HIGHER ORDER PROTEIN DNA COMPLEXES IN GENE REGULATION

描述(改编自申请人摘要)：本提案的目标 就是发现基因调控蛋白是如何组装到特定位置的 在哺乳动物染色质中，扰乱核小体并形成局部 激活基因表达的高阶结构。工具现在已经 手头上解决这个问题的发展方面的 调节的血清白蛋白基因转录增强子：必需的 增强子的序列已经确定；反式作用结合蛋白 以及关于增强子核小体的基本信息 获得了肝细胞的结构。调查员提出了 以下旨在揭示潜在的控制机制：1.提供 核小体结构和核小体的详细分子特征 天然肝染色质中与白蛋白增强子结合的蛋白质和 控制组织。2.确定核小体的排列顺序 在肝细胞发育过程中，白蛋白增强子发生重排。 3.体外将白蛋白增强子重组为核小体片段 并测试相关调节蛋白的结合、干扰能力 核小体结构，并允许结合其他因子 不能自行捆绑。4.分级核抽提物和筛选 促进增强子重组的非DNA结合活性 染色质片段在体外。该提案旨在揭示 调节因子与染色质结合并激活基因的层次结构 转录；这些层次结构控制着可能是 对人类的正常生长发育至关重要。

DESCRIPTION (Adapted from Applicant's Abstract): The goal of this proposal is to discover how genetic regulatory proteins assemble onto specific sites in mammalian chromatin, perturbing nucleosomes and forming local higher-order structures that activate gene expression. The tools are now in hand to address this question with regard to the developmentally regulated transcriptional enhancer of the serum albumin gene: Essential sequences of the enhancer have been defined; trans-acting binding proteins have been cloned; and basic information about the enhancer's nucleosome structure in liver cell has been obtained. The investigator proposes the following aims to reveal the underlying mechanisms of control: 1. Provide a detailed molecular characterization of the nucleosomal structure and proteins binding to the albumin enhancer in native liver chromatin and control tissues. 2. Determine the order with which nucleosomal rearrangements occur at the albumin enhancer during hepatocyte development. 3. Reconstitute the albumin enhancer into nucleosomal segments in vitro and test relevant regulatory proteins for their ability to bind, perturb nucleosome structure, and to permit the binding of other factors that cannot bind on their own. 4. Fractionate nuclear extracts and screen for non-DNA binding activities that facilitate the reorganization of enhancer chromatin fragments in vitro. The proposal is designed to reveal hierarchies by which regulatory factors bind to chromatin and activate gene transcription; these hierarchies govern basic processes that may be essential for normal growth and development in humans.