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.