This project will investigate the mechanisms that allow organisms to survive after exposure to high temperatures. Using baker's yeast (Saccharomyces cerevisiae), experiments will focus on understanding how cells coordinate genome-wide changes in gene expression following heat stress. Because the heat stress response is evolutionarily conserved, research using yeast as the model is expected to provide insights into the fundamental mechanisms employed in more complex organisms. Additional broader impacts of this research include the training of two graduate students and summer research opportunities for three high school biology teachers from a school district with a large, underrepresented minority population.
The dynamic regulation of stress-responsive genes is critical for viability of all eukaryotes. In S. cerevisiae, the gene-specific and evolutionarily conserved activator, Heat Shock Factor 1 (Hsf1), plays a central role in stimulating both basal and induced transcription of HSP genes. These genes encode molecular chaperones that are critical for maintaining protein homeostasis and in combating proteotoxic stress. Whether Hsf1 is also responsible for the global down-regulation of non-HSP gene expression following exposure to thermal stress is unknown. Aim 1 employs chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) to investigate genome-wide occupancy of Hsf1 under both non-stressful and stressful conditions, and determine the activator's role in directing the genome-wide occupancy of Mediator, a central coactivator and signal integrator of Pol II transcription. In addition, comparative dynamic transcriptome analysis will be used to measure global transcription rates and correlate these with genome-wide Hsf1, Mediator and Pol II occupancy. Aim 2 employs chromosome conformation capture techniques to characterize the in vivo conformation and nuclear organization of Hsf1-regulated genes under both non-inducing and heat shock-inducing conditions. A key goal is to solidify evidence that HSP genes loop, 'crumple' (accordion-style) and coalesce into transcriptionally active foci following exposure of cells to heat shock, and determine the kinetics with which they form and dissipate looped DNA structures. Additionally, through use of looping, crumpling and coalescence-deficient mutants, the biological significance of these phenomena will be explored. Results are expected to advance understanding of how gene regulatory changes enable cell survival following heat stress.
