Differential control of Zap1-regulated genes in response to zinc deficiency in Saccharomyces cerevisiae

Abstract

Background: The Zap1 transcription factor is a central player in the response of yeast to changes in zinc status. We previously used transcriptome profiling with DNA microarrays to identify 46 potential Zap1 target genes in the yeast genome. In this new study, we used complementary methods to identify additional Zap1 target genes.

Results: With alternative growth conditions for the microarray experiments and a more sensitive motif identification algorithm, we identified 31 new potential targets of Zap1 activation. Moreover, an analysis of the response of Zap1 target genes to a range of zinc concentrations and to zinc withdrawal over time demonstrated that these genes respond differently to zinc deficiency. Some genes are induced under mild zinc deficiency and act as a first line of defense against this stress. First-line defense genes serve to maintain zinc homeostasis by increasing zinc uptake, and by mobilizing and conserving intracellular zinc pools. Other genes respond only to severe zinc limitation and act as a second line of defense. These second-line defense genes allow cells to adapt to conditions of zinc deficiency and include genes involved in maintaining secretory pathway and cell wall function, and stress responses.

Conclusion: We have identified several new targets of Zap1-mediated regulation. Furthermore, our results indicate that through the differential regulation of its target genes, Zap1 prioritizes mechanisms of zinc homeostasis and adaptive responses to zinc deficiency.

Figure 1

New strategies to identify genes regulated by Zap1. A) Identifying genes affected by moderate zinc deficiency. Experiments E1, E2, and E3 were combined to identify genes that showed increased expression in zinc-limited cells (E1), wild type cells vs. zap1Δ mutant cells in low zinc (E2), and Zap1^TC-expressing cells in high zinc (E3). B) The ZRE sequences in the promoters of the previously identified 46 Zap1 target genes [3] were aligned and a logo was built using WebLogo. C) Experiments E3 and E4 were combined to identify Zap1 targets that respond to severe zinc deficiency. Regulatory Sequence Analysis Tools (RSAT) was used to identify potential ZREs in the promoters of co-regulated genes.

Figure 2

Confirmation of the microarray results for potential Zap1 target genes. S1 nuclease protection assays were performed using RNA isolated from cells grown under the same conditions as microarray experiments E3 and E4. A) ZRT1 and CMD1 were used as positive and loading controls, respectively. Results with candidate genes from Table 1 (B), Table 2 (C) and Table 3 (D) are shown. The band intensities were quantified and normalized to CMD1 levels, and the fold increase in E3 and E4 conditions is reported. These data confirmed the microarray results for these genes.

Figure 3

Evidence that ZRE sequences identified by RSAT are functional Zap1 binding sites. A) Electrophoretic mobility shift assay of candidate ZREs. Radiolabeled double-stranded oligonucleotides (0.5 pmol, 10,000 cpm) containing potential ZRE sequences from the indicated promoters were used as probes. The probes were mixed with 0 (-), 0.2 (±), 0.4 (+), or 0.8 (‡) μg per reaction of purified Zap1 DNA binding domain (Zap1_DBD). The arrow indicates the Zap1_DBD-DNA complex. The bona fide ZRE from TSA1 was used as a positive control and a mutant nonfunctional allele of that sequence (TSA1m) was used as a negative control. B) Nonrandom distribution of ZRE-like sequences in candidate Zap1 target gene promoters. In the upper panel, the positions of ZRE-like sequences in candidate Zap1 target promoters are plotted relative to the distance from the ATG start codon of the corresponding ORF. In the lower panel, the positions of ZRE-like sequences identified in the promoters of genes not showing zinc- and/or Zap1-responsive gene expression are plotted.

Figure 4

Differential regulation of Zap1 target genes in response to zinc. A) Microarray studies were performed with cells grown over a range of zinc (left panel) or over time after zinc withdrawal (right panel). For the dose-response analysis, cells were grown in LZM + 3 mM ZnCl₂, or LZM + 300, 100, 30, 10 or 3 μM ZnCl₂. Transcript levels were assayed using microarrays in which each sample was paired with the zinc-replete (LZM + 3 mM ZnCl₂) control. For the time-course studies, cells were grown to exponential phase in a zinc-replete medium (LZM + 1 mM ZnCl₂) and then transferred to a zinc-limiting medium (LZM + 1 μM ZnCl₂) for 8 hours. RNA was isolated at the indicated times and transcript levels were assayed using microarrays in which each sample was paired with the zinc-replete T_o control. Genes were grouped by related function and the results are displayed using the Java Treeview program . A narrow color intensity scale (yellow, increased expression relative to control; blue decreased expression) is used to show the conditions under which changes in gene expression were first detectable. B) The data for highly expressed genes in panel A were plotted with a broader scale to better show the differences in gene expression. The complete dose-response and time-course analyses were performed twice with similar results and the data, presented as the ratio relative to control, are provided in Additional file 5.

Figure 5

Differences in ZREs among Zap1 target genes. All ZREs from genes that respond to mild zinc deficiency and those that respond only to severe deficiency were divided into quartiles based on their RSAT scores. In the upper panel, the percentage of ZREs in each quartile for the genes responding to mild zinc deficiency are plotted. In the lower panel, the percentage of ZREs in each quartile for the genes responding to severe zinc deficiency are plotted. Chi-square analysis indicated that the different distributions among quartiles obtained with the two sets of ZREs are significant (P < 0.001).

Figure 6

The relationship between gene function and regulation by Zap1 in response to zinc status. The top line indicates the range of zinc levels in our experiments ranging from mild (LZM + 300 μM ZnCl₂) to severe (LZM + 3 μM ZnCl₂) zinc deficiency. The bars below indicate the range of zinc over which each functional response occurs. Sample data from Figure 4A are included to illustrate the patterns of differential regulation for genes from different functional categories. See Table 4 for details regarding the various adaptive responses and the particular genes associated with both homeostatic and adaptive responses.