Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow catfish, Pelteobagrus fulvidraco, using in vitro cultured HEK293T model cells. The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors’ binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The −2017 bp/−2004 bp MRE in the zip10 promoter, the −360 bp/−345 bp MRE in the zip13 promoter, and the −1457 bp/−1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The −606 bp/−594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The −1383 bp/−1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates.

Here, we characterized the function of ctr1, ctr2 and atox1 promoters in yellow catfish Pelteobagrus fulvidraco, a common freshwater teleost in Asian countries. We obtained 1359 bp, 1842 bp and 1825 bp sequences of ctr1, ctr2 and atox1 promoters, and predicted key transcription factor binding sites on their promoters, including MRE, SREBP1, NRF2, KLF4 and STAT3. Cu differentially influenced the activities of ctr1, ctr2 and atox1 promoters from different regions. We found that the −326/−334 bp and −1232/−1240 bp locus in the atox1 promoter were functional NRF2 binding sites, which negatively controlled the activity of the atox1 promoter. The −91/−100 bp locus in the ctr1 promoter and −232/−241 bp and −699/−708 bp locus in the atox1 promoter were functional SREBP1 binding sites, which positively controlled the activities of ctr1 and atox1 promoters. Cu inhibited the NRF2 binding ability to the atox1 promoter, but promoted the SREBP1 binding ability to the ctr1 and atox1 promoters. Dietary Cu excess significantly down-regulated hepatic mRNA and total protein expression of CTR1, CTR2 and ATOX1 of yellow catfish, compared to the adequate dietary Cu group. The subcellular localization showed that CTR1 was mainly localized on the cell membrane, CTR2 in the cell membrane and the lysosome, and ATOX1 in the cytoplasm. In conclusion, we demonstrated the regulatory mechanism of three Cu transporters at the transcription levels, and found the functional NRF2 and SREBP1 response elements in ctr1, ctr2 and atox1 promoters, which provided new insights into their roles in the regulation of Cu homeostasis in fish.

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