Global transcriptional response of wild type and transcription factor deletion strains of Saccharomyces cerevisiae to the environmental stress of cold shock and subsequent recovery. Saccharomyces cerevisiae

Previous studies on the global transcriptional response of budding yeast, Saccharomyces cerevisiae, to cold shock have revealed that the response can be divided into a set of early response genes (after 15 minutes to 2 hours of cold temperatures) and late response genes (after 12 to 60 hours of cold temperatures). The late response genes include the ESR genes induced by many environmental stresses and are regulated by the Msn2 and Msn4 transcription factors, as they are during other environmental stresses (Kandror et al. 2004 PMID:15053871; Schade et al. 2004 PMID:15483057). However, the transcription factors responsible for the induction of the early response genes, the overall regulatory mechanism governing this early response, and the transcriptional response to recovery after cold shock remain largely unknown. Thus, we measured the early transcriptional response of S. cerevisiae to cold shock and subsequent recovery using DNA microarrays. To determine which transcription factors were responsible for these changes in expression, the same cold shock and recovery microarray experiments were then performed on six strains individually deleted for the transcription factors Cin5, Gln3, Hap4, Hmo1, Swi4, and Zap1. Overall design: Yeast cells were grown to early log phase at 30°C, then shifted to 13°C for 60 minutes (cold shock), and then shifted back to 30°C for another 60 minutes (recovery). Samples from independently grown replicate cultures (flasks) were collected before cold shock (t0), after 15 (t15), 30 (t30), and 60 minutes (t60) of cold shock, and after 60 minutes of cold shock followed by 30 (t90) and 60 minutes (t120) of recovery at 30°C. Data was collected for the following strains: wild type (BY4741 or BY4739), BY4741 Δcin5, BY4741 Δgln3, BY4741 Δhap4, BY4741 Δhmo1, BY4741 Δswi4, and BY4741 Δzap1. Generally, 4 independent biological replicates were performed for each strain (independent culture flasks), although for some timepoints and strains there are 3 or 5 replicates instead. Note that the t15 sample was not collected for the BY4741 Δswi4 strain. The sample from each experimental timepoint (t15, t30, t60, t90, and t120) was labeled with Cy5, and the t0 sample (before cold shock control) was labeled with Cy3. Experimental and control samples from the same flask were co-hybridized onto the same microarray slide. The dye orientation was swapped for two of the biological replicates per timepoint per strain (experimental sample labeled with Cy3 and control sample labeled with Cy5), except in one instance when there was only one dye swap performed for a particular strain and timepoint. Technical replicates were not performed. There were a total of 137 microarrays hybridized in this study.

过往针对酿酒酵母（*Saccharomyces cerevisiae*）冷激全局转录响应的研究表明，该响应可划分为早期响应基因（低温处理15分钟至2小时）与晚期响应基因（低温处理12至60小时）两类。晚期响应基因包含受多种环境胁迫诱导的环境胁迫响应（Environmental Stress Response, ESR）基因，且正如其他环境胁迫情境中一样，其表达受Msn2与Msn4转录因子调控（Kandror等人，2004，PMID:15053871；Schade等人，2004，PMID:15483057）。然而，介导早期响应基因诱导的转录因子、调控该早期响应的整体调控机制，以及冷激后恢复过程中的转录响应，迄今仍未明确。为此，我们利用DNA微阵列（DNA microarray）技术检测了酿酒酵母（*S. cerevisiae*）对冷激及后续恢复过程的早期转录响应。为明确哪些转录因子介导了这些表达变化，我们针对6个分别敲除了Cin5、Gln3、Hap4、Hmo1、Swi4及Zap1转录因子的菌株，开展了相同的冷激与恢复微阵列实验。

整体实验设计如下：酵母细胞于30℃下培养至对数生长期早期，随后转移至13℃处理60分钟（冷激），再转回30℃继续培养60分钟（恢复阶段）。从独立培养的重复培养物（摇瓶）中，于冷激前（t0）、冷激15分钟（t15）、30分钟（t30）及60分钟（t60）时，以及冷激60分钟后于30℃恢复30分钟（t90）、60分钟（t120）时收集样本。本研究检测的菌株包括：野生型（BY4741或BY4739）、BY4741 Δcin5、BY4741 Δgln3、BY4741 Δhap4、BY4741 Δhmo1、BY4741 Δswi4及BY4741 Δzap1。通常每个菌株设置4次独立生物学重复（独立培养摇瓶），但部分时间点与菌株的重复数为3或5次。需注意，BY4741 Δswi4菌株未收集t15样本。每个实验时间点（t15、t30、t60、t90及t120）的样本用Cy5标记，冷激前的t0样本（对照）用Cy3标记。将同一摇瓶的实验样本与对照样本共杂交至同一张DNA微阵列芯片上。对于每个菌株的每个时间点的2次生物学重复，交换染料标记方向（即实验样本用Cy3标记，对照样本用Cy5标记）；仅在个别情况下，某一菌株的某一时间点仅进行了1次染料交换。本研究未设置技术重复，共计完成137张DNA微阵列芯片的杂交实验。