Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2011;6(7):e22535. doi: 10.1371/journal.pone.0022535. Epub 2011 Jul 22.
The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA binding domain indicating other parts of Zap1 are unnecessary for this control. Overexpression of Zap1 overrode DNA binding regulation and resulted in constitutive promoter occupancy. Under these conditions of constitutive binding, both the zinc dose response of Zap1 activity and cellular zinc accumulation were altered suggesting the importance of DNA binding control to zinc homeostasis. Thus, our results indicated that zinc regulates Zap1 activity post-translationally via three independent mechanisms, all of which contribute to the overall zinc responsiveness of Zap1.
酿酒酵母的 Zap1 转录因子通过控制参与锌代谢的基因的表达，在锌稳态中发挥核心作用。Zap1 在缺锌细胞中活跃，在锌充足的细胞中受到抑制。在转录水平上，Zap1 通过正自调控来控制自身的表达。此外，Zap1 的两个激活结构域通过锌直接结合这些区域并抑制激活功能，彼此独立地受到调节。在本报告中，我们表明锌也抑制了 Zap1 的 DNA 结合。DMS 足迹法显示，Zap1 靶基因启动子的占有率受到转录自调控或不受其调控的调节。使用染色质免疫沉淀法证实了这些结果。锌对 DNA 结合活性的调节定位在 DNA 结合域上，表明 Zap1 的其他部分不需要这种控制。Zap1 的过表达会破坏 DNA 结合调控，导致组成型启动子占有率。在这些组成型结合的条件下，Zap1 的活性的锌剂量反应和细胞内锌积累都发生了改变，这表明 DNA 结合控制对锌稳态的重要性。因此，我们的结果表明，锌通过三种独立的机制对 Zap1 活性进行翻译后调节，所有这些机制都有助于 Zap1 的整体锌反应性。
