HIGHER ORDER ORGANIZATION OF IN VIVO-ASSEMBLED CHROMATIN

染色质研究的主要目标之一是确定染色质的高阶组织(即超出核小体本身的组织)如何影响核功能，如转录、复制和细胞周期控制。目前还不能做到这一点，因为没有技术手段来严格表征体内组装的染色质的高阶结构特征。在R21“高风险/高影响研究的探索性研究”提案中，这一限制性的技术障碍将通过开发定量琼脂糖凝胶电泳来克服，作为一种手段来表征两个不同的、定义良好的体内组装的染色质系统的高阶折叠程度、灵活性和表面电荷密度。在具体目标1中，定量电泳法将用于分析酵母微染色体的高阶组织结构，这些微染色体是由天然核心组蛋白和在其生物重要N末端区域表达各种突变的核心组蛋白在体内组装而成的。在特定的目标2中，将检测染色质高级组织对转录调控的影响。这将通过首先测定小鼠乳腺肿瘤病毒长末端重复序列(MMTV-LTR)启动子在类固醇激素激活MMTV-LTR转录前后的高阶折叠、柔韧性和表面电荷密度来实现。同样在特定的目标2中，从酵母a细胞中分离的具有转录活性的TALS微染色体的高阶折叠、柔韧性和表面电荷密度将与从α细胞中分离的相同微染色体的结构特性进行比较，在阿尔法细胞中，它被转录抑制。拟议的研究将是第一次直接表征体内组装的染色质的高阶结构性质的任何类型的实验，这样做将克服染色质研究面临的一大障碍。随后将有可能研究体内组装的许多功能重要的染色质区域(例如启动子、增强剂)的结构性质，以及其他体内组装的功能重要的多组分复合体的结构性质。

One of the major objectives in chromatin research is to ascertain how the higher order organization of chromatin (i.e., that beyond the nucleosome per se) influences nuclear functions such as transcription, replication and cell-cycle control. This currently cannot be accomplished because there is no technical means to rigorously characterize the higher order structural features of in vivo-assembled chromatin. In this R21 "Exploratory Studies for HighRisk/High Impact Research" proposal, this limiting technical barrier will be overcome by developing quantitative agarose gel electrophoresis as a means to characterize the extent of higher order folding, flexibility, and surface charge density of two different well defined in vivo- assembled chromatin systems. In Specific Aim 1, the quantitative electrophoretic approach will be applied to the analysis of the higher order organization of yeast minichromosomes that have been assembled in vivo from both native core histones as well as core histones expressing a variety of mutations in their biologically important N-terminal domains. In Specific Aim 2, the effect of chromatin higher order organization on transcriptional regulation will be examined. This will be accomplished by first determining the higher order folding, flexibility, and surface charge density of the mouse mammary tumor virus long-terminal repeat (MMTV-LTR) promoter before and after steroid hormone activation of MMTV-LTR transcription. Also in Specific Aim 2, the higher order folding, flexibility, and surface charge density of the transcriptionally active TALS minichromosome isolated from yeast a cells will be compared with the structural properties of the same minichromosome isolated from alpha cells, where it is transcriptionally repressed. The proposed studies will be the first experiments of any type to directly characterize the higher order structural properties of in vivo-assembled chromatin, and in doing so will overcome a major barrier facing chromatin research. It subsequently will become possible to study the structural properties of numerous functionally important in vivo- assembled chromatin regions (e.g., promoters, enhancers), and other functionally important in vivo-assembled multi-component complexes as well.