HIGHER ORDER PROTEIN DNA COMPLEXES IN GENE REGULATION

描述（改编自申请人摘要）：本提案的目标 是发现基因调节蛋白如何在特定位点上组装 在哺乳动物染色质中，干扰核小体并形成局部 激活基因表达的高级结构。 工具现在 为了解决这一问题， 血清白蛋白基因转录调控增强子：必需的 增强子的序列已经确定;反式作用结合蛋白 已被克隆;和有关增强子的核小体的基本信息 获得了肝细胞的结构。 调查人员建议， 以下目的是揭示控制的潜在机制：1。 提供 核小体结构的详细分子表征， 与天然肝染色质中的白蛋白增强子结合的蛋白质， 对照组织。 2. 确定核小体 在肝细胞发育过程中，白蛋白增强子处发生重排。 3. 体外重组白蛋白增强子核小体片段 并测试相关的调节蛋白结合、干扰 核小体结构，并允许结合其他因子， 无法自行绑定。 4. 破碎细胞核提取物和筛选 促进增强子重组的非DNA结合活性 体外染色质片段。 该提案旨在揭示 调节因子与染色质结合并激活基因等级 转录;这些层次结构管理可能是 对人类的正常生长和发育至关重要。

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.