Functional profiling in yeast with zinc-limitation. Saccharomyces cerevisiae

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. As a result, we identified over 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Overall design: Pools of homozygous diploid deletion mutants (n = 4,607 strains) were grown in zinc defined media (LZM) at 2 concentrations for 5 and 15 generations (5g, 15g). Each strain has a deletion in a different gene. In each strain, the gene was replaced by a deletion cassette containing a antibiotic resistance gene and two BARCODES, up and down tags (Please see Giaever et al, 2002, Nature). These tags in the DNA are specific to each strain. We pool all deletion strains and grow them under a selective condition; extract DNA; amplify barcodes using universal primers and hybridize to arrays containing complemetary sequences to the up and down tags. In this way, we can look at growth of each of the strains. Our strategy is to analyze separately the up and down tags. Therefore, for each array (CEL file), we generate two data files, one for all ups and another one for all downs. In these files, the list of genes are the same but the data come from different set of probes, up or down. The values are log2 averages of replicate probes for the same tag. These pre processed files were used to identify strains with differential growth in 1uM zinc by comparing to the 100uM zinc arrays.

锌是一类必需营养素，作为众多酶类与转录因子的必需辅因子发挥功能。为发掘酵母在锌限制条件下生长所必需的基因与生物学过程，我们采用了全基因组功能谱分析（genome-wide functional profiling）。本研究共鉴定出400余个锌限制条件下酵母最优生长所必需的基因，其中包含多个锌响应转录因子Zap1（Zap1 zinc-responsive transcription factor）的靶基因，其必要性与低锌条件下Zap1对它们的上调调控作用相符。本研究同时鉴定出一批与Zap1非依赖型生物学过程相关的基因，涵盖内质网（endoplasmic reticulum）功能、氧化应激抗性、囊泡运输、过氧化物酶体生物发生及染色质修饰等过程。本研究还证实了巨自噬（macroautophagy）在低锌生长条件下的关键作用。最后，通过本研究分析，我们发现了ICE2基因在维持内质网锌稳态中此前未被报道的功能。

实验总体设计：将包含4607株纯合二倍体缺失突变株的混合菌株，在两种浓度的锌限定培养基（LZM）中分别培养5代与15代（记为5g、15g）。每株突变株仅缺失一个特定基因，该基因被一段包含抗生素抗性基因与两段分子条形码（BARCODES）的缺失盒替换，即上游标签与下游标签（详见Giaever等人2002年发表于《Nature》的研究）。DNA中的这些标签具有菌株特异性。我们将所有缺失突变株混合后，在选择性条件下培养；随后提取基因组DNA，利用通用引物扩增条形码序列，并将扩增产物与包含上游、下游标签互补序列的芯片进行杂交。通过该策略，我们可解析每株突变株的生长情况。本研究采用分别分析上游标签与下游标签的实验策略，因此针对每一张芯片（CEL文件），我们将生成两份数据文件：一份对应所有上游标签，另一份对应所有下游标签。两份文件中的基因列表完全一致，但数据分别来自上游标签探针组与下游标签探针组。文件中的数值为同一标签重复探针的log2均值。我们利用这些预处理后的文件，通过与100μM锌浓度组的芯片数据进行比对，筛选出1μM锌浓度下生长存在差异的突变株。