Epigenetic regulation of metabolic renal shutdown and recovery under freezing and ischemia-reperfusion stress in a stress-tolerant vertebrate (Lithobates sylvaticus)

Freeze tolerance is an extraordinary adaptation that allows certain vertebrates, such as the wood frog (Lithobates sylvaticus formerly called Rana sylvatica), to endure the freezing of up to 70% of their body water during overwintering. Survival under such conditions requires an intense depression of metabolism, suppression of energy-expensive cellular processes, and activation of protective mechanisms that preserve genome integrity and organ viability. This thesis investigates the epigenetic regulation of freeze tolerance in the wood frog, focusing on how chromatin modifications rearrange transcriptional control during freezing and thawing cycles. Using a combination of biochemical and molecular assays, this research characterizes changes in histone methylation, acetylation, and DNA methylation across key tissues, the kidney and adrenal gland, that play central roles in osmoregulation, energy balance, and stress response. The studies reveal a dynamic reprogramming of the chromatin structure driven by reversible posttranslational modifications of histones and DNA cytosine residues. In the kidney, freezing induced a coordinated repression of transcription-associated histone methyl marks alongside increased activity of repressive methyltransferases and suppression of demethylases. Conversely, thawing reactivated transcriptional marks, suggesting a controlled reawakening of gene expression needed for cellular repair and recovery. Parallel studies on histone arginine methylation identified tissue-specific regulation of protein arginine methyltransferases PRMT1/3/5 and marks H3R17me2a and H3R26me2a, highlighting a layer of epigenetic tweaking during hypometabolism. Investigations into histone acetylation and deacetylation revealed that KATs and HDACs are differentially expressed under freezing stress, maintaining a chromatin state that balances gene silencing with the activation of stress-responsive loci. Complementary analysis if DNA methylation and TET family of demethylases showed that global cytosine methylation patterns shift reversibly between frozen and thawed states, reinforcing long-term transcriptional stability. Together, these findings establish that freeze tolerance is the L. sylvatica is epigenetically mediated through a suite of chromatin modifications that regulate transcriptional shutdown and reactivation in a reversible, tissue specific manner. The work provides novel insights into the molecular foundation of metabolic rate depression, offering broader implications for understanding extreme stress survival, ischemia tolerance and cryopreservation strategies in vertebrates.

Transcriptional and Post-Transcriptional Gene Regulation of Anoxic Trachemys scripta elegans Liver and Muscle

Life without oxygen, termed anaerobiosis, is uncommon among vertebrate life given the heavy energy reliance from mitochondrial oxidative catabolic processes. In a subset of freshwater turtles (genera: Trachemys, Chrysemys) however, anoxia tolerance is incredibly developed, enabled via profound metabolic rate depression (MRD) and impressive buffering capacities. Translational suppression (> 90%) is a principal contributor to anoxia-mediated MRD in Trachemys scripta elegans turtles. Preliminary epigenetic control has been implicated in T.s. elegans anoxic survival, while transcriptome analysis has identified many anoxia-responsive genes—paving the way for additional investigations into transcriptional and post-transcriptional gene regulation in anoxic phenotypes. Here, I expand the histone methyl-epigenome of anoxic T.s. elegans, profiling transcriptionally-relevant histone modifications, along with key enzyme families (protein arginine methyltransferases, PRMT; lysine demethylases, KDM) that regulate them. In liver, I performed immunoblotting on isolated histones for epigenome profiling and on total protein for effector expression across anoxic states. I also utilized RT-qPCR to identify suitable reference genes and explore transcriptional regulation of hypoxia-responsive KDMs in early anoxic hepatic and muscular tissues. This work suggests a gene silencing role for the H3R8me2a-H3K9me2/3-H3S10ph motif on histone H3 during anaerobiosis, as well as opposing trends for Type II (PRMT5, SND1) and Type III (PRMT7, PADI4) proteins. At the mRNA level, I identify beta-actin and 14-3-3ζ as the optimal reference gene pair in anoxic tissues, while showing dynamic kdm expression suggestive of transcriptional activation, in liver, but not muscle. Post-transcriptional regulation was assessed through RNA m6A methylation and miRNAs. Immunoblotting of m6A effectors reveal conserved anoxia-mediated reductions in key demethylases and m6A-binding proteins (ALKBH5, eIF3D), albeit some differences. Enzyme expression and demethylase activity didn’t correlate with increased global m6A content, though m6A was > 10-fold greater in liver RNA versus red muscle. Separately, small RNA sequencing identified upregulated (let-7f-5p, miR-2114-5p) and downregulated (miR-1260b, miR-5100) miRNAs in anoxic T.s. elegans red muscle. Downstream KEGG and GO analyses predict miRNA-mediated inhibition of the cell cycle and protein/RNA turnover while suggesting alleviated miRNA action in muscle atrophy resistance. Together, this thesis significantly enhances our understanding of tissue-specific gene regulatory controls in T.s. elegans hypometabolic anoxia responses.

