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级组织（即，超出核小体本身的）影响核功能，如转录、复制和细胞周期控制。 这目前无法实现，因为没有技术手段来严格表征体内组装的染色质的高级结构特征。 在本R21“高风险/高影响研究的探索性研究”提案中，将通过开发定量琼脂糖凝胶电泳来克服这一限制性技术障碍，以表征两种不同的明确体内组装染色质系统的高阶折叠程度、柔韧性和表面电荷密度。 在具体目标1中，定量电泳方法将应用于分析酵母微型染色体的高阶组织，所述酵母微型染色体已在体内从天然核心组蛋白以及在其生物学上重要的N-末端结构域中表达各种突变的核心组蛋白组装。 在具体目标2中，将检查染色质高级组织对转录调控的影响。 这将通过首先确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转录之前和之后的高阶折叠、柔性和表面电荷密度来实现。 此外，在具体目标2中，将从酵母α细胞分离的转录活性TALS微型染色体的高阶折叠、柔性和表面电荷密度与从α细胞分离的相同微型染色体的结构特性进行比较，其中所述微型染色体被转录抑制。 拟议的研究将是直接表征体内组装染色质的高阶结构特性的任何类型的第一个实验，这样做将克服染色质研究面临的主要障碍。 随后将有可能研究许多功能上重要的体内组装的染色质区域的结构特性（例如，启动子、增强子），以及其它在体内组装的多组分复合物的重要功能。

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.