Molecular signatures and cellular responses underlying hypoxic adaptation in sea turtles

Hypoxia-inducible factors (HIFs) are transcriptional regulators that orchestrate the canonical response to low-oxygen tension in animal cells. Vertebrates possess three HIF- isoforms, which arose from two gene duplication events of the ancestral HIF-1 gene. Here, we examined whether the HIF gene family (HIF-1, HIF-2, HIF-3, and HIF-1N inhibitor) shows evidence of positive selection in hypoxia-tolerant reptiles (Testudines), compared evolutionary patterns within the family, and assessed the transcriptional response to hypoxia in primary cells derived from a hypoxia-tolerant (Caretta caretta) and a non-tolerant (Sceloporus occidentalis) reptile. We found that HIF-1, HIF-2, and HIF-1N are highly conserved across reptiles, whereas HIF-3 is under positive selection in Testudines. We also identified multiple novel regulatory motifs unique to Testudines. Transcriptional signatures of hypoxia exposure indicated stark differences between lizards and turtles. Whereas lizard cells exhibited a canonical response to hypoxia, characterized by enriched cell-survival pathways, sea turtle cells exhibit a robust, distinctive transcriptional response involving enriched pathways related to protein synthesis, quality maintenance, and mitochondrial integrity. Surprisingly, cis-regulatory element analysis did not show HIFs as key regulators of the transcriptional response in either species. Instead, TFDP1 in lizard cells and E2F1 in sea turtle cells emerged as potential key regulators. TFDP1 regulates the cell cycle, specifically DNA synthesis and cell cycle progression, while E2F regulates DNA-damage response, apoptosis, metabolism, and fatty acid biosynthesis. These results suggest that the reptilian response to hypoxia is shaped by transcriptional plasticity, while highlighting key regulatory mechanisms driving hypoxic adaptation in sea turtle cells. However, positive selection of HIF-3 and novel HIF motifs suggest a combined, but yet to be uncovered, contribution of regulatory and coding sequence evolutionary mechanisms shaping hypoxia tolerance in Testudines.