Hibernation vs. hypoxia: an underlying epigenetic,
microRNA, and signaling framework

Abstract:

A myriad of survival strategies have evolved in the animal kingdom to tolerate extreme environmental stress, ranging from freezing temperatures to oxygen deprivation to extreme heat/dehydration. All of these conditions, and the strategies to combat them, necessitate extensive yet reversible phenotypic changes to survive – changes grouped under overarching molecular themes of metabolic rate depression. In this thesis, I focused on cardiac tissue of two mammalian species with robustly evolved survival strategies: hibernation in thirteen-lined ground squirrel Ictidomys tridecemlineatus; and hypoxia tolerance in naked mole-rat Heterocephalus glaber. I examined a representative mode of regulation occurring at  each level of the central dogma, illustrating the vast interplay of coordinating molecular mechanisms required for hypometabolism. These included: epigenetic modification via RNA m6A methylation of mRNA transcripts; post-transcriptional regulation via miRNA; protein degradation via the ubiquitin-proteasome system and cullin-RING E3 ligases; and signaling modulation via SMAD proteins. The data I collected forms a network of intricate crosstalk between cardio-metabolic reorganization and cardioprotection during stress. Hypoxia is a subcomponent stress of hibernation; and as such, both species displayed cytoprotective adaptations to hypoxia-induced oxidative stress and ROS generation. Ground squirrels with increased DNA repair capacity to combat oxidative stress during hibernation had an additional layer of crosstalk between hibernation and hypothermia, increasing sensitivity to UV-irradiated DNA which is recognized and facilitated through proteins of the ubiquitin-proteasome system. Naked mole-rats demonstrated increased levels of protein degradation and proteasomal activity, incorporating hypoxia-related oxidative stress tolerance with their extreme longevity. Altered cellular signaling in both species included SMAD, MAPK, mTOR, AMPK, and NFκB pathways. NFκB dysregulation during oxidative stress linked with hypoxia champion HIF-1, and its degradation through the ubiquitin-proteasome system. Continuous inhibition of mTOR was proposed through the torpor-arousal cycle, facilitated by noncanonical eIF3-m6A translation which also serves as an energy saving mechanism. Altered mitochondrial dynamics were a recurring theme in both species and stresses; facets of their regulation were suggested via miRNA targeting and ubiquitin proteasome regulation. Taken together, my work both highlights the complexity of hypometabolic adaptation in cardiac tissue, and suggests globally conserved themes for regulation reflected across the central dogma of biology.

Enzymatic regulation of hepatic carbohydrate metabolism in freeze-tolerant wood frog, Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a widely researched vertebrate species due to their ability to endure natural freeze tolerance. These frogs can survive months of sub-zero temperatures during winter, even when 65-70% of their total body water is frozen as extracellular ice. However, this freezing results in the cessation of blood circulation, heartbeat, and breathing, leading to limited oxygen supply. Wood frogs must depend on anaerobic glycolysis for energy production during this time. Two of the basic mechanisms underlying freeze tolerance in wood frogs are metabolic rate depression (MRD) and the production of high concentrations of glucose as a cryoprotectant by the liver. This thesis aimed to investigate the regulation of key enzymatic checkpoints in hepatic carbohydrate metabolism in wood frogs. The research revealed the downregulation of pyruvate kinase (PK) during freezing, leading to the inhibition of glycolysis. The study also shed light on the suppression of fructose-1,6-bisphosphate (FBPase) and citrate synthase (CS), which subsequently inhibited flux through gluconeogenesis and TCA, respectively. This suppression is likely to aid in the survival of MRD during severe winters. Moreover, it was found that glycerol-3-phosphate dehydrogenase (G3PDH)—an enzyme linking lipid and carbohydrate metabolism—is upregulated despite the hypometabolic conditions during freezing. This upregulation of G3PDH activity likely plays a vital role in supporting the metabolic survival strategies of wood frogs. Overall, this thesis uncovered an intricate yet synchronized network of enzymes that support MRD and initiate hepatoprotective mechanisms allowing wood frogs to endure prolonged freezing and maintain cellular homeostasis.

