Miller, Dana L. et al. (2009) International Worm Meeting "Hydrogen Sulfide Protects Against Hypoxia."

Roth, Mark B., Miller, Dana L.

[International Worm Meeting, 2009]

It has been recently demonstrated that hydrogen sulfide (H2S) can protect animals against damage and death associated with decreased O2 availability. For example, H2S protects mammals against otherwise lethal hypoxia and improves outcome in mammalian models of severe blood loss, myocardial infarction, aortic occlusion and hepatic ischemia/reperfusion. The mechanisms by which H2S exerts beneficial effects in mammals are unknown. We have developed a C. elegans model investigate the genetic factors that contribute to damage associated with ischemia in animals and to define the molecular mechanisms that mediate the beneficial effects of H2S. Here we show that, as in mammals, H2S protects against hypoxia in C. elegans. We have found that specific hypoxic conditions induce aggregation of polyglutamine-containing proteins. The range of O2 concentrations that cause aggregation is expanded by mutations in the highly conserved hypoxia-inducible transcription factor, hif-1. These data suggest that perturbations in protein homeostasis occur when the cellular response to hypoxia is overwhelmed. Adaptation to H2S protects against hypoxia-induced disturbances of protein homeostasis. Even transient exposure to H2S early in development is sufficient to protect against hypoxia-induced aggregation of polyglutamine proteins. The H2S-induced changes that protect against hypoxia are distinct from those that cause increased lifespan and thermotolerance. These data show that exposure to H2S results in persistent physiological effects that can influence responses to changing environmental conditions.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Dana L Miller et al. (2004) West Coast Worm Meeting "Responses to Oxygen Deprivation in C. elegans"

Dana L Miller, Mark B Roth

[West Coast Worm Meeting, 2004]

Oxygen deprivation is a major cause of cellular damage associated with many pathological states. We are interested in understanding the cellular mechanisms that are required to sense and respond to changing levels of oxygen. C. elegans can survive in a wide range of oxygen tensions. There are at least two genetically independent responses to reduced oxygen tensions that have been characterized in embryos. At very low oxygen tensions, embryos enter a state of suspended animation and halt development. At slightly higher levels of oxygen, a hif-1 mediated response allows embryos to remain animated and continue through development. Intermediate oxygen tensions are lethal, suggesting that neither response can protect the embryo from hypoxic damage in this range. We have been characterizing the response of post-embryonic stages of C. elegans to various oxygen tensions. These observations have been used to design a forward genetic screen for mutants that are unable to sense and respond to changing oxygen tensions.

Horsman, Joe et al. (2013) International Worm Meeting "Translational Effect of Hydrogen Sulfide and a Novel Role for HIF-1."

Horsman, Joe, Miller, Dana

[International Worm Meeting, 2013]

The conserved transcription factor hypoxia inducible factor-1 (hif-1), responsible for responding to low concentrations of oxygen (hypoxia), is critical in the initial response to hydrogen sulfide (H2S). Hypoxia induces a dramatic decrease in translation. It has been shown that H2S can affect translation in specific mammalian tissues, suggesting mechanistic overlap. By studying translation in H2S we hope to uncover the mechanisms by which H2S elicits phenotypic changes in C. elegans. We have shown that exposure to low levels of H2S do not change global levels of translation in C. elegans. However, hif-1 is required to maintain translation in H2S. The role of hif-1 in modulating translation rates suggests a new and unique role for this key transcription factor. We hypothesize C. elegans actively maintains translation in H2S. While hif-1 is necessary for both survival and to maintain translation, our data suggests these two phenomena can be uncoupled. Preliminary results show that hif-1 can affect translation in a cell non-autonomous manner, as rescuing hif-1 only in the neurons is able to dramatically affect how the organism adapts to H2S. Our findings are a preliminary look at the the molecular mechanisms by which H2S affects C. elegans and suggests a novel hif-1 function in modulating translation rates.

Fawcett, Emily et al. (2013) International Worm Meeting "Adaptation to hydrogen sulfide induces a reversible developmental plasticity in C. elegans."

Miller, Dana, Fawcett, Emily

[International Worm Meeting, 2013]

