Heat shock response (HSR) is an ancient and highly conserved signaling pathway in cells that regulates the expression of heat shock proteins (HSPs) in the presence of thermal and other environmental stresses. HSPs function to prevent the formation of non-specific protein aggregates and to assist proteins in acquiring their native structures. Although HSR has been extensively studied, key aspects of this pathway remain a mystery. In particular, how HSR is activated and regulated by the master transcription factor HSF1 is not well understood. The broad goal of this thesis is to develop a quantitative framework aimed at elucidating the HSF1-mediated activation of HSR in yeast cells. Understanding this process has important implications for development, physiology and disease. Indeed, HSF1 is conserved from yeast to human, has been shown to play an important role in stress resistance, health and disease, and is a therapeutic target for neurodegenerative diseases. Broadly, there are two putative (not mutually exclusive) models for activation in response to heat shock: (1) HSF1 dissociation from chaperone proteins and (2) hyper-phosphorylation and the subsequent activation of HSF1. However the relative contribution of each of these events in the activation process is not characterized. Thus far, there is no direct evidence linking either of these two events to activation, and the relative contribution of each mechanism to the activation process has not been quantitatively characterized. To address these issues, we develop a quantitative model of HSR in yeast cells. We use the model to make a series of quantitative predictions and, in a collaborative effort, experimentally test these predictions in a yeast model of HSR. Critically, we provide the first direct evidence for chaperone dissociation of HSF1 in response to heat shock. Moreover, we find that HSF1 phosphorylation is dispensable for activation of HSR, but is able to modulate its activity. Taken together, our work leads to a model for two “orthogonal” mechanisms regulating HSR in yeast, in which chaperone dissociation acts as an ON/OFF switch, whereas phosphorylation functions to tune the gain of the response. Finally, to complement and further test this quantitative model, we develop a novel microfluidic system to explore in more depth the behavior of individual cells in the presence of heat shock inputs. This includes (1) a microfluidic device with microscale on-chip heaters enabling programmable thermal perturbations and (2) a custom image analysis platform to follow single cells through heat shock time courses. In preliminary single-cell studies, we find a relationship between HSF1 phosphorylation state and cell-to-cell variability in HSR activation level (as measured by a transcriptional reporter). These preliminary results suggest that HSF1 phosphorylation may be generating and tuning noise in the HSR in order to promote phenotypic plasticity and increased survivability of a cell population in the face of stress.

Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.

Introduction: Myocardial Infarction (MI) is the leading cause of premature death worldwide. During MI-induced ischemia, the release of heat shock proteins (HSPs), a classic damage-associated molecular pattern (DAMP), by severely injured cells leads to prolonged inflammation through their activation of innate pattern recognition receptors, fibrosis, and subsequent contractile dysfunction. The regulation of HSPs is orchestrated by its master transcription factor, Heat Shock Factor 1 (HSF1). However, it is unknown if HSF1 is a potential integrated functional target to improve MI outcomes. We addressed this question by asking if the coordinated modulation of HSPs via genetic deletion of Hsf1 can be beneficial in MI. Hypothesis: We hypothesized that genetic deletion of Hsf1 can lead to improved survival and left ventricle (LV) remodeling through reduction of pro-inflammatory pathway activation in a murine model of MI-induced coronary artery ligation. Methods and Results: Eleven to thirteen-week-old male Hsf1-/- mice and Hsf1+/+ littermate controls were subjected to MI by left anterior descending (LAD) coronary artery ligation or sham operation. Hsf1-/- mice subjected to induced-MI had a significant higher survival rate (74%) at 28 days than WT mice post-MI in the same time frame (34%, p<0.001). Echocardiography at 3, 7, and 28 days post-MI; however, did not identify any difference in LV function between Hsf1+/+ and Hsf1-/- mice. Masson Trichrome and Picro Sirius Red staining of heart tissue sections following 7 days of sham or MI-operation indicated that MI-operated Hsf1-/- hearts had a significant smaller infarct size than Hsf1+/+ hearts at 19% compared to 32% (p<0.05), respectively; and less collagen deposition when compared to WT littermates. Cardiac expression of heat shock proteins was significantly lowered in the Hsf1-/- hearts compared to Hsf1+/+ hearts following 3 and 7 days of MI. However, no significant difference was observed in number of immune cells, cardiac gene expression of pro-inflammatory cytokines and chemokines, cardiac protein expression of NF-κB and MAPK-ERK1/2 signaling proteins, and serum IL-6 concentration between Hsf1+/+ and Hsf1-/- mice 3 days post-MI. Following 7 days of MI, there is a significant increase in the gene expression of pro-inflammatory cytokines, such as Il1b, and chemokines, such as Ccl2, in Hsf1-/- hearts than Hsf1+/+ hearts. Conclusion & Future Directions: Overall, the loss of Hsf1 improved survival and reduced infarct size following MI. However, its deletion did not affect inflammatory processes until 7 days post-MI or improved cardiac function in our specific murine MI model.

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.