In order to explore the regulatory mechanism of zip4, zip5 and zip9 in zinc metabolism of grass carp (Ctenopharyngodon idella), the effects of zinc (Zn) on the mRNA expression of zip4, zip5 and zip9 were investigated. Compared to the control, the mRNA levels of zip4 and zip9 were significantly reduced under low and high zinc in L8824 cells
the mRNA expression level of zip5 was significantly increased under low and high zinc incubation. Then, their promoter sequences were cloned, which were 2361 bp, 2004 bp and 2186 bp sequences for zip4, zip5 and zip9 promoters, respectively. The transcriptional activities of the three promoters had different responses to Zn treatment. The transcriptional factor signal transducer and activator of transcription 3 (STAT3) had specific binding sites at -1111/-1121 bp of zip5 promoter and at -1679/-1689 bp of zip9 promoter. Similarly, krüppel-like factor 4 (KLF4) could specifically bind to the -599/-609 bp sequence on the zip5 promoter and the -261/-272 bp sequence on the zip9 promoter. The results of electrophoretic mobility-shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) indicated that Zn incubation increased DNA binding capacity of STAT3 to zip5 and zip9 promoters, and decreased DNA binding capacity of KLF4 to zip5 and zip9 promoters. This study provides a good basis for elucidating the regulatory mechanism of zinc metabolism in the vertebrates.