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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

基本信息

批准号：
9298654
负责人：
Thomas Gregor
金额：
$ 37万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
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/9298654
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 Dimensions 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 Goals Homeostasis Homologous Gene Image Imagery Individual Investigation Label Lateral Libraries Light Malignant Neoplasms Mammalian Cell Mammals Maps Measurement Measures Methods Microscope Microscopy Modeling Modernization Multipotent Stem Cells Mus Noise Nuclear Nucleic Acid Regulatory Sequences Optics Process RNA Regulator Genes Research Resolution Role Sampling Signal Transduction Stem cells System Technology Testing Time Tissues Titrations Transcriptional Regulation base experimental study fly functional gain genetic information genome editing high resolution imaging imaging modality in vivo interest live cell imaging new technology novel programs promoter prototype public health relevance stem tool user-friendly whole genome

项目摘要

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.