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.

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V–FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.

The steroid hormones are required for gonadal development in fish. The present study was undertaken to characterize the cDNA and promoter sequences of TSPO and SMAD4 genes in yellow catfish Pelteobagrus fulvidraco, explored the mRNA tissue expression and deciphered their promoter regions. Yellow catfish TSPO and SMAD4 shared the similar domains to the corresponding genes from other vertebrates. The TSPO and SMAD4 mRNAs were widely expressed in the detected tissues, but at different levels. Several transcription factors were predicted, such as Sp, GATA, AP1, SOX1, SRY, STAT, HNF4α, PPARγ, Pu.1 and FOXL2. PPARγ overexpression increased but STAT3 overexpression reduced TSPO promoter activity, and FOXL2 overexpression inhibited the promoter activity of TSPO and SMAD4. The site mutation and EMSA analysis indicated that TSPO promoter possessed STAT3 and FOXL2 sites. Overall, our provided the novel understanding into the transcriptionally regulatory mechanisms of TSPO and SMAD4 in fish.

Zinc (Zn) is a crucial trace element in vertebrates, fulfilling a range of physiological functions, whose metabolism and homeostasis are manipulated by Zn transporter proteins. SUMOylation, a reversible post-translational modification (PTM), extensively participates in various biological processes in the body, yet its underlying mechanism in regulating Zn transporters remains unexplored. Our findings indicate that high dietary Zn substantially elevated intestinal Zn content and modulated the expression profiles of Zn transporter-related genes and proteins, including ZIP8 transporter. In addition, high Zn diet tended to inhibit the SUMOylation modification and upregulate deSUMOylation modification in the intestine and intestinal epithelial cells. Furthermore, we found that the ZIP8 protein undergoes SUMOylation modification; UBC9 upregulated but SENP1 and Zn downregulated the SUMOylation level of ZIP8, and the K24 and K222 positions are the primary SUMOylation modification sites of ZIP8 protein in yellow catfish. Mechanistically, SENP1 modulates the deSUMOylation modification of ZIP8 by sensing Zn-induced oxidative stress. In summary, for the first time, we have uncovered a unique regulatory mechanism of ZIP8 mediated by SUMOylation modification in vertebrates and demonstrate that SENP1 is capable of sensing oxidative stress to reduce the SUMOylation modification of ZIP8 at K24 and K222 sites.