Mechanistic Problems in Eukaryotic Gene Regulation

描述(由申请人提供)：我的研究计划的长期目标是发展对体内基因调控的量化理解。这个项目集中在基因调控的最早步骤，即基因调控蛋白如何进入染色质中的DNA靶点。对核小体内靶点的访问可以自发发生，并且可以被依赖于ATP的核小体重塑因子催化。我们正在研究这两种机制。目标1的研究将描述在模型染色质纤维中，特定部位的DNA结合蛋白自发侵入核小体的平衡和动力学。目标2的研究试图阐明Snf2类ATP依赖的重塑机器改变包括核小体在内的蛋白质-DNA复合体结构的详细分子机制。我们把重点放在酵母Snf2家族成员Mot1上，因为我们相信它的活性--TBP从DNA上的置换--是Snf2家族成员中最简单的，因此也是目前最容易进行详细机制分析的。Mot1本身也是非常重要的，因为TBP在包含和不包含TATA的启动子上都可以限制基因表达的速率。对Mot1结构域的研究有助于加深对Mot1结构域组织和功能的理解，明确Mot1作用力化学偶联的作用机制 与公共健康相关：对基因的适当调控对所有生物体的发育和健康至关重要。我们的期望是，从长远来看，了解调控蛋白如何找到并与其DNA靶点结合将导致更好地理解疾病过程中的基本机制，并由此产生新的诊断方法。我们还预计，该项目目标的进展将导致改进设计人工转基因应用于生物技术和基因治疗的能力。对ATP依赖的核小体重塑因子的研究与人类发育和健康具有特殊的相关性，因为这些因子的突变导致了广泛的人类发育疾病和癌症，包括：Williams综合征、Schimke免疫性骨发育不良、Cockayne综合征、X-连锁1地中海贫血症、智力低下、恶性横纹肌肉瘤、慢性髓系白血病以及前列腺癌和肺癌。

DESCRIPTION (provided by applicant): The long-term aim of my research program is to develop a quantitative understanding of gene regulation in vivo. This project focuses on the earliest steps in gene regulation, namely, how gene regulatory proteins gain access to their DNA target sites in chromatin. Access to target sites inside nucleosomes can occur spontaneously, and can be catalyzed by ATP-dependent nucleosome remodeling factors. We are studying both of these mechanisms. Studies in Aim 1 will characterize the equilibria and kinetics for spontaneous invasion of nucleosomes by site-specific DNA binding proteins, in model chromatin fibers. Studies in Aim 2 seek to elucidate the detailed molecular mechanisms by which the Snf2 class of ATP-dependent remodeling machines act to alter the structure of protein-DNA complexes including nucleosomes. We focus on the yeast Snf2 family member Mot1 because we believe its activity- the displacement of TBP from DNA - to be the simplest of any of the Snf2 family members, and thus the most amenable to detailed mechanistic analysis at this time. Mot1 is also of great importance in its own right, since TBP occupancy at both TATA-containing and TATA-less promoters can be rate-limiting for gene expression. Studies in this aim will advance our understanding of Mot1 domain organization and function, and define the mechanism of mechanochemical coupling in Mot1 action PUBLIC HEALTH RELEVANCE: The proper regulation of genes is essential for the development and health of all organisms. Our expectation is that understanding how regulatory proteins find and bind to their DNA target sites will lead, over the long term, to a better understanding of fundamental mechanisms in disease processes, and from there, to new diagnostics. We also expect that progress in the aims of this project will lead to an improved ability to engineer artificial transgenes for applications in biotechnology and gene therapy. Studies of ATP-dependent nucleosome remodeling factors have a specific relevance to human development and health, as mutations in these factors are responsible for a wide range of human developmental diseases and cancers, including: Williams syndrome, Schimke immunoosseous dysplasia, Cockayne syndrome, X-linked 1 thalassaemia, mental retardation, malignant rhabdoid tumors, chronic myeloid leukemia, and prostate and lung carcinomas.