Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast

Abstract

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

Figure 1

A diagrammatic representation of our approach to identify Zap1p target genes. WT, wild type.

Figure 2

The probability-based ZRE motif derived from meme analysis. The plot shows the letter-probability matrix (17) of the ZRE based on the elements found within the 46 potential Zap1p target genes. The scale indicates the degree of shading that corresponds to the probability of each possible base occurring at each position of the motif multiplied by 10 and rounded to the nearest integer. The most probable form of the ZRE (i.e., the meme consensus) derived from the probability matrix is shown.

Figure 3

Zinc responsiveness of intact promoter regions fused to the lacZ gene. (Left) β-Galactosidase activity in wild-type cells (DY1457) in response to zinc in CSD. (Center) The same fusions assayed in wild-type (DY1457) and zap1 mutant (ZHY6) cells grown in CSD with (+Zn) or without (−Zn) 10 μM ZnCl₂ added. (Right) The activity of the same fusions assayed in the wild-type (DY1457) and ZAP1-1^up (ZHY7) strains grown in SD. Each graph is a representative experiment, and error bars represent 1 SD. WT, wild type.

Figure 4

Average expression ratios, as determined by microarray analysis, for several genes involved in fermentation and alcohol use. The dotted line denotes 2-fold changes. WT, wild type.