Hypoxic Response And Promoter Activity Analysis Of Zebrafish HO-1 Gene

Heme oxygenase-1 (HO-1) is the initating and rate-limiting enzyme of heme degradation. HO-1 has protective effects through anti-inflammatory, anti-apoptotic and anti-oxidative in many tissues and cells. HO-1 is the most inducible enzyme of the HO family. A variety of chemical and physical stress can induce its expression. Previous studies have found that hypoxia treatment can increase HO-1 mRNA level significantly in zebrafish embryos. In this paper we studied the hypoxia response and promoter activity of zebrafish HO-1 (DrHO-1).DrHO-1 encodes 272 amino acids. The structure is similar to mammalian, containing heme oxygenase domain and heme oxygenase signature. Multiple amino acid sequence alignment found that the DrHO-1 is relatively conservative. Real-Time PCR showed that hypoxia can increase the expression level of DrHO-1 gene in many tissues, such as brain, liver and gill. Knock-down of DrHO-1 gene can significantly retard the embryo development compared with control under hypoxic conditions.Co-microinjection DrHO-1 mRNA and DrHO-1 morpholino can restore the phenotypes. These results show that DrHO-1 is a hypoxia-responsive gene and plays an important role in hypoxia adaption of zebrafish.Moreover, we isolated and sequenced 5'-flanking sequences 1765bp of the DrHO-1 gene for the regulation mechanism investigation. Sequence analysis showed many potential binding sites on the promoter fragment for many transcription factors, such as AP-1, CREBP, SOX5, STRE, GATA family, HRE, and so on. We amplified a series of deletion fragments of the promoter and performed in vitro luciferase assays for DrHO-1 promoter activities.The data showed that the regulation region from -740 to -220 might contain major c/s-acting elements, whereas the region from -1615 to -740 might contain suppression elements. To reveal the role of DrHO-1 promoter in HO-1 transcription regulation, we cloned the deletion fragments of the promoter into Tol2 plasmid and microinject the recombinant vectors into zebrafish embryos. Green fluorescence analysis showed that the -1615/+150 and -1215/+150 fragments have the strongest promoter activity in vivo.EPC cells were co-transfected with pCS2-zfHIF-1a/HIF-3aPPL-EGFP and 5' deletion constructs of DrHO-1 promoter. In vitro luciferase assays showed that HIF-1a binding sites on HO-1 promoter sequence might mainly locate in the region from -1215 to -740 which has two forward HRE sites, and HIF-3a binding sites might mainly located in the region from -740 to -220 which has a reverse HRE site. Moreover, zebrafish embryos were microinjected with HIF-la/HIF-3a mRNA. Real-Time PCR analysis showed that the expression of the DrHO-1 gene increased significantly after DrHIF-1?/HIF-3? overexpressed. These results showed that HIF-1/HIF-3 might regulate the expression of DrHO-1.