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.

Project Report

The malfunction of transcriptional regulation is at the core of a majority of medical conditions and diseases. A critical step in the activation and repression of genes is the modification of the chromatin structure at gene promoters. These chromatin rearrangements include posttranslational modifications of histones and in extreme cases, which nevertheless often happen during the activation of gene transcription, the displacement of nucleosomes from gene promoters. The factors involved in chromatin remodeling associated with gene activation and repression have been a focus of research efforts of a large number of research teams for several decades. Since the basic principles of chromatin rearrangements are the same for all eukaryotes, we utilized the yeast model system as the most genetically pliable and well-studied. The genes we analyzed as a model are heat shock genes. Heat shock genes’ activity is very easy to manipulate just by changing the temperature, and yet, these genes are preserved for all eukaryotes and in humans are shown to be a critical part of the development of such diseases as cancer, diabetes, neurodegeneration, and others. To execute this project, we investigated the enzymatic factors and intermediate steps of the gene activation process at three reporter gene promoters – HSP12, HSP82 and SSA3. The experiments were done primarily by utilizing gene knockout strains, which were either created by us or purchased from commercial sources. We found that the removal of nucleosomes at the HSP12, HSP82 and SSA3 promoters upon gene activation strongly depends on the activity of the RSC complex, which is one of the major ATP-dependent chromatin remodeling protein complexes, and to a lesser extent depends on the function of the other similar complex – SWI/SNF (Figure 1). We also found that both RSC and SWI/SNF complexes cooperated functionally with other protein factors – histone chaperones. The histone chaperones are recently discovered proteins or protein complexes which are transiently interacting with nucleosomal histones participating in assembly/disassembly of nucleosomes at specific DNA loci. We tested several histone chaperones—ASF1, SPT6 and SPT16—and found that the inactivation of any single histone chaperone created a temperature-sensitive phenotype. That finding indicates that all of these histone chaperones are likely involved in the regulation of the expression of heat shock genes responsible for survival at higher temperatures. Interestingly, for one histone chaperone – SPT16 (which is a part of the larger protein complex called FACT) – we found that while at a normal temperature without SPT16, the growth rate was low, with an increase of temperature, the growth rate was restored to that of the wild type cells (Figure 2). That further indicates the involvement of SPT16 in the regulation of heat shock genes. We tested what happens at the promoters of HSP12, HSP82 and SSA3 genes and found that inactivation of SPT16 creates a defect in the nucleosome assembly at these promoters. This defect creates a more open promoter readily accessible for the transcription activator, HSF, which, in turn, recruits RNA polymerase II and leads to the overexpression of heat shock genes. This overexpression is likely the reason for the synthetic thermo tolerance of the SPT16 depleted strain. The other important outcome of our project is the formulation of the new transcription activation mechanism which involves interactions between the activation domains of transcriptional activators and histones of promoter nucleosomes. According to this hypothesis, the activation domains, which function similar to histone chaperones, initially contact promoter nucleosomes causing a nucleosome distortion which, in turn, initiates downstream histone modifications and chromatin remodeling events leading to nucleosome removal and initiation of transcription (Figure 3). We based our hypothesis on the results of our genetic screen, which indicated that interacting targets of activation domains are positively charged hydrophobic moieties, suggesting histones as possible interacting partners for activation domains. Replacing native activation domains in such activators as Gal4 and HSF with known histone-binding modules creates a functional transcriptional activator capable of nucleosome remodeling and transcriptional activation. We also demonstrated that the histone binding module of histone chaperone Nap1, while fused to Gal4 DNA binding domain, creates an activator of transcription comparable in strength to the native Gal4. This hypothesis and data supporting it were delivered as an oral presentation at the Keystone Symposium on Molecular and Cellular Biology (January 9-15, 2011, Utah) and attracted significant attention. The experimental work was carried out by two faculty members with the participation of two graduate and seven undergraduate students. The results and ideology of the experimental work became part of several undergraduate and graduate courses at Butler University. These courses are: Clinical Biochemistry, Pharmaceutical Biotechnology, and Biopharmaceutical Analysis. A new course, Molecular Biology/Pharmacology, was also created and implemented for students of the departments of Biology, Chemistry, and Pharmaceutical Sciences at Butler University. In addition, the results were disseminated through invited seminars inside and outside of Butler University and participation in multiple international conferences.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
1029254
Program Officer
Susannah Gal
Project Start
Project End
Budget Start
2009-12-01
Budget End
2013-03-31
Support Year
Fiscal Year
2010
Total Cost
$395,621
Indirect Cost
Name
Butler University
Department
Type
DUNS #
City
Indianapolis
State
IN
Country
United States
Zip Code
46208