Regulation of the Heat Shock Response and HSF-1 Nuclear Stress Bodies in C. elegans

The Heat Shock Response (HSR) is a highly conserved stress responsive molecular pathway that functions to promote appropriate protein folding in the cell. The HSR accomplishes this primarily through the use of molecular chaperones that serve to bind to misfolded or unfolded proteins to assist in stabilizing and folding proteins back to their native functional state. The master regulator of this pathway is a transcription factor known as Heat Shock Factor 1 (HSF1). HSF1 regulates molecular chaperone expression in the cell’s basal state, but can also be stress induced by diverse biotic and abiotic signals including thermal shock, oxidative stress, osmotic imbalance, pathogenic invasion, cell transformation, and other pathological disease states. Thus, it is essential to understand how HSF1 function is regulated to better appreciate how the compromise of protein homeostasis (proteostasis) underlies many clinical disease pathologies. Evidence from invertebrates suggests that the HSR undergoes a rapid decline very early in adulthood and may explain the physiological effect of aging across many cell and tissue types. To better understand this process, we sought to develop an endogenously tagged fluorescent model of HSF1 in C. elegans. We utilized CRISPR/Cas9 mediated transgenesis and found that our tagged model behaves very similar to wildtype animals and displays similar phenotypes to previously published low-copy fluorescent models of HSF-1 which is the C. elegans homolog of mammalian HSF1. Using this novel model, we find that HSF-1 is capable of responding to novel cell stressors including Juglone, Peroxide, Paraquat, Osmotic stress, and UV exposure. This model also displays tissue-specific localization changes during the transition to adulthood. This time period of around 24 hours has been shown to be the critical window where the HSR collapses. The formation of these age-related HSF-1::GFP nuclear stress bodies (nSBs) is typically correlated with an increase in HSR activity, yet multiple measurements of proteostasis in the worm suggest that HSF-1 cannot mount an adequate response to meet acute stress demands. Genetic loss of the germline that has been shown previously to enhance longevity and stress resistance was able to suppress the formation of nSBs suggesting that the HSR remains robust and retains the youthful phenotype. Our data suggests that most cells in the worm form HSF-1:GFP nSBs during this early timepoint except the neurons. We hypothesized that this may be due to physical contact and examined the effect of ensheathment defective mutants, but found no difference in the appearance of nSBs after the transition to adulthood. Recent data in the literature suggested that chromatin remodel may underlie the abrupt decline in the HSR has previously stated. To identify other candidate chromatin remodeling genes we performed a targeted RNAi subscreen to search for other regulators of the HSR across the transition to adulthood. Our work identified pyp-1, an inorganic pyrophosphatase, that when suppressed is capable of enhancing the activity of HSR transcriptional reporters and can also support metastable protein folding reporter animals. Interestingly, we did not find a subsequent benefit in longevity due to this increased HSF-1 dependent activity. Additionally, the effect of pyp-1 knockdown on our reporter animals appeared to require initiation of RNAi prior to the transition of adulthood. Taken together, this data may suggest that pyp-1 performs a specific function during the transition to adulthood and that when this process is suppressed it results in increased HSF-1 activity in adulthood, but it is not sufficient to more broadly enhance proteostasis. This suggests further investigation into pyp-1 expression and activity to better understand its role in regulating the HSR. The literature suggests that mammalian HSF1 can be post-translationally modified by O-GlcNAclyation. This modification, which is similar to phosphorylation, is thought to be very dynamic and highly dysregulated in many metabolic disorders including diabetes, cancer, and neurodegeneration. The overall effect of O-GlcNAclyation on the HSR at the organismal level is still unknown. To investigate the role of O-GlcNAclyation in C. elegans, we utilized knockdown of the two O-GlcNAclyation modifying enzymes, oga-1 and ogt-1, to examine the effect of hyper-O-GlcNAclyation and hypo-O-GlcNAclyation on the HSR in the worm. We found that in larval animals disruption O-GlcNAc cycling typically results in the enhancement of proteostasis. However, in adults, we found that knockdown of oga-1 typically resulted in increased HSF-1 activity and ogt-1 knockdown compromised proteostasis. Interestingly, we found that modulating O-GlcNAc cycling appeared to alter HSF-1::GFP localization specifically in the intestine suggesting further research. Intriguingly, when performing experiments to confirm the modulation of O-GlcNAc cycling on the HSR was HSF-1 dependent we found a dramatic reversal of the phenotypes of oga-1 and ogt-1 genetic dosage. This conflict may suggest that the bacterial food source, RNAi pathway activation, or other factors may synergize with O-GlcNAclyation to specifically regulate the HSR and suggests future experimentation. Previous research from our lab suggests that HSF-1 may regulate a number of collagen and cuticle genes in C. elegans both in basal conditions and during acute stress. It was suggested that these collagen and cuticle genes may themselves regulate the HSR. To address this, we performed a RNAi subscreen of all available cuticle and collagen genes using a hsf-16.2 fluorescent transcriptional reporter. We found a number of candidate genes that both enhanced and suppressed stress induction relative to control knockdown. Further research is required to determine if these candidates also regulate endogenous HSF-1 target gene expression and by what mechanism this is performed with. Lastly, we utilized our validated HSF-1::GFP CRISPR/Cas9 model to examine the genetic regulation of HSF-1:GFP nSBs. It has been shown that the formation of HSF1 nSBs are typically correlated with an increase in HSR activity, but prolonged HSF1 nSBs is associated with a compromise in proteostasis. Similar to our previous research we find that longevity enhancing genetic backgrounds typically suppress HSF-1::GFP nSB formation during the transition to adulthood. Previously, we found that genetic loss of the germline conferred by a glp-1 mutation blocked the formation of these nSBs. Here we found that this effect requires p38 MAPK signaling as a pmk-1 mutant in the glp-1 mutant background reversed the effect of glp-1. Next, we found that SIR-2.1 overexpression also suppressed nSBs that form during the transition to adulthood and that this required the lysine demethylase jmjd-3.1. We also examined the role of disrupting insulin signaling which is well known to dramatically enhance longevity and stress resistance. Interestingly, we did find less nSBs relative to wildtype but the effect was not completely suppressed as seen in glp-1 and SIR-2.1 OE genetic backgrounds. Finally, we examined the role of hsb-1 in regulating HSF-1::GFP nSBs and found that hsb-1 mutants typically had increased nSBs and a delay in restoring the basal level of nSBs after acute stress. Also, in the hsb-1 mutant background, we found that jmjd-3.1 expression is enhanced which has been previously shown to regulate HSF-1’s chromatin accessibility to its target hsps. Taken together, this entire work establishes an endogenously tagged whole-organism model of HSF-1 and expands upon the knowledge of stress conditions that regulate HSF-1. We also identify novel genetic pathways at the whole organism level to regulate the HSR including age-specific modulation of inorganic pyrophosphatase, post-translation modification pathways, and manipulation to the worm cuticle. These identified signaling cascades require further research work to fully understand how each contributes to HSF-1 regulation and in what tissue types this regulation is present in