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体内基因调控的定量理解。该项目侧重于基因调控的最早阶段，即基因调控蛋白如何进入染色质中的DNA靶位点。进入核小体内的靶位点可以自发发生，并且可以由ATP依赖性核小体重塑因子催化。我们正在研究这两种机制。目标1中的研究将描述在模型染色质纤维中，位点特异性DNA结合蛋白自发侵入核小体的平衡和动力学。目的2中的研究试图阐明Snf 2类ATP依赖性重塑机器用于改变蛋白质-DNA复合物（包括核小体）结构的详细分子机制。我们专注于酵母Snf 2家族成员Mot 1，因为我们相信它的活性-从DNA中置换TBP-是Snf 2家族成员中最简单的，因此最适合于详细的机制分析。Mot 1本身也非常重要，因为TBP在含TATA和无TATA启动子处的占据可以限制基因表达的速率。这方面的研究将有助于我们进一步了解Mot 1结构域的组织和功能，并明确Mot 1作用中的机械化学偶联机制 公共卫生相关性：基因的适当调节对所有生物体的发育和健康至关重要。我们的期望是，了解调控蛋白如何找到并结合到它们的DNA靶位点，从长远来看，将导致更好地了解疾病过程中的基本机制，并从那里获得新的诊断方法。我们还期望，该项目目标的进展将导致提高在生物技术和基因治疗中应用人工转基因的能力。对ATP依赖性核小体重塑因子的研究与人类发育和健康具有特定的相关性，因为这些因子中的突变是导致广泛的人类发育疾病和癌症的原因，包括：威廉姆斯综合征、Schimke免疫骨发育不良、Cockayne综合征、X连锁1型地中海贫血、精神发育迟滞、恶性横纹肌样肿瘤、慢性髓性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.