Mechanistic Problems in Eukaryotic Gene Regulation

描述（由申请人提供）：我的研究项目的长期目标是对体内基因调控进行定量理解。这个项目的重点是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靶位点。进入核小体内的目标位点可以自发发生，并可以由atp依赖性核小体重塑因子催化。我们正在研究这两种机制。Aim 1的研究将描述核小体在模型染色质纤维中由位点特异性DNA结合蛋白自发入侵的平衡和动力学。Aim 2的研究试图阐明Snf2类atp依赖性重塑机器改变包括核小体在内的蛋白质- dna复合物结构的详细分子机制。我们将重点放在酵母Snf2家族成员Mot1上，因为我们相信它的活性——从DNA中置换TBP——是Snf2家族成员中最简单的，因此最适合进行详细的机制分析。Mot1本身也很重要，因为TBP在含tata和不含tata的启动子上的占用都可能限制基因表达的速率。该研究将有助于进一步认识Mot1结构域的组织和功能，明确Mot1作用中的机械化学耦合机制

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.