Abstract
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.
Highlights
As a naturally occurring and evolutionarily conserved physiological process, apoptosis plays an important role in tissue development, physiology and homeostasis [1]
Our study found that the promoter regions of apoptosis-related genes B-cell lymphoma 2 (Bcl-2) and Cycs have the functional metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α) binding sites that can respond directly to Zn, which provides an important basis for elucidating the transcriptional regulation of apoptosis by Zn
Studies show that HNF4α, Kruppel-like factor 4 (KLF4), STAT3 and FOXO4 mediate the regulation of cell apoptosis [37,38,39,40]
Summary
As a naturally occurring and evolutionarily conserved physiological process, apoptosis plays an important role in tissue development, physiology and homeostasis [1]. Cytochrome c is an important component of the apoptosome, which initiates the caspase cascade to induce apoptosis [2]. Bcl-2 is an antiapoptotic protein and protects cells from apoptosis by inhibiting the cytochrome c release from the mitochondria [3,4]. Bcl-2 and Cycs play very essential roles in the regulation of apoptosis. Bcl-2 and Cycs promoters have been studied extensively in mammals but have rarely been reported in fish [5,6]. Previous studies show that Zn inhibits apoptosis through three pathways: inhibiting the activity of Ca2+/Mg2+dependent endonuclease responsible for nuclear DNA fragmentation, inhibiting the activity of effector caspase-3 and increasing the Bcl-2/Bax ratio [13,14,15].
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