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.