人提供）：现代生物学中最基本的问题之一是了解活细胞中天然染色体背景下的动态基因活性。拟议研究的目标是测量活细胞中由定义的长程染色体相互作用产生的转录水平。传统的实时成像方法缺乏空间分辨率来准确地确定基因活性的动态，而使用固定材料的批量测定强烈限制了时间动态的调查。在这里，我们建议通过开发新的显微镜和计算分析方法来克服这些限制。大多数研究将利用早期果蝇胚胎的独特优势，开发定量活细胞成像方法。以前的研究已经确定了数百种这样的相互作用，我们将对其中的几种进行取样，以提供不同距离的“滴定”，从几十到几百种酶， 如在哺乳动物系统中所见。有两个具体目标：1。开发高分辨率成像方法和相关的计算算法，用于在活胚胎中选择内源性基因座的动态增强子-启动子相互作用的可视化和定量。2.标记调控区和相关的转录单位的个别遗传基因座表现出长距离的相互作用，包括在Hox基因座的transvection过程中的trans-homolog协会，以测量在体内的影响，染色体拓扑结构的转录活性。我们计划将这种方法扩展到包括在遗传工程果蝇品系库中的数百个荧光DNA焦点的可视化，以建立对果蝇的动态的总体概述。 活胚胎中的整个染色体及其对转录的影响。这些研究的成功实现将极大地增强我们目前将基于固定组织的全基因组图谱复制到活细胞的动态染色体上的能力。由此产生的技术将立即应用于哺乳动物组织中染色体动态的可视化，特别是多能祖细胞，如小鼠成肝细胞。