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

知识价值。染色质重塑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。在温度诱导过程中，酵母热休克基因启动子的染色质变化强度超过了其他已确定的基因启动子的染色质重塑事件，但彼此之间存在显著差异。这些特征体现了热休克基因作为研究染色质重塑机制的强大模型。酵母细胞的应激反应受两类激活因子HSF和Msn2/4调控，它们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，以及即使是密切相关和共调控的热休克基因，染色质也会发生巨大变化的原因。本项目将探讨组蛋白伴侣蛋白的功能及其与已确定的atp依赖性染色质重塑蛋白在调节热休克蛋白基因表达中的可能合作。由于组蛋白伴侣的一些组蛋白相互作用域可以作为反式激活域，如果反过来是正确的，那么激活域的功能包括与组蛋白的相互作用，这将被测试。另一个方向将是研究Msn2/4降解率是否以及如何受到介质复合体成分的调节，以及这是否对染色质重塑事件有影响。方法方法是基于使用针对染色质成分的抗体和转录装置，可从各种来源获得，用于染色质免疫沉淀，然后是高通量实时PCR。这种方法允许在一段时间内监测特定于启动子的特性的变化。染色质重塑和转录起始的研究将利用酵母菌株的基因组收集，系统地删除或标记细胞蛋白质组的不同成分。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。更广泛的影响。这个项目的影响将不仅在真核基因表达的科学领域，而且在开发研究生课程：基因的分子生物学，医学生物化学和生物医学科学的基础在桑福德医学院在美元。现代分子生物学的方法，包括上面提到的，将被纳入基础生物医学科学部和南达科他州Vermillion校区的研究生和本科课程的实验课程。过去，来自南达科他州印第安部落学校的学生参加了暑期课程，预计未来还会继续这样做。目前参与该项目的有研究生和本科生。该项目的科学成果将通过出版物和国际会议和接触进行讨论和传播。

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.