Characterization of Kruppel-like factor 6 in Epinephelus coioides: The role in viral infection and the transcriptional regulation on Peroxisome proliferator-activated receptor δ

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of EcKLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys2-His2) domain in the C-terminal region. Basing on the detection of the mRNA levels of viral genes, western blotting of MCP protein, and morphological CPEs, we found that the overexpression of EcKLF6 suppressed the replication of Singapore grouper iridovirus (SGIV), exerting its antiviral activity against fish virus. Moreover, promoter analysis was performed to investigate whether EcKLF6 was a regulator of EcPPAR-δ. The luciferase reporter assay and real time PCR results indicated a negative regulatory role of EcKLF6 on EcPPAR-δ transcription in grouper. Further experimental analysis shows that the potential EcKLF6 binding sites may locate in the EcPPAR-δ-4-M3 (+133 to +154) and EcPPAR-δ-4-M4 (+354 to +368) region of the EcPPAR-δ promoter. Electrophoretic mobile shift assays (EMSAs) verified that EcKLF6 interacted with the binding site of the EcPPAR-δ-4-M4 promoter region. In addition, we also found that KLF6 promotes inflammatory responses in GS cells. Considering that KLF6 and PPAR-δ play opposite roles in regulating inflammatory responses, we speculated the promoting effect of KLF6 on inflammatory response may be related to its negative regulation on EcPPAR-δ. In conclusion, the present study provides the first evidence of the negative regulation of EcPPAR-δ transcription by EcKLF6 and contributes to a better understanding of the transcriptional mechanisms of EcKLF6 in fish.