This project directly addresses the fundamental biological question as to how genes are turned on and off in response to an environmental stimulus. Yeast is used as the model system and the response to oxidative stress as the stimulus. The yeast system allows for the identification and characterization of important players in living cells (in vivo), followed by expansion and refinement of these studies at the mechanistic level (in vitro). This multidisciplinary approach involving genetic, molecular, and biochemical techniques provides research opportunities for students at various skill levels. In addition, the oxidative stress response is a critical physiological response that is extremely important to cell survival in an oxygen-rich environment. Indeed, oxidative stress is also highly relevant to the human condition because it plays fundamental roles in aging, neurodegenerative diseases, and cancer. With the goal of understanding transcriptional themes in response to oxidative stress, this project explores two main areas: the role of regulatory factors in the response to oxidative stress and the mechanism(s) by which a single activator can differentially regulate a wide array of genes. Taken together, these combined studies will advance the understanding of the transcriptional response to oxidative stress, and will add to the current knowledge of the ways in which genes are turned on and off in response to an environmental change.
In addition to the intellectual merit, the project will have broad impact in at least four ways. First, many aspects of these studies will provide suitable projects for undergraduate trainees, who may have time constraints as well as limited laboratory experience. Second, studies with higher technical and time demands will provide outstanding cross-training experiences for graduate students in a number of different disciplines (biochemistry, genetics, molecular biology). It is important to note that a majority of these techniques are versatile. Trainees could certainly utilize these techniques to investigate other critical genetic mechanisms (DNA repair, replication, recombination, RNA metabolism, epigentics, etc.) in their future careers. Third, to propagate the tradition of outreach, both undergraduate and graduate students will be involved in taking gene expression studies into elementary school classrooms. Fourth, results from this project will be published in broad based scientific journals, and presented at local and international meetings. Taken together, this project directly supports the mission of the NSF to advance discovery and understanding, while promoting teaching, training and learning.
Intellectual Merit One exciting emerging theme in the gene expression field is that many genes are regulated at a step after recruitment of the general transcription machinery. TATA-binding protein (TBP) and RNA polymerase II (RNAPII) occupy these "poised" promoters even when the gene is not expressed. Poised promoters are found across the evolutionary spectrum from yeast to humans. They are especially important during development and embryogenesis, although little is known about the transcription components required for expression of poised promoters in vivo. We have found that genes in yeast regulated in response to oxidative stress have poised promoters. Using a genetic screen, as well as characterization of a genuine poised promoter, we find that the Spt-Ada-Gcn5-acetyltransferase (SAGA) and Mediator coactivator complexes play distinct and essential roles in the regulation of gene expression at these poised promoters. Several transcription factors (including TBP, Spn1, RNAPII, and SAGA) occupy the promoter under both uninduced and induced conditions but in contrast, Mediator is recruited only after transfer to inducing conditions (Figure 1). Since all of these proteins are conserved from yeast to humans, continued studies using yeast as a model organism will enhance our understanding of the universal features of gene activation. Broader Impacts This project has offered training opportunities to a wide assortment of researchers. Three postdoctoral fellows, three PhD candidates (two of which have already earned their degrees and currently have faculty positions), six undergraduate students and two REU students have contributed to the research efforts of the project. In addition, we have partnered with fifth graders and their teachers at an Elementary School in an outreach project called "Biochemistry is Elementary". We created seven separate 90 minute sessions involving hands-on activities designed to introduce specific concepts in gene expression, biochemistry, yeast genetics and scientific reasoning to fifth graders (Figure 2). During each session, elementary students used our ~50 page custom-made workbook to complete their experiments, record their results and share their data with each other; all of which are vital components for scientific discovery. The direct participation in science as an inquiry-oriented experience positively influences everyone that is exposed to it (students, teachers, parents, and graduate student and postdoctoral volunteers). Our program was powerful because over 300 fifth graders were introduced to the potential for science as a career, to basic concepts in gene expression, and to the value of studying model systems (an important concept that is not effectively conveyed to the general public). In addition, the graduate students and postdoctoral fellows (~20) that assisted in this program are our next generation of science educators. These enriching experiences are key in developing their sense of contribution not just to the research community but also to the general public and "students" of all ages.
