Investigation of Chromatin Remodeling Mechanisms at the Promoters of Heat Shock Genes

智力上的优点。染色质重构是真核生物基因激活的基本前提。尽管经过几十年的深入研究，了解染色质变化的机制仍然是分子生物学领域的一个关键挑战。已经确定的是，在温度诱导过程中，酵母热休克基因启动子处的染色质变化的强度超过了其他充分表征的基因启动子处的染色质重塑事件，但彼此之间存在显著差异。这些特点概括了热休克基因作为一个强大的模型，研究染色质重塑机制。酵母细胞中的应激反应受两类激活剂HSF和Msn 2/4的调节，这两类激活剂对启动子染色质重塑的影响不同。本项目的重点是研究酵母热休克基因启动子染色质重塑的分子机制，以及为什么染色质变化差异巨大的原因，即使是密切相关的和共调控的热休克基因。本项目将探讨组蛋白伴侣蛋白的功能及其与ATP依赖的染色质重塑因子在HSP基因表达调控中的可能合作。由于组蛋白伴侣的一些组蛋白相互作用结构域可以作为反式激活结构域起作用，因此将测试激活结构域功能包括与组蛋白的相互作用是否匡威亦然。 一个额外的方向将是调查是否和如何Msn 2/4降解率调节的调解人复杂的组件，如果这对染色质重塑events.The方法学的方法是基于使用抗体对染色质和转录装置的组件，可从各种来源，染色质免疫沉淀，然后通过高通量实时PCR。该方法允许监测启动子特异性特征随时间的变化。染色质重塑和转录起始的研究将利用酵母菌株的基因组集合进行，所述酵母菌株具有细胞蛋白质组的不同组分的系统性缺失或标记。标准的基因工程技术也将用于操纵基因和基因启动子区域。该项目的影响不仅将影响真核基因表达的科学领域，还将影响USD桑福德医学院的基因分子生物学、医学生物化学和生物医学科学基础等研究生课程的开发。现代分子生物学的方法，包括上面提到的那些，将被纳入研究生和本科生课程的实验室课程在基础生物医学科学和USD校园在Vermillion，南达科他州的司。来自南达科他州印第安部落学校的学生过去参加过暑期项目，预计今后将继续这样做。研究生和本科生目前都参与了该项目。该项目的科学成果将通过出版物、国际会议和接触加以讨论和传播。

Intellectual merit. Chromatin remodeling is a fundamental prerequisite to eukaryotic gene activation. Despite intensive study over the course of decades, understanding the mechanisms that underlie chromatin changes remains a key challenge in the field of molecular biology. It has been established that the intensity of chromatin changes at promoters of yeast heat shock genes during temperature induction surpass the chromatin remodeling events at other well characterized gene promoters, yet significantly differ from each other. These features epitomize heat shock genes as a powerful model for studying chromatin remodeling mechanisms. Stress response in yeast cells is regulated by two classes of activators, HSF and Msn2/4, which differentially affect promoter chromatin remodeling. The focus of this project is to investigate the molecular mechanisms of chromatin remodeling at yeast heat shock gene promoters and the reasons why the chromatin changes vary drastically even for closely related and co-regulated heat shock genes. This project will address questions about the function of histone chaperones and possible cooperation between them and the identified ATP-dependent chromatin remodelers in regulating HSP gene expression. Since some histone interacting domains of histone chaperones can function as trans-activation domains, it will be tested if the converse is true, that activation domain function includes interaction with histones. An additional direction will be to investigate if and how the Msn2/4 degradation rate is regulated by components of the Mediator complex and if this has an effect on chromatin remodeling events.The methodological approach is based on using antibodies against components of chromatin and the transcriptional apparatus, available from a variety of sources, for chromatin immune-precipitations followed by high throughput real-time PCR. This approach allows the monitoring of changes in promoter-specific characteristics over a time course. Investigation of chromatin remodeling and transcription initiation will be done utilizing genomic collections of yeast strains with systematic deletion or tagging of diverse components of the cellular proteome. Standard genetic engineering techniques will be utilized as well for manipulating genes and gene promoter regions.Broader impact. The impact of this project will not only be on the scientific area of eukaryotic gene expression but also on developing graduate courses: the Molecular Biology of the Gene, Medical Biochemistry, and Foundations of Biomedical Sciences at the Sanford School of Medicine at USD. Methods of modern Molecular Biology, including those mentioned above, will be incorporated into the laboratory courses of graduate and undergraduate programs at the Division of Basic Biomedical Sciences and USD campus at Vermillion, South Dakota. Students from Indian tribal schools of South Dakota have participated in summer programs in the past and this is expected to continue in the future. Graduate and undergraduate students are currently involved in the project. Scientific results of the project will be discussed and disseminated via publications and international meetings and contacts.