Chromatin Domain Structure and Function

描述(由申请人提供)：目前的基因调控模型表明，间期染色体被划分为大的、功能不同的染色质区域，便于对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。目前只有间接结构分析方法可用于分析这些结构域的结构，其关键限制是与直接可视化结构域大规模染色质折叠相关的技术困难。我们的长期目标是剖析特定基因座位的大规模染色质折叠，分析这些折叠基序的顺式和反式决定因素，并确定这种水平的染色质组织在转录调控方面的功能意义。 这个项目期的具体目标将是解决以下问题：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.转录激活剂通过什么机制修改大规模染色质结构？4.什么顺式因素控制大规模染色质结构？利用新的可视化方法，可以在体内直接观察转录激活或沉默过程中特定基因和染色体位点的动态变化。光学显微镜和电子显微镜的结合将显示在转录激活或抑制期间这些染色体区域的大规模染色质组织和核定位的变化，并监测各种转录辅助因子顺序地招募到这些位置。将使用几种方法开始从机械上剖析观察到的大规模染色质结构重组背后的潜在生化机制，并将评估大规模染色质组织相对于转录激活潜力的功能重要性。这些研究的洞察力应该有助于指导未来用于基因治疗的结构和人造染色体的设计。

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.