Developmental plasticity, a phenomenon where early environmental conditions dictate adult phenotypes, predicts future environmental conditions to increase the likelihood of survival. However, there may be negative consequences if the future environment is different than predicted. Understanding the molecular underpinnings of plastic phenotypes, and how they may be reversed, may provide insight into how individuals respond differently to changes in the environment. We discovered that transient exposure to low levels of the gas hydrogen sulfide (H2S) during embryogenesis allows for survival of otherwise lethal H2S concentrations as adults in C. elegans. We have found that memory of adaptation to H2S requires the conserved SWI/SNF chromatin-remodeling complex, suggesting that the response to H2S stimulates epigenetic adaptations with long-lasting consequences. Remarkably, we have also demonstrated that H2S memory is reversible, suggesting that these epigenetic changes are malleable. The memory of H2S adaptation is erased by brief periods of fasting, but not by exposure to other environmental stresses. These results suggest that there is a specific interaction between the fasting response and the memory of adaptation to H2S. Consistent with this model, we show that the insulin/IGF1-like signaling (IIS) pathway modulates the persistence of H2S memory in fasting. Intriguingly, IIS is also required for the dauer developmental decision, another example of developmental plasticity in C. elegans. We found that well-fed animals with loss-of-function mutations in the FOXO transcription factor DAF-16 maintained H2S memory. However, adaptation to H2S was lost even faster in daf-16 mutant animals after fasting than wild-type controls. In contrast, animals with constitutively active DAF-16 are insensitive to fasting - they retain the memory to H2S significantly better than wild-type. Our results demonstrate that the cellular response to nutrient availability through the insulin-like signaling pathway is required for the persistence of H2S memory upon subsequent nutritional stress. We are currently working to understand the molecular underpinnings that integrate IIS signaling with the epigenetic memory of adaptation to H2S.

Iranon, Nicole et al. (2013) International Worm Meeting "AMPK regulates protein homeostasis in response to hypoxia and nutrient deprivation."

Miller, Dana, Iranon, Nicole

[International Worm Meeting, 2013]

In order to survive in changing environmental conditions, organisms must be able to successfully sense and integrate diverse environmental signals to respond appropriately. We are interested in how the energy sensor AMP-activated protein kinase (AMPK) integrates environmental cues regarding oxygen and nutrient availability to regulate protein homeostasis. Protein homeostasis (proteostasis) involves the coordination of protein synthesis, folding, degradation, and quality control in order to maintain a functional proteome. Maintaining proteostasis is required for the successful completion of development, healthy aging, and the ability to resist stressful environmental conditions. We have found that specific concentrations of low oxygen (hypoxia) cause a disruption of protein homeostasis in C. elegans, as measured by increased aggregation of polyglutamine proteins. Here, we show that nutritional cues regulate the effect of hypoxia on proteostasis. Animals that are fasted develop dramatically fewer protein aggregates compared to their fed counterparts when exposed to hypoxia. The effects of hypoxia and nutrient deprivation on protein aggregation are mediated through AMPK, a cellular nutrient sensor that coordinates energy homeostasis. Both hypoxia and a lack of nutrients can activate AMPK. We discovered that, when fed, AMPK mutant animals do not display increased protein aggregation in hypoxia. Moreover, fasting does not protect against hypoxia-induced aggregation in these mutant animals. We previously found that AMPK activity is required for developmental progression in hypoxia. Taken together, our results suggest that AMPK synthesizes input from various environmental cues in order to coordinate energy-consuming processes including proteostasis and development, which maximizes survival in a changing environment.

Fawcett, Emily et al. (2015) International Worm Meeting "Integrating environmental cues during development: An epigenetic bookmark of hydrogen sulfide."

Fawcett, Emily, Miller, Dana, Johnson, Jenna

[International Worm Meeting, 2015]

Fluctuations in environmental conditions can be deadly. The ability to rapidly and appropriately respond to stressful conditions can mean the difference between life and death. One strategy animals use to survive is to predict the onset and pre-emptively respond to stressful conditions based on prior life experiences. There is emerging evidence that this strategy, known as cellular bookmarking, is established through changes to the epigenetic landscape. However, accumulation of such modifications and their effects on gene expression could be detrimental to the success of a species if not tightly controlled. We have discovered that in C. elegans the response to the toxic gas hydrogen sulfide (H2S) forms a cellular bookmark that is maintained throughout development. Hydrogen sulfide is a common workplace toxin that has rapid and powerful physiological effects. We show that animals transiently exposed to sublethal levels of H2S survive subsequent exposure to H2S concentrations that are lethal to naive controls. Animals that have established the H2S bookmark exhibit a more robust transcriptional reactivation of H2S-inducible genes when re-exposed to H2S. We discovered that machinery involved in H3K4me2 (SPR-5 and SET-2) is required for the maintenance of a bookmark of H2S, including the elevated transcriptional reactivation required for survival of subsequent exposure. Surprisingly, the H2S bookmark is readily reversed by short periods of fasting, but not by other stresses. This reversal of H2S bookmarking also requires SPR-5, suggesting that H2S bookmarking formation and reversal are tightly controlled by H3K4me2 status. We are leveraging this uniquely tractable system to define mechanisms that allow animals to code changes of environmental conditions into chromatin modifications in a tightly controlled manner. We propose that these mechanisms will help to explain, at least partially, differences in sensitivity between individuals to drugs, stress, and even aging. .