Complex yet coordinated: regulation of transcriptional factors and cell signaling pathways to endure anoxia in Rana sylvatica

Abstract:

Wood frogs (Rana sylvatica) are a well-studied vertebrate model of natural freeze tolerance, surviving several months of winter subzero temperatures with 65-70% of total body water frozen as extracellular ice. Freezing halts blood circulation, heartbeat and breathing, restricting oxygen availability throughout the body and requiring a switch to anaerobic glycolysis for energy production, with its much lower ATP yield. To survive, wood frogs suppress their metabolic rate by about 90% to match ATP availability from glycolysis alone. Multiple cellular processes are regulated and suppressed, sustaining only pro-survival pathways until thawing occurs. Episodes of anoxia/reoxygenation also elevate reactive oxygen species (ROS) production that can surpass the antioxidant capacity of cells causing oxidative stress and tissue damage. This thesis examined a network of stress-responsive transcription factors (NRF2, OCT1, OCT4, YAP/TEAD, and RBPJ) and their associated pathways to determine their response and regulation over the anoxia/reoxygenation cycle. Decreased binding of transcriptional complexes to the promoter regions of target genes indicated a global reduction in transcription/translation processes. The data show also “functional switching” of OCT1, OCT4, and MAML while selectively upregulating antioxidants in a stress/organ specific manner. The present studies also shed new light on tissue repair mechanisms by demonstrating upregulation of selected pathway proteins. An increase in AHCY levels in liver also suggests maintenance of redox control, and elevated JMJD2C, TAZ, and MAML in skeletal and cardiac muscles indicates a potential increase in the expression of MyoD for muscle regeneration. Overall, the findings of this thesis document a complex yet coordinated network of transcriptional factors that support metabolic rate depression during freezing, combat oxidative stress, and initiate tissue repair mechanisms to endure prolonged anoxia and maintain cellular homeostasis in frozen wood frogs.

Role of glucose-induced transcription factor signalling and mitochondrial epigenetics in stress tolerant wood frog, Rana sylvatica

Abstract:

The freeze-tolerant wood frogs, Rana sylvatica are one of only a few vertebrate species in the animal kingdom, which are extensively studied to understand vertebrate freeze tolerance. They undergo whole-body freezing during winter and become ice solid with no heartbeat, brain activity and blood flow but amazingly come back to life during spring unharmed without any major changes in their body. Freeze survival is challenging, with wood frogs facing ischemia due to freezing of blood, dehydration via cell volume reductions due to loss of 60-70% of total body water into extracellular space as well as hyperglycemia, producing a huge amount of glucose as a cryoprotectant. Interestingly, wood frogs can also tolerate these stresses independent of freezing. Also, winter survival by wood frogs is associated with a metabolic reorganization to reduce their energy demands to a bare minimum by globally suppressing energy-expensive pathways and selectively regulating genes to prioritize available energy use for pro-survival pathways. This thesis examined the effects of freezing and dehydration-induced hyperglycemic response in selectively inducing transcription factor MondoA in regulating glucose-induced transcription and activating an adaptive transcriptional response to induce stress response via inflammasome activation, mitochondrial dysfunction and mitochondrial epigenetics. The current findings establish MondoA in guiding an adaptive transcriptional response to activate genes regulating glucose homeostasis and circadian rhythm in a tissue-specific manner in the liver during the freeze/thaw cycle. Also, the role of TXNIP (downstream to MondoA) and its PTMs, in activating inflammasome via NLRP-3 in stress-specific way during freezing was shown. Moreover, the higher mitochondrial presence of TXNIP did not correlate to protein expression of its downstream targets in inducing mitochondrial dysfunction in any of the stresses, which were attributed to its low/weak binding to TRX-2. Investigating the role of mitochondrial methylation suggests its tissue-specific regulation in the liver and potential role in maintaining a tight regulation of mitochondrial transcriptional and gene expression response. Altogether, findings from this thesis demonstrate that a highly synchronized and intricate control via multiple levels of regulation is present in activating mechanisms that are involved in maintaining cellular milieu during stress in wood frogs.

