Chromatin Domain Structure and Funcion

项目概述：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物接近靶序列、控制远端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，转录活性基因位点的实际大规模染色质组织是未知的，教科书模型主要基于间接的分子分析。我们的长期目标是确定大规模的染色质折叠和特定基因位点的核内定位，确定它们的顺式和反式决定因素，并了解这一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大。我们已经开发了一种方法，可以直接可视化大规模的染色质去浓缩和核内运动，伴随着BAG转基因的基因激活-首先在活细胞中，然后在超微结构水平上使用一种新的免疫金标记程序。这些方法应该适用于内源基因位点。本项目期间的具体目标是：(1)直接确定基因激活过程中三维大尺度染色质超微结构的变化；(2)直接观察与基因激活/抑制相关的基因位点在核内定位的变化，并测试这些运动对肌动蛋白/肌球蛋白的依赖性；(3)确定大规模染色质结构变化的顺式和反式决定因素以及与基因激活/抑制相关的基因位点的核内定位；(4)剖析果蝇多烯染色体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理解不完全。我们的研究成果将有助于指导未来基因构建和基因治疗中使用的人工染色体的设计。

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.