Johnson, Jenna et al. (2015) International Worm Meeting "Histone modifications are required for the persistence of hydrogen sulfide memory in C. elegans."

Fawcett, Emily, Miller, Dana, Johnson, Jenna

[International Worm Meeting, 2015]

The formation of cellular memories allows animals to increase their probability of survival upon future stressors. The mechanisms by which this cellular memory persists in the absence of stimuli, however, remain unclear. To address this question, we developed a model by which the nematode Caenorhabditis elegans acclimates to the toxic gas hydrogen sulfide (H2S). We discovered that transient acclimation to sub-lethal concentrations of H2S allows for survival when exposed to otherwise lethal concentrations. Acclimated animals then have a more robust transcriptional activation upon subsequent H2S exposure. The SWI/SNF chromatin remodeling complex is known to play a role in the mechanism by which H2S memory persists in the absence of stimuli. We hypothesized that chromatin modifiers in addition to the SWI/SNF complex contribute to the formation and persistence of H2S memory. To test this we performed a candidate screen of mutant animals that had defects in chromatin modifications. We identified two genes, set-2, a histone methyl transferase, and spr-5, a histone demethylase, which are required for the persistence of H2S memory. Importantly, set-2 and spr-5 were also required for enhanced transcriptional activation when acclimated animals were subsequently exposed to H2S. Our data suggests that H3 lysine 4 dimethylation (H3K4me2) may be significant in H2S memory as both set-2 and spr-5 play a role in it. Based on our findings, we propose a mechanism by which acclimation to H2S and recruitment of the SWI/SNF complex changes methylation of histones at H2S-inducible promoters, thereby modifying the transcriptional response in the event that H2S is encountered again. This study identifies one fundamental mechanism that contributes to the persistence of cellular memories of environmental stressors. Additionally, our findings show potential conserved aspects of how environmental stress early in life influences chromatin structure and function. .

Alvares, Stacy et al. (2015) International Worm Meeting "Examining interactions between AMPK and HIF-1 during C. elegans development in hypoxia."

Alvares, Stacy, Miller, Dana, Clough, Courtnee

[International Worm Meeting, 2015]

Long-lived cells, including stem cells and cancer stem cells, often reside in a low oxygen (hypoxic) niche. Accumulating evidence suggests that the activation of genetic programs by hypoxia-inducible factor 1 (HIF-1) is important for growth, survival, and the self-renewal capability of these cells. C. elegans uses simple diffusion for gas exchange, allowing us to both deliver precise amounts of oxygen to all cells in the organism and study the effects of hypoxia on different cell types. In wild-type animals, development and reproduction continue when animals are exposed to 0.5% O2. In contrast, we have found that loss of function mutations in either hif-1 or aak-2, the catalytic subunit of AMP-activated protein kinase (AMPK), cause a reversible arrest of cell divisions and postembryonic development in larvae, and reproductive development ceases in adults. Our data indicate that AMPK and HIF-1 act in different pathways to regulate developmental arrest in hypoxia. Activating AMPK by metformin in a hif-1 mutant was not sufficient to sustain reproductive activity in 0.5% O2, and developmental arrest was not perturbed in a vhl-1 (ok161); aak-2(ok524) strain. Through an EMS screen, we isolated lines that exhibit perturbed development in hypoxia. The identification of the causative genetic lesions in these strains will provide insight into the genetic pathways responsible for promoting C. elegans development in hypoxia and may offer insight into what long-lived cells require in order to survive. .

Fawcett, Emily et al. (2011) International Worm Meeting "Adaptation to hydrogen sulfide modulates protein homeostasis."

Stoll, Kate, Fawcett, Emily, Miller, Dana L.

[International Worm Meeting, 2011]

Hydrogen sulfide (H2S), which is naturally produced in animal cells, has dramatic effects on physiology that can improve survival in changing environmental conditions. We are using C. elegans as a model to define molecular factors that mediate the physiological response to H2S in animals. We have defined transcriptional changes in mRNA abundance upon exposure to H2S using a microarray approach. These data reveal a rapid and progressive transcriptional response to H2S. We show that the hif-1 and skn-1 transcription factors coordinate the transcriptional response to H2S and are each essential to survive exposure to H2S. Functional analysis of gene products that accumulate in H2S suggests that adaptation to H2S alters protein turnover pathways. We are currently exploring the possibility that these factors mediate the effects of adaptation to H2S on protein homeostasis. Together, our data suggest that H2S has multiple effects on protein metabolism from translation to turnover. These changes in protein homeostasis may underlie the beneficial effects of H2S that result in increased lifespan and thermotolerance. Moreover, this work will provide unique insight into the mechanisms by which adaptation to H2S is integrated with the response to hypoxia at the cellular and organismal level.