Regulation of citric acid cycle enzymes and related pathways in the skeletal muscle of hibernating Richardson’s ground squirrels, Urocitellus Richardsonii

Abstract:

Richardson’s ground squirrels (Urocitellus richardsonii) are small rodents inhabiting western Canada that spend a large portion of their life in hibernation. Hibernation is accompanied by a profound drop in body temperature to a minimum of 2-3 °C and a notable shift from carbohydrate to lipid consumption that involves large-scale rearrangements of central metabolic processes. This thesis investigated the regulation of key enzymatic checkpoints in the citric acid cycle (CAC) as well as enzymes that shuttle substrates into the CAC in skeletal muscle of ground squirrels during hibernation. Initial work investigated regulation of the pyruvate dehydrogenase complex (PDC) that bridges glycolysis and the CAC. Muscle PDC showed few changes in properties in terms of activity and inhibitory phosphorylation of the enzyme. This was in stark contrast to liver where strong suppression of PDC activity occurred during hibernation correlated with increased inhibitory phosphorylation on serine-300. This then led to investigation of two crucial irreversible regulatory steps of the CAC in the muscle: citrate synthase (CS) and the α-ketoglutarate dehydrogenase complex (KGDC). CS activity decreased significantly during hibernation. This correlated with decreased lysine succinylation of CS that reflected increased SIRT5 levels, the enzyme responsible for desuccinylase activity in mitochondria. KGDC also showed decreased affinity for coenzyme A in hibernating squirrels and marked differences in posttranslational modifications including increased tyrosine phosphorylation on all three enzyme subunits and increased serine phosphorylation on E2 subunit. Stimulating the action of endogenous protein kinases demonstrated decreased affinity for coenzyme A. Finally, regulation of muscle glutamate dehydrogenase (GDH) was analyzed to ascertain how GDH regulation mediated the flow of α-ketoglutarate into the CAC from amino acid catabolism. Most GDH kinetic parameters were unaffected between hibernating and euthermic states, except that glutamate affinity was substantially lower at 8 °C (a physiologically relevant temperature) for the enzyme from hibernating squirrels. GDH from hibernating animals also exhibited significantly higher ADP-ribosylation, suggesting a regulatory mechanism for modulating GDH. Taken together these findings suggest that enzymatic regulation in Richardson’s ground squirrel muscle is actively mediated by a variety of posttranslational mechanisms of the CAC and related enzymes to coordinate metabolic suppression during hibernation.

Molecular adaptations of mammalian hypoxia tolerance: Regulation of oxidative damage, neuroprotection, and microRNA

Abstract:

Prolonged exposure to limited oxygen can be lethal. Investigating the biological consequences of oxygen-deprivation in a hypoxia tolerant mammalian model can provide us with novel insights that could be applied to alleviate the ischemic insults experienced during stroke, or to better tolerate the hypoxia of high-altitude. Naked mole-rats (Heterocephalus glaber) represent nature’s solution to the problem of both acute and chronic oxygen limitation among mammals, solutions that have developed over evolutionary time. In this thesis I investigate their unique adaptations. The data I collected paints a picture of intricate signalling mechanisms in place to facilitate metabolic reorganization and protection during hypoxia. I determine that naked mole-rats are not as vulnerable to hypoxia-induced oxidative damage, as compared to hypoxia intolerant animals, and that brains appear to be the most resilient. The cell-survival proteins I profile implicate the induction of mechanisms responsible for conserving energy and maintaining neural integrity under low oxygen levels. Next, I perform the first microRNA-sequencing analysis in naked mole-rats, focusing on the hypoxic brain. Hypoxia-induced microRNAs suppress ATP-expensive processes, activate central signalling pathways, and coordinate a shift to non-fructose based anaerobic glycolysis. I then examine global metabolic reorganization and characterize a microRNA-mediated, AMPK-driven shift to carbohydrate metabolism in hypoxic skeletal muscles that may support tissue-specific prioritization of energy for more essential organs. Taken together, these findings advance our understanding of mammalian hypoxia tolerance and highlight the molecular mechanisms and complex layered regulatory controls required to endure frequent hypoxia exposures, as well as provide directions for future studies.

