Imaging chromosome dynamics and measuring its impact on transcriptional activity

 描述(申请人提供)：现代生物学中最基本的问题之一是在活细胞中天然染色体的背景下理解动态的基因活动在时间和空间上的活动。这项拟议的研究的目标是测量活细胞中特定的远程染色体相互作用产生的转录水平。传统的活体成像方法缺乏空间分辨率来准确确定基因活性的动力学，而使用固定材料的批量分析强烈限制了时间动力学的研究。在这里，我们建议通过发展新的显微镜和计算分析方法来克服这些限制。大多数研究将利用果蝇早期胚胎的独特优势，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采样，以提供不同距离的“滴定”，从几十到几百个千基， 正如在哺乳动物系统中看到的那样。有两个具体的目标：1.发展高分辨率成像方法和相关的计算算法，以可视化和定量的动态增强子-启动子相互作用在选定的内源基因座在活体胚胎。2.标记具有长距离相互作用的个体基因座的调节区和相关的转录单位，包括在Hox基因座的转导过程中的反式同源关联，以在体内测量染色体拓扑对转录活性的影响。我们计划将这一方法扩展到包括在基因工程苍蝇品系库中的数百个荧光DNA焦点的可视化，以建立对 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叠加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的染色体动力学可视化，特别是多能祖细胞，如小鼠肝母细胞。

 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.