Identification Of HIF-1α Promoter And Expression Regulation Of HIF-1α Gene By Lps And Hypoxia In Zebrafish

The ubiquitously expressed hypoxia inducible factor-1α (HIF-1α) acts as a key transcription factor in regulating metabolism, development, cellular survival, proliferation and pathology under hypoxia condition. So far, the researches about HIF-1α usually focus on cardiovascular and cerebrovascular diseases, cancer and embryonic development among mammals. Compared to mammals, fish are more vulnerable to hypoxia stress and contamination, however, the regulation of HIF-1α in fish remains obscure. Thus, we chose zebrafish, the model animal as study subject, for cloning and bioinformatic analysis of HIF-1α romoter, and then study the HIF-1α expression by LPS (lipopolysaccharide) and hypoxia induction. The main results are as follows:1. Bioinformatic analysis of zebrafish HIF-1α promoterThe bioinformatic softwares were used to analyze zebrafish HIF-1α promoter, gene2promoter procedure predicated that the sequence from-651bp to+102bp was the promoter region. We then designed primers and chose-1578to+97, the whole1675bp promoter region for further study. CpG Island Searcher predicated CpG island was located at-97bp to+403bp, MethPrime predicated CpG island was located at-173bp to+38bp, indicating that zebrafish HIF-1α promoter contained a CpG island. Multiple potential transcription factors were subsequently identified, such as the essential regulation elements CCAAT/enhancer binding protein (CEBP) and enhancer box (E-box), moreover, the stress and immune response elements, cAMP-responsive element binding proteins (CREB), signal transducer and activator of transcription (STAT), heat shock factors (HSF) and nuclear factor kappa B (NF-κB) were also found. Aligning240bp HIF-1α proximal promoter region of zebrafish with other vertebrates showed more than82%identity with cyprinid fishes. Alignment with transcription factors (TFs) further revealed the common CCAAT binding factors (CAAT), STAT, hypoxia inducible factor (HIFF) and some other TFs in the same region.2. Construction of reporter plasmids of zebrafish HIF-1α promoter and luciferase assays Cloning six zebrafish HIF-1a promoters and reporter plasmids were constructed. Cells transfected with pGL1-1showed the highest luciferase level, while luciferase activity of pGL1-2and pGLl-3were almost abolished. Moreover, cells transfected with pGL2-1, pGL2-2and pGL2-3revealed considerable luciferase activity, indicating that the cis-acting elements required for basal promoter activity of the zebrafish HIF-1α gene were mainly located in-134to+97(pGL2-3). Sequence analysis of the core minimal promoter region-134to+97revealed a cluster of cis-elements, such as E-box, CEBP, STAT, HSF, CREB, etc. While the region between-1578and-930might contain mild negative regulatory elements interferon regulatory factors (IRF) and positive regulatory domain I binding factor (PRDI).3. Zebrafish HIF-1a transcriptional expression of LPS and hypoxia inductionAfter LPS stimulation only pGL2-2and pGL2-3were moderately activated, while other constructs were almost remain unchanged, suggesting LPS failed to lead to a significant change of the reporter plasmids under normoxic condition. Zebrafish larvae at3dpf were treated with various concentrations of LPS under normoxic condition, nevertheless, HIF-1α mRNA level did not show significant change in LPS-treated groups compared to the control group. It was also found that hypoxia alone (2.2mg/L of DO) could not activate the transcriptional level of HIF-1α either. In contrast, treatment with different concentrations of LPS under hypoxia condition caused a gradual increase of the HIF-la mRNA, LPS at a dose of10μg/mL exerted the significant increase. Time-course studies of LPS stimulation under hypoxia condition to HIF-1α in zebrafish larvae were then performed, the mRNA levels were moderately up-regulated within3h after treatment and reached the peak at8h after treatment, suggesting LPS and hypoxia regulated zebrafish HIF-1α transcriptional activity in a synergistic pattern. This synergistic effect was closely related to the living environment of fish, indicating this mechanism would be more conducive to fish survival.