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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.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

Prokaryotic and eukaryotic organisms respond to various stresses with the production of heat shock proteins (HSPs). HSPs are molecular chaperones that bind to unfolded proteins and inhibit their aggregation as well as maintaining their solubility until they can be refolded to their original conformation. Stress-inducible hsp gene transcription is mediated by the heat shock element (HSE), which interacts with heat shock transcription factor (HSF). In this study, we examined the effect of KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam), a benzylidene lactam compound, on heat shock, sodium arsenite, cadmium chloride and herbimycin A-induced hsp gene expression in Xenopus laevis A6 kidney epithelial cells. In studies limited to mammalian cultured cells, KNK437 has been shown to inhibit HSE-HSF1 binding activity and stress-induced hsp gene expression. In the present study, western and northern blot analysis revealed that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP30 protein and hsp30 mRNA, respectively. Western blot analysis also determined that exposure of A6 cells to heat shock, sodium arsenite, cadmium chloride and herbimycin A induced the accumulation of HSP70 protein. Pre-treatment of A6 cells with 100 µM KNK437 inhibited stress-induced hsp30 mRNA as well as HSP30 and HSP70 protein accumulation. Immunocytochemistry and confocal microscopy were used to confirm the results gained from western blot analysis as well as determine the localization of HSP30 accumulation in A6 cells. A 2 h heat shock at 33°C resulted in the accumulation of HSP30 in the mostly in the cytoplasm with a small amount in the nucleus. Heat shock at 35°C resulted in substantial HSP30 accumulation in the nucleus. This is in contrast with A6 cells treated for 14 h with 10 µM sodium arsenite, 100 µM cadmium chloride and 1 µg/mL herbimycin A, where HSP30 accumulation was found only in the cytoplasm and not in the nucleus. A 6 h pre-treatment with 100 µM KNK437 completely inhibited the accumulation of HSP30 in A6 cells heat shocked at 33 or 35°C as well as cells treated with 1 µg/mL herbimycin A. The same pre-treatment with KNK437 resulted in a 97-100% decrease in HSP30 accumulation in A6 cells treated with 10 µM sodium arsenite or 100 µM cadmium chloride. These results show that KNK437 is effective at inhibiting both heat shock and chemical induced hsp gene expression in amphibian cells.

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.

<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.

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.

Small ubiquitin-like modifier (SUMO) regulates numerous biological functions. In a previous study we found that sumoylation of HSF2 is involved in regulating HSF2 bookmarking function, but the mechanism that mediates this regulation was unknown. The results in my work support the intriguing hypothesis that polycomb protein, Mel-18, actually functions as an anti-SUMO E3 protein, interacting both with HSF2 and the SUMO E2 Ubc9, but acting to inhibit Ubc9 activity and thereby decrease sumoylation of the HSF2. This study also suggested that Mel-18 negatively regulates the sumoylation of other cellular proteins, and we extend its targets to RanGAP1 protein. The results also show that RanGAP1 sumoylation is decreased during mitosis, and that this is associated with increased interaction between RanGAP1 and Mel-18. Previous studies showed little evidence of anti-SUMO E3 proteins, however, my study, taken together, found Mel-18 actually functions as a novel anti-SUMO E3 protein, interacting both with substrates and the SUMO E2 Ubc9 but acting to inhibit Ubc9 activity to decrease sumoylation of target proteins and also provide an explanation for how mitotic HSF2/RanGAP1 sumoylation is regulated. This finding also gives a clue for a future study direction in Mel-18 as a tumor suppressor: the anti-SUMO E3 function. Additionally, we identify a single-nucleotide polymorphism in another human PcG protein, Bmi-1, that changes a cysteine residue within its RING domain, cysteine 18, to a tyrosine. This C18Y polymorphism is associated with a significant decrease in levels of the Bmi-1 protein. Furthermore, the C18Y Bmi-1 protein exhibits a very high level of ubiquitination compared to wild-type Bmi-1, suggesting that that the low levels of this form of Bmi-1 are due to its destruction by the ubiquitin-proteasome system. Consistent with this hypothesis, treatment of cells with the proteasome inhibitor MG-132 results in a significant increase in levels of C18Y Bmi-1. This is the first example of a polymorphism in human Bmi- 1 that reduces levels of this important protein.

The ability to accurately predict danger and implement appropriate protective responses is critical for survival. Environmental fluctuations can cause damage at the cellular level, leading to the misfolding and aggregation of proteins. Such damage is toxic to cells: in age-related neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s and Huntington’s Diseases, the accumulation of damaged proteins in the brain ultimately leads to neuronal cell death and disease onset. To date, there is still no cure to combat the progressive degeneration and cell death seen in the brains of patients. Cells within an animal possess defense programs to minimize protein damage. One such defense mechanism is the activation of a program called the Heat Shock Response, which increases production of protective proteins known as heat shock proteins (HSPs). These HSPs act as molecular chaperones to assist with the clearing out of damaged proteins. This program is implemented by a conserved transcription factor, Heat Shock Factor 1 (HSF-1). However, in brains of patients with degenerative diseases, this protective mechanism, for reasons yet unknown, is not constantly activated. My thesis has involved the discovery of innate mechanisms that exist in organisms to activate this cellular protective mechanism against protein misfolding. My research, using the model organism Caenorhabditis elegans, has shown that the protective heat shock response in the cells of the animal can be triggered through neurohormonal signaling. The neurohormonal signaling that I am studying is one that is highly conserved across all organisms from plants to insects to mammals – serotonergic signaling. The stimulation of serotonergic signaling appears sufficient to activate the Heat Shock Response, even in the absence of real damage. In fact, the neuronal release of serotonin facilitates a pre-emptive upregulation of protective genes in the animal, which we have observed to be able to reduce the accumulation of damaged proteins in a C. elegans model of Huntington’s Disease. Additionally, I have seen that anticipating danger can enhance the animal’s stress response in a serotonin-dependent manner, thus facilitating better survival against a subsequent insult that can cause protein damage. Together, these studies present the novel possibility of protection against neurodegenerative disease via modulation of neurotransmission and/or neurosecretion. They also allow for understanding how sensory inputs are coupled to gene expression under stressful conditions. I hope to understand the mechanism by which animals adapt to changes in their environment by coordinating their sensory input with changes in behavior and gene expression.