Rong-Mullins Xiaoqing, Ayers Michael C, Summers Mahmoud, Gallagher Jennifer E G
Department of Biology, West Virginia University, Morgantown, West Virginia 26506.
Department of Biology, West Virginia University, Morgantown, West Virginia 26506
G3 (Bethesda). 2018 Feb 2;8(2):607-619. doi: 10.1534/g3.117.300138.
Cellular metabolism can change the potency of a chemical's tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of alone was sufficient to overcome 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including In addition to a known Yrr1 target , Yrr1 also bound upstream of ; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the purine pathway, and the ribonuclease reductase pathways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the genes in the purine biosynthetic pathway, the mutant Yrr1 increased expression of genes even in the absence of 4NQO. These same genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization.
细胞代谢可改变化学物质的致癌潜能。4-硝基喹啉-1-氧化物（4NQO）是一种广泛用于癌症研究动物模型的致癌药物。转录因子Yrr1的多态性赋予对4NQO不同水平的抗性。为了研究不同的Yrr1等位基因如何调节基因表达从而导致抗性，通过RNA测序（RNA-Seq）和染色质免疫沉淀测序（ChIP-Seq）对携带不同Yrr1等位基因的三个同基因菌株在存在和不存在4NQO的情况下进行转录组分析。响应4NQO，Yrr1的所有等位基因都驱动（一种多药转运蛋白）的表达，在赋予4NQO抗性的等位基因存在时该表达最高，单独过表达就足以克服对4NQO敏感的生长。使用形状指标来优化ChIP-Seq峰，Yrr1与三个位点强烈相关，包括 除了已知的Yrr1靶标 外，Yrr1还结合在 的上游；然而，这些基因的过表达并未赋予4NQO抗性。RNA-Seq数据还涉及核苷酸合成途径，包括嘌呤途径，并且核糖核酸还原酶途径在响应4NQO时下调。通过单点突变将4NQO敏感等位基因转化为4NQO抗性等位基因在表型上模拟了4NQO抗性等位基因，并且虽然4NQO抗性等位基因增加了嘌呤生物合成途径中 基因的表达，但突变的Yrr1即使在不存在4NQO的情况下也增加了 基因的表达。这些相同的 基因仅在存在4NQO时在野生型等位基因中增加，表明该点突变激活了Yrr1以上调通常仅在应激反应中激活的途径。各种Yrr1等位基因还通过改变线粒体的功能影响在不同碳源上的生长。因此，对4NQO抗性的补充是在不可发酵碳源上生长不良，这又因同基因酵母中表达的Yrr1等位基因而异。酵母的氧化状态通过改变细胞代谢产生的活性氧（ROS）来影响4NQO毒性。RNA-Seq和ChIP-Seq的整合阐明了Yrr1如何响应4NQO调节全局基因转录以及各种Yrr1等位基因如何赋予对4NQO的差异抗性。这项研究为进一步研究Yrr1如何调节细胞对4NQO的反应提供了指导，也为进一步分析转录因子变异对碳源利用的影响提供了转录组资源。