Roles of inflammatory signaling and microRNA in the adipose stress response of hibernating Ictidomys tridecemlineatus

Abstract:

Hibernating ground squirrels have an interesting ability to avoid organ dysfunction despite months of obesity, starvation, and low body temperature. However, pro-inflammatory signaling and conserved miRNA expression patterns have yet to be investigated in white and brown adipose tissues (WAT, BAT), organs with roles in fat storage and heat production, respectively. The inflammasome was activated in BAT during torpor and arousal relative to the control, as evidenced by increased inflammasome priming, elevated protein levels of NLRP3, AIM2, cleaved gasdermin D and IL-18, as well as increased caspase-1 activity. By contrast, caspase-1 activity, the ultimate indicator of inflammasome activation, was decreased during torpor and arousal in WAT relative to the euthermic control. Pro-inflammatory cytokines, matrix metalloproteinases (MMPs), and their inhibitors were also investigated to determine if cytokines and tissue remodeling proteins could be important in the stress response in hibernator adipose tissue. An increase in IL-1α during torpor in BAT furthered the idea that BAT may use pro-inflammatory pathways as part of the response to cell stress. By contrast, the only change in WAT was a decrease in the total protein levels of MMP2, suggesting tissue remodeling may not be important in the maintenance of WAT homeostasis. Finally, conserved BAT and WAT miRNAs were analyzed. There was an association between the BAT miRNA expression profile and condition (control or torpor), but no association between the two variables in WAT. Consistently, fewer miRNAs were differentially expressed in WAT than BAT, with more being downregulated than upregulated. As expected, microRNAs were predicted to inhibit energy expensive pathways during torpor in both tissues, suggesting an important role for non-coding RNAs in the regulation of metabolic rate suppression. Unexpectedly, KEGG pathway analysis suggested miRNAs were less likely to target pathways involved in damage sensing and wound repair in BAT, and DNA damage repair in WAT. Together, the data in this thesis suggest an upregulation of stress sensing and response in BAT in torpid and arousing ground squirrels through the regulation of inflammasomes, inflammatory signaling, and miRNA expression. By contrast, DNA repair may be increased in WAT but generally, pro-inflammatory pathways were suppressed.

Regulation and modification of peripheral circadian molecular clocks in 13-lined ground squirrels during hibernation

Abstract:

During winter, hibernators are able to conserve energy during times of limited resources through the virtual cessation of energetically expensive processes that are thought to be intrinsic to the cell in homeostasis. During prolonged hibernation, these mammals, such as the 13-lined ground squirrel (Ictidomys tridecemlineatus), shut down the bulk of transcription and translation in order to preserve resources yet still require the expression of subsets of genes to assist with the challenges encountered during hibernation. Hibernators provide a unique opportunity for examining the dynamics of circadian clock activation in a system that requires the selection of groups of transcripts against a backdrop of suppressed cellular activity. This research shows that peripheral circadian clocks are regulated and have adapted to function in a tissue-specific manner that is congruent with the tissues functions during hibernation.

In addition, substantial transcriptional and post-transcriptional machineries are required to endure deep torpor and low body temperature, including increased regulation over genomic activity by epigenetic enzymes. Both RNA adenosine and protein arginine methylation act to regulate activity within the circadian clock via epigenetic mechanisms and provide novel opportunities to uncover information about the post-translational modifications used during hibernation. RNA N6-methyladenosine (m6A) dynamics were maintained during hibernation and levels of m6A were increased on mRNA transcripts during torpor in liver. Responses by protein arginine methyltransferase (PRMT) enzymes were tissue-specific and within liver and white adipose, revealed responses that characterized metabolic reprogramming, whereas skeletal muscle PRMT activity was centered around transcriptional regulation. This research suggests that dynamic epigenetic modifications provide a mechanism for maintaining translation of selected groups of necessary transcripts during hibernation, including core circadian clock genes, against a backdrop of stunted transcript processing. These data also provide evidence that the circadian clock is an important and integral regulator of peripheral tissues within the mammalian hibernation phenotype.