Zhao H, Butler E, Rodgers J, Spizzo T, Duesterhoeft S, Eide D
Nutritional Sciences Program, University of Missouri, Columbia, Missouri 65211, USA.
J Biol Chem. 1998 Oct 30;273(44):28713-20. doi: 10.1074/jbc.273.44.28713.
Zinc homeostasis in yeast is controlled primarily through the regulation of zinc uptake. Transcription of the ZRT1 and ZRT2 zinc transporters increases in zinc-limited cells, and this induction is dependent on the ZAP1 gene. We hypothesized previously that ZAP1 encodes a zinc-responsive transcriptional activator. Expression of ZAP1 itself increases in zinc-limited cells. This response is also dependent on ZAP1 function through a potential positive autoregulatory mechanism. In this report, we describe the characterization of zinc-responsive elements (ZREs) in the promoters of the ZRT1, ZRT2, and ZAP1 genes. A ZRE consensus sequence, 5'-ACCYYNAAGGT-3', was identified and found to be both necessary and sufficient for zinc-responsive transcriptional regulation. We also demonstrate that ZREs are DNA binding sites for ZAP1. First, a dominant ZAP1 mutation, ZAP1-1(up), which causes increased expression of ZAP1-regulated genes in zinc-replete cells, exerted its effects specifically through the ZREs. Second, electrophoretic mobility shift assays and in vitro DNase I footprint analyses indicated that ZAP1 binds to ZREs in a sequence-specific fashion. These studies demonstrate that ZAP1 plays a direct role in controlling zinc-responsive gene expression in yeast by binding to zinc-responsive elements in the promoters of genes that it regulates.
酵母中的锌稳态主要通过锌摄取的调节来控制。在锌限制的细胞中，ZRT1和ZRT2锌转运蛋白的转录增加，并且这种诱导依赖于ZAP1基因。我们之前假设ZAP1编码一种锌响应转录激活因子。ZAP1自身的表达在锌限制的细胞中增加。这种反应也通过一种潜在的正自调节机制依赖于ZAP1的功能。在本报告中，我们描述了ZRT1、ZRT2和ZAP1基因启动子中锌响应元件（ZREs）的特征。一个ZRE共有序列5'-ACCYYNAAGGT-3'被鉴定出来，并且发现它对于锌响应转录调节既是必需的也是充分的。我们还证明ZREs是ZAP1的DNA结合位点。首先，一个显性的ZAP1突变体ZAP1-1(up)，它在锌充足的细胞中导致ZAP1调节基因的表达增加，其作用是通过ZREs特异性发挥的。其次，电泳迁移率变动分析和体外DNase I足迹分析表明ZAP1以序列特异性方式结合到ZREs上。这些研究表明ZAP1通过结合到它所调节基因启动子中的锌响应元件上，在控制酵母中锌响应基因表达方面发挥直接作用。
