Genome-wide Response of Saccharomyces cerevisiae upon Arsenate Exposure. Saccharomyces cerevisiae

Arsenic metalloid is a double-edge sword. On the one hand it is a very toxic and powerful carcinogen, and on the other it has been successfully used in the treatment of acute promyelocytic leukemia. In order to prevent the deleterious effects caused by arsenic compounds, almost all living organisms have developed mechanisms to eliminate it. In this study genome-wide response of S. cerevisiae to arsenic shows that this metal interferes with genes involved in the iron homeostasis including those encoding proteins that function in iron uptake, incorporation into Fe–S clusters, and more. In addition our data indicate that Yap1 transcriptionally controls the iron homeostasis regulator AFT2 as well as its direct target, MRS4. Most importantly in response to arsenate exposure Yap1 strongly regulates the expression of several genes involved in the Fe-S proteins biosynthesis, namely NBP35 and YFH1. Interestingly mRNA levels encoding Fet3, Ferro-O2-oxidoreductase required for high-affinity iron uptake, are drastically destabilized upon arsenic exposure. Such destabilization is due to the 5’ to 3’ exonuclease Xrn1 localized in the P Bodies. Moreover FET3 mRNA decay is not mediated by Cth2 and is independent on the formation of ROS responsible for the toxic effects of arsenic compounds. Strikingly, in presence of arsenate fet3 mutant shows resistance over the wild-type which leads us to suggest that Fet3 has a role in arsenic toxicity. Unexpectedly arsenic treatment seems to activate the non-reductive iron uptake in order to maintain the cellular iron homeostasis. Furthermore our genetic, biochemical, and physiological analysis demonstrate that aft1 mutant is sensitive to arsenic compounds and such phenotype is reversible upon addition of iron. We also show that arsenic exposure induces iron deficiency in aft1 mutant. In conclusion this work shows for the first time that arsenic, a chemotherapy drug used to treat a specific type of acute promyelocytic leukemia (APL), disrupts iron homeostasis and our results suggest that this disruption is independent on ROS generation. Finally we provide preliminary data confirming that such disruption also takes place in mammalian cells, an observation that can be very relevant in term of clinical applications. Overall design: yap1yap8 mutant cells independently transformed with pRS416 and YcpLac111, YcpLac111-YAP1, or pRS416-YAP8 were grown in triplicates in SC-URA-LEU containing 2mM of AsV until exponential growth phase, and RNA was extracted, labeled, and hybridized to Affymetrix Yeast Genome S98 arrays.

类砷（arsenic metalloid）实为一把双刃剑：一方面它是剧毒且强致癌的致癌物，另一方面却已成功应用于急性早幼粒细胞白血病（acute promyelocytic leukemia, APL）的临床治疗。为抵御砷化合物引发的有害效应，几乎所有生物体均演化出了排砷机制。

本研究针对酿酒酵母（S. cerevisiae）的砷暴露全基因组应答分析显示，该金属会干扰参与铁稳态（iron homeostasis）的基因，包括编码参与铁摄取、铁硫簇（Fe–S clusters）组装等过程的蛋白质的相关基因。此外，本研究数据表明，Yap1可通过转录调控铁稳态调节因子AFT2及其直接靶基因MRS4。尤为关键的是，在砷酸盐暴露条件下，Yap1会强力调控数个参与铁硫蛋白生物合成的基因的表达，具体包括NBP35与YFH1。

值得注意的是，编码高亲和力铁摄取所需的亚铁-O₂-氧化还原酶（Ferro-O2-oxidoreductase）Fet3的mRNA水平，在砷暴露后会发生显著不稳定。该不稳定现象由定位于P小体（P Bodies）的5'→3'核酸外切酶Xrn1介导。进一步研究发现，FET3 mRNA的降解并不依赖Cth2，且与砷化合物毒性相关的活性氧（reactive oxygen species, ROS）生成无关。

令人意外的是，砷酸盐存在时，fet3突变体相较于野生型菌株表现出抗性，这一结果提示Fet3在砷毒性过程中发挥了特定作用。更出人意料的是，砷处理似乎会激活非还原性铁摄取途径，以维持细胞内的铁稳态。此外，本研究的遗传、生化与生理分析证实，aft1突变体对砷化合物敏感，且该表型可通过添加铁元素实现逆转。我们还发现，砷暴露会诱导aft1突变体产生铁缺乏症状。

综上，本研究首次证实，砷——一种用于治疗特定类型急性早幼粒细胞白血病（APL）的化疗药物——会破坏细胞铁稳态，且本研究结果提示该破坏过程与ROS生成无关。最后，我们提供了初步数据证实，这种铁稳态破坏现象同样存在于哺乳动物细胞中，这一发现对于临床应用具有重要参考价值。

实验设计：将yap1yap8突变体细胞分别转化pRS416、YcpLac111、YcpLac111-YAP1或pRS416-YAP8，于含2mM砷酸盐的SC-URA-LEU培养基中以三重复样本培养至指数生长期，随后提取RNA、完成标记并与Affymetrix酵母基因组S98芯片进行杂交。