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

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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.