Transcriptional regulation of Znt family members znt4, znt5 and znt10 and their function in zinc transport in yellow catfish (Pelteobagrus fulvidraco)

The study characterized the transcriptionally regulatory mechanism and functions of three zinc (Zn) transporters (znt4, znt5 and znt10) in Zn²⁺ metabolism in yellow catfish (Pelteobagrus fulvidraco), commonly freshwater fish in China and other countries. We cloned the sequences of znt4 promoter, spanning from −1217 bp to +80 bp relative to TSS (1297 bp); znt5, spanning from −1783 bp to +49 bp relative to TSS (1832 bp) and znt10, spanning from −1923 bp to +190 bp relative to TSS (2113 bp). In addition, after conducting the experiments of sequential deletion of promoter region and mutation of potential binding site, we found that the Nrf2 binding site (−607/−621 bp) and Klf4 binding site (−5/−14 bp) were required on znt4 promoter, the Mtf-1 binding site (−1674/−1687 bp) and Atf4 binding site (−444/−456 bp) were required on znt5 promoter and the Atf4 binding site (−905/−918 bp) was required on znt10 promoter. Then, according to EMSA and ChIP, we found that Zn²⁺ incubation increased DNA affinity of Atf4 to znt5 or znt10 promoter, but decreased DNA affinity of Nrf2 to znt4 promoter, Klf4 to znt4 promoter and Mtf-1 to znt5 promoter. Using fluorescent microscopy, it was revealed that Znt4 and Znt10 were located in the lysosome and Golgi, and Znt5 was located in the Golgi. Finally, we found that znt4 knockdown reduced the zinc content of lysosome and Golgi in the control and zinc-treated group; znt5 knockdown reduced the zinc content of Golgi in the control and zinc-treated group and znt10 knockdown reduced the zinc content of Golgi in the zinc-treated group. High dietary zinc supplement up-regulated Znt4 and Znt5 protein expression. Above all, for the first time, we revealed that Klf4 and Nrf2 transcriptionally regulated the activities of znt4 promoter; Mtf-1 and Atf4 transcriptionally regulated the activities of znt5 promoter and Atf4 transcriptionally regulated the activities of znt10 promoter, which provided innovative regulatory mechanism of zinc transporting in yellow catfish. Our study also elucidated their subcellular location, and regulatory role of zinc homeostasis in yellow catfish.