Chromatin Domain Structure and Funcion

描述(由申请人提供)：项目摘要：基因调控模型表明，大规模染色质组织通过限制大蛋白复合体对靶序列的可及性、控制远距离调控序列之间的相互作用和/或调节核内基因定位来调控转录。然而，转录活性基因座的实际大规模染色质组织尚不清楚，教科书模型主要基于间接分子分析。我们的长期目标是确定特定基因座的大规模染色质折叠和核内定位，识别它们的顺式和反式决定因素，并了解这种水平的染色质组织在转录调控方面的功能意义。最近的几项实验进展表明，进行富有成效的研究的可能性很高。我们已经开发出一种方法，可以直接显示伴随Bag转基因基因激活的大规模染色质解缩和核内运动-首先是在活细胞中，然后是在超微结构水平上使用一种新的免疫金标记程序。这些方法应适用于内源基因座。本项目期间的具体目标将是：(1)直接确定伴随基因激活的三维大规模染色质超微结构的变化；(2)直接可视化与基因激活/抑制相关的基因座位的核内定位变化，并测试这些运动对肌动蛋白/肌球蛋白的依赖；(3)确定与基因激活/抑制相关的大规模染色质结构变化和核内定位的顺式和反式决定因素；(4)剖析决定果蝇多线染色体条带和带间组织的分子序列决定因素。公共卫生相关性：目前，基因治疗方法发展的一个主要障碍是我们对确保转基因高水平和持续表达的要求不完全。我们的研究洞察应该有助于指导未来用于基因治疗的基因结构和人造染色体的设计。

DESCRIPTION (provided by applicant): Project Summary: Models of gene regulation suggest large-scale chromatin organization regulates transcription by restricting accessibility of large protein complexes to target sequences, controlling interactions between distant regulatory sequences, and/or modulating intranuclear gene positioning. However, the actual large-scale chromatin organization of transcriptionally active gene loci is unknown, with textbook models based largely on indirect molecular assays. Our long-term objectives are to determine the large-scale chromatin folding and intranuclear positioning of specific gene loci, to identify their cis and trans determinants, and to understand the functional significance of this level of chromatin organization with regard to transcriptional regulation. Several recent experimental developments indicate a high probability for productive investigations. We have developed methods allowing direct visualization of large-scale chromatin decondensation and intranuclear movements accompanying gene activation of BAG transgenes- first in live cells, and then at the ultrastructural level using a novel immunogold labeling procedure. These methods should be applicable to endogenous gene loci. The specific aims for this project period will be to: (1) Directly determine changes in 3-D large-scale chromatin ultrastructure accompanying gene activation; (2) Directly visualize changes in intranuclear positioning of gene loci associated with gene activation / repression and test the dependence of these movements on actin / myosin; (3) Identify the cis and trans determinants of changes in large-scale chromatin structure and intranuclear positioning of gene loci associated with gene activation / repression; (4) Dissect the molecular sequence determinants which determine Drosophila polytene chromosome band and interband organization. Public Health Relevance: Currently a major impediment to development of gene therapy methods is our incomplete understanding of the requirements for ensuring high and sustained levels of expression from transgenes. Insight from our studies should be useful in guiding the design of future gene constructs and artificial chromosomes used in gene therapy.