Organization and promoter analysis of the zebrafish (Danio rerio) chemokine gene (CXC-64) promoter

Zebrafish CXC-64, a chemokine representing a superfamily of chemotactic cytokines present in fish, is involved in recruitment, activation, and response to inflammatory stimulation. We cloned and sequenced the genomic DNA of the zebrafish CXC-64 chemokine; it was most similar to CXCL11 from humans and CXCL10 from a catfish. The zebrafish CXC-64 gene is approximately 4.0 kb long and has a four-exon, three-intron structure common to the human CXCL11 gene. However, the promoter region includes a typical TATA box and multi-transcription factor-binding sequences. To understand the roles of lipopolysaccharide (LPS), poly I:poly C, and tumor necrosis factor (TNF)-alpha in regulating zebrafish CXC-64 expression, serial deletions were made in the promoter region of this clone. Different fragments of the zebrafish CXC-64 5'-flanking region were transfected into RAW264.7 (mouse macrophage; Abelson murine leukemia virus transformed) and zfl (zebrafish liver) cells and then treated with 0, 10, 50, 100, and 200 ng/ml LPS, poly I:poly C, or TNF-alpha. The results showed that the promoter activity presented dose-dependent effects in LPS-treated RAW264.7 cells, TNF-alpha-treated RAW264.7 cells, and LPS-treated zfl cells. These results reveal that the zebrafish CXC-64 chemokine gene promoter region can be induced by LPS in both human and fish cell lines, which suggests that it plays an important role in regulating LPS.