DDI2 and DDI3 (DDI2/3) are two identical genes in Saccharomyces cerevisiae encoding cyanamide (CY) hydratase. They are not only highly induced by CY, but also by a DNA-damaging agent methyl methanesulfonate (MMS), and the regulatory mechanism is unknown. In this study, we performed a modified genome-wide genetic synthetic array screen and identified Fzf1 as a zinc-finger transcriptional activator required for CY/MMS-induced DDI2/3 expression. Fzf1 binds to a DDI2/3 promoter consensus sequence CS2 in vivo and in vitro, and this interaction was enhanced in response to the CY treatment. Indeed, experimental over production of Fzf1 alone was sufficient to induce DDI2/3 expression; however, CY and MMS treatments did not cause the accumulation or apparent alteration in migration of cellular Fzf1. To test a hypothesis that Fzf1 is activated by covalent modification of CY and MMS, we performed mass spectrometry of CY/MMS-treated Fzf1 and detected a few modified lysine residues. Amino acid substitutions of these residues revealed that Fzf1-K70A completely abolished MMS-induced and reduced CY-induced DDI2/3 expression, indicating that the Fzf1-K70 methylation activates Fzf1. This study collectively reveals a novel regulatory mechanism by which Fzf1 is activated by chemical modifications and in turn induces the expression of its target genes for detoxification.

DDI2和DDI3 ( DDI2/3 ) 是酿酒酵母中编码氰胺 (CY) 水合酶的两个相同基因。它们不仅受到 CY 的高度诱导，而且还受到 DNA 损伤剂甲磺酸甲酯 (MMS) 的高度诱导，且调控机制尚不清楚。在这项研究中，我们进行了修改后的全基因组遗传合成阵列筛选，并将 Fzf1 鉴定为 CY/MMS 诱导的DDI2/3表达所需的锌指转录激活剂。Fzf1 在体内和体外与DDI2/3启动子共有序列 CS2结合，并且这种相互作用因 CY 治疗而增强。事实上，仅凭实验过量产生 Fzf1 就足以诱导DDI2/3表达；然而，CY 和 MMS 处理并没有引起细胞 Fzf1 的积累或迁移的明显改变。为了检验 Fzf1 通过 CY 和 MMS 的共价修饰激活的假设，我们对 CY/MMS 处理的 Fzf1 进行了质谱分析，并检测到了一些修饰的赖氨酸残基。这些残基的氨基酸取代表明，Fzf1-K70A 完全消除了 MMS 诱导的并减少了 CY 诱导的DDI2/3表达，表明 Fzf1-K70 甲基化激活了 Fzf1。这项研究共同揭示了一种新的调节机制，通过化学修饰激活 Fzf1，进而诱导其解毒靶基因的表达。