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.