DELETION OF METHYLTRANSFERASE SET9 ENHANCES HYPOXIA TOLERANCE IN ZEBRAFISH

As a group of the lower vertebrates, fishes live in water for almost their whole life and have evolved gill as a specific organ to absorb oxygen dissolved in water but not in air. Compared to land environment, oxygen in water is much lower. In addition to altitude, oxygen in water is also influenced by temperature, sun light, season, water mobility, aquatic life, etc. Thus, fish has evolved a variety of species with a high range of hypoxia adaptation. For aquaculture, due to the influence of oxygen dissolved in water on survive, growth, reproduction and disease-resistant, oxygen is one of the major limiting factors. With the development of aquaculture, in order to obtain high yields in a limited body of water, high density cultivation has become a pursuing tendency. So, oxygen is becoming more and more critical for improving aquaculture constantly and healthily. Similar to mammalians, during speciation, fishes have evolved sophisticated cellular sensors and systematic physiological systems responding to O₂ gradients. It is well-known that the hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) are master regulators of the cellular response to O₂. HIF-1α and HIF-2α orchestrate this cellular response to hypoxia by regulating the expression of a wide set of genes involved in multiple biological processes.　　In order to study the function of fish methyltransferase set9 (SET domain containing (lysine methyltransferase) 9, also called set7 or setd7) in hypoxic tolerance, we used zebrafish as the model organism. We targeted the first exon of set9 gene, and obtained a mutant line with 8 base pairs deletion of set9 gene using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technology. The putative transcript from this mutant line was predicted truncated peptide due to premature termination. The predicted protein encoded by set9^−/− was 14aa residues starting from the amino terminus and semi-quantitative RT-PCR assays showed that set9 mRNA was reduced in the mutant line as compared with the WT, indicating that set9 had been successfully knocked out in the mutant line. Then, a Ruskinn Invivo2 I-400 work station was used for hypoxia treatment. For hypoxia treatment of zebrafish embryo and larvae at 3 days postfertilization (dpf), the oxygen concentration in the Ruskinn Invivo2 I-400 work station was set to 2%. During preliminary experiments, we noticed that zebrafish adults were less tolerant of hypoxic conditions than larvae (3 dpf) and the zebrafish body weight significantly affected hypoxia tolerance. Then, we selected adult zebrafish (3 mpf) with similar body weights to test their hypoxia tolerance and the oxygen concentration in the Ruskinn Invivo2 I-400 work station was set to 5% instead of 2%. The apoptotic cells in the brains of set9-null zebrafish and their WT siblings subjected to hypoxia (5% O₂) were detected by TUNEL assay using the Apoptag Peroxidase In Situ Apoptosis Detection Kit (Millipore) following the manufacturer’s instructions. After 6h of hypoxia, there were significantly more apoptotic cells in the WT zebrafish brains than in the set9-null zebrafish brains. The results indicated that set9-deficient zebrafish showed significantly enhanced hypoxic tolerance and significantly reduced apoptosis in brain tissue. This study provides clues for further reveal the function and molecular mechanism of fish methyltransferase set9 in hypoxic tolerance, and provides a candidate target for breeding new fish species with enhanced hypoxia tolerance.