Chromatin Domain Structure and Function

描述（由申请人提供）：目前的基因调控模型表明，间期染色体被分为大的，功能不同的染色质结构域，促进邻近基因的独立调控。建立这些结构域的结构和分子机制仍然知之甚少。只有间接的结构测定已可用于分析这些结构域的结构，与直接可视化结构域大规模染色质折叠相关的技术困难是一个关键的限制。我们的长期目标是解剖特定基因位点的大规模染色质折叠，分析这些折叠图案的顺式和反式决定因素，并确定这一水平的染色质组织的转录调控的功能意义。 本项目期间的具体目标是解决以下问题：1.大规模染色质结构的变化伴随着转录激活/抑制？2.大规模染色质凝聚是否限制转录激活？3.转录激活因子通过什么机制改变大规模染色质结构？4.什么样的顺式因子控制着大规模的染色质结构？使用新的可视化方法，在转录激活或沉默期间，将直接观察特定基因和染色体位点的体内动态。光学和电子显微镜的组合将可视化的变化，在大规模的染色质组织和核定位的这些染色体区域在转录激活或抑制，并监测这些网站的各种转录辅因子的顺序招聘。几种方法将被用来开始mechanisticdissection潜在的生化机制所观察到的大规模染色质结构的重组，和功能的重要性大规模染色质组织方面的潜在转录激活将被评估。从这些研究中获得的见解应该有助于指导未来基因治疗中使用的构建体和人工染色体的设计。

DESCRIPTION (provided by applicant): Current models of gene regulation suggest that interphase chromosomes are divided into large, functionally distinct chromatin domains, facilitating independent regulation of neighboring genes. The structural and molecular mechanisms establishing these domains remain poorly understood. Only indirect structural assays have been available to analyze the structure of these domains, with a key limitation being the technical difficulty associated with directly visualizing domain large-scale chromatin folding. Our long term objectives are to dissect the large-scale chromatin folding of specific gene loci, to analyze the cis and trans determinants of these folding motifs, and to determine the functional significance of this level of chromatin organization with regard to transcriptional regulation. The specific aims for this project period will be to address the following questions:1. What changes in large-scale chromatin structure accompany transcriptional activation/repression?2. Does large-scale chromatin condensation constrain transcriptional activation?3. By what mechanisms do transcriptional activators modify large-scale chromatin structure?4. What cis factors control large-scale chromatin structure? Using novel visualization methods, in vivo dynamics of specific gene and chromosome loci will be observed directly during transcriptional activation or silencing. A combination of light and electron microscopy will visualize changes in large-scale chromatin organization and nuclear positioning of these chromosome regions during transcriptional activation or repression and monitor the sequential recruitment of various transcription cofactors to these sites. Several approaches will be used to begin mechanisticdissection of the underlying biochemical mechanisms underlying observed reorganization of large-scale chromatin structure, and the functional importance of large-scale chromatin organization with respect to potential for transcriptional activation will be assessed. Insight from these studies should be of help in guiding the design of future constructs and artificial chromosomes used in gene therapy.