Carnitine palmitoyltransferase I (CPT I) is a key enzyme involved in the regulation of lipid metabolism and fatty acid β-oxidation. To understand the transcriptional mechanism of CPT Iα1b and CPT Iα2a genes, we cloned the 2695-bp and 2631-bp regions of CPT Iα1b and CPT Iα2a promoters of grass carp (Ctenopharyngodon idella), respectively, and explored the structure and functional characteristics of these promoters. CPT Iα1b had two transcription start sites (TSSs), while CPT Iα2a had only one TSS. DNase I foot printing showed that the CPT Iα1b promoter was AT-rich and TATA-less, and mediated basal transcription through an initiator (INR)-independent mechanism. Bioinformatics analysis indicated that specificity protein 1 (Sp1) and nuclear factor Y (NF-Y) played potential important roles in driving basal expression of CPT Iα2a gene. In HepG2 and HEK293 cells, progressive deletion analysis indicated that several regions contained cis-elements controlling the transcription of the CPT Iα1b and CPT Iα2a genes. Moreover, some transcription factors, such as thyroid hormone receptor (TR), hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor (PPAR) family, were all identified on the CPT Iα1b and CPT Iα2a promoters. The TRα binding sites were only identified on CPT Iα1b promoter, while TRβ binding sites were only identified on CPT Iα2a promoter, suggesting that the transcription of CPT Iα1b and CPT Iα2a was regulated by a different mechanism. Site-mutation and electrophoretic mobility-shift assay (EMSA) revealed that fenofibrate-induced PPARα activation did not bind with predicted PPARα binding sites of CPT I promoters. Additionally, PPARα was not the only member of PPAR family regulating CPT I expression, and PPARγ also regulated the CPT I expression. All of these results provided new insights into the mechanisms for transcriptional regulation of CPT I genes in fish.

Fish gill tissue is a primary organ responsive to acute oxygen deprivation or dissolved oxygen (DO) fluctuations in aquatic environments. However, the adaptive mechanism of crucian carp to hypoxic stress remains largely unknown. Here, we investigated gill physiological and transcriptomic changes of crucian carp exposed to hypoxic conditions (dissolved oxygen concentration of 0.6 ± 0.3 mg/L) for different durations (0 d, 1 d, 2d, 3d, 4 d, and 5d). Transcriptomic analysis revealed that the hypoxia group (0.6 ± 0.3 mg/L DO) exhibited a reduction in interlamellar cell mass (ILCM) on the gill filaments, compared with the control group (6.6 ± 0.3 mg/L DO). With prolonged hypoxia stress, the epithelial cells in the gill lamellae became sparse at 3 d to 5 d, and gill vacuoles were increased. A total of 3,502 differentially expressed genes (DEGs) were identified, and 3 hypoxia-specific modules were screened through differential expression analysis, weighted gene co-expression network analysis (WGCNA), and Bayesian network analysis. The apoptosis, necroptosis, efferocytosis and FoxO signaling pathways were significantly enriched based on the KEGG enrichment pathway analysis. The VEGF pathway genes are significantly expressed, enhancing the generation of microvessels in the gill filaments, and improving the capacity to carry oxygen, thus enabling the crucian carp to adapt to hypoxia stress. Hypoxia activated glycolysis, enhanced anaerobic metabolism, promoted β-oxidation of fatty acids, providing energy and maintaining normal physiological metabolism, eventually improving antioxidant and immune capabilities in crucian carp. In summary, this study reveals the molecular mechanism by which crucian carp adapt to hypoxic stress. Our findings provide valuable references for promoting the healthy aquaculture of hypoxic-sensitive fish and breeding hypoxia-tolerant fish varieties.

We characterized the promoters of target genes of the signal transducer and activator of transcription 3, STAT3 (carnitine palmitoyltransferase I, CPT Iα1b, acetyl-CoA carboxylase alpha, ACCα; fatty acid synthase, FAS; and peroxisome proliferator-activated receptor gamma, PPARγ) in a teleost Pelteobagrus fulvidraco. Binding sites of STAT3 were predicted on these promoters, indicating that STAT3 probably mediated their transcriptional activities. Leptin had no effect on the activity of ACCα and PPARγ promoters, but increased CPT Iα1b promoter activity and decreased FAS promoter activity. The −979/−997 STAT3 binding site of CPT Iα1b and the −794/−812 STAT3 binding site of FAS were functional binding loci responsible for leptin-induced transcriptional activation. The study provided direct evidence that STAT3 regulated the expression of CPT Iα1b and FAS at the transcription level, and determined the STAT3 response element on promoters of CPT Iα1b and FAS under leptin signal.