By analysis of whole-genome expression, we have identified a set of 150 genes that were upregulated in the nrg1Δ nrg2Δ
double mutant, relative to wild type, when cells were grown in rich medium containing glucose. These genes are involved in mitochondrial function, carbon utilization and signaling, nitrogen utilization, transport of nutrients and ions, cell wall and cell surface function, transcriptional control, mating, and other cellular processes. Of the 150 genes, 94 are glucose repressed and show increased expression when cells are limited for glucose, in accord with studies implicating Nrg1/Nrg2 in glucose repression of several specific genes (21
). Analysis of the upstream regions of the Nrg1/Nrg2 targets revealed that STRE and STRE-like elements are overrepresented, and a search of available expression data sets showed that many of these genes are regulated by a variety of stresses and by the stress-responsive transcriptional activators Msn2/Msn4. Thus, these studies implicate Nrg1/Nrg2 in regulation of a set of stress-responsive genes.
We also note that Nrg1/Nrg2 may regulate a set of stress-responsive genes larger than that identified here. Our genomic expression analysis identified genes that, in the absence of Nrg1/Nrg2, exhibit increased expression during exponential growth in rich medium containing glucose (unstressed conditions). It seems likely that some, and perhaps many, Nrg1/Nrg2 target genes are activated primarily by stress-responsive activators and are simply not activated under such optimal growth conditions, despite release of Nrg-mediated repression. Thus, it is possible that Nrg1/Nrg2 repress additional stress-responsive genes. It is also possible that the effects of Nrg1/Nrg2 on some of the 150 genes identified in this study are indirect. Only 14 of the 150 genes were among the 128 targets of Nrg1 identified by genome-wide location analysis (P
value < 0.001) carried out in cells exposed to hydrogen peroxide (SGA1
, and YGR050C
). However, there are at least two possible explanations for this minimal overlap: our study was carried out in unstressed cells, and the binding of Nrg1 to some promoters may not be easily detected by genome-wide location analysis. In accord with this view, the 128 identified targets did not include three genes shown to bind Nrg1 (CYC7
, and FLO11
, which has an upstream region nearly identical to that of STA1
; see references 2
, and 39
The relationship of Nrg1/Nrg2 to Msn2/Msn4 is not clear. Many Nrg-repressed genes contain STRE and STRE-like elements. The footprint of Nrg1 in the STA
) and the binding of CaNrg1 to the STRE (30
) suggest that the binding specificity of Nrg1/Nrg2 overlaps that of Msn2/Msn4, and it is possible that Nrg1/Nrg2 compete directly with Msn2/Msn4 for binding to some promoters. The individual roles of Nrg1 and Nrg2 are also unclear. For most of the 150 Nrg-repressed genes, RNA levels in glucose-grown nrg1Δ
single mutants were similar to those of the double mutant, indicating that both Nrg1 and Nrg2 are required for full repression under unstressed conditions. Levels of the two proteins are similar in unstressed cells (in both wild types and single mutants; C. D. Berkey, unpublished data), and twofold-reduced levels of total Nrg1/Nrg2 protein may be insufficient for repression at most promoters. In response to stress, the level of NRG1
RNA increases more than that of NRG2
); this differential regulation suggests that Nrg1 is particularly important for adaptation to stress.
The phenotypes of mutants lacking Nrg1 and/or Nrg2 support the physiological importance of these repressors in the regulation of stress responses. Previous studies showed that the nrg1Δ
mutation increases ion tolerance and suppresses the rim101Δ
defect in alkaline pH tolerance (22
) and that the nrg1Δ nrg2Δ
mutant exhibits increased invasive and pseudohyphal growth in response to carbon and nitrogen limitation, respectively (21
). Here we have documented increased resistance to salt and oxidative stress, consistent with the increased expression of stress-induced genes in the mutants. Conversely, we showed that the nrg1Δ nrg2Δ
mutant is more sensitive to freezing temperatures, in accord with evidence that the mutant exhibits increased expression of genes that are repressed in response to cold shock.
We have examined the expression of selected Nrg-repressed, stress-induced genes in the nrg1Δ nrg2Δ mutant during salt stress. In the cases of CTT1, CYC7, RSB1, ENA, XBP1, and SGA1, the RNA was upregulated more strongly in the mutant than in the wild type, indicating that Nrg1/Nrg2 limit the amplitude of induction, presumably by counteracting the function of stress-responsive activators. Based on our initial identification of Nrg-repressed genes by virtue of their increased expression in glucose-grown nrg1Δ nrg2Δ cells, Nrg1/Nrg2 clearly function to keep some stress-induced genes securely off in the absence of stress. It is also possible that the repressive function of Nrg1/Nrg2 effectively raises the threshold for induction of the stress response.
Nrg1/Nrg2 alter the resistance of cells to various stresses in growth assays, and yet these repressors make only modest contributions to the regulation of the genes we have examined here. Although Nrg1/Nrg2 may play more substantial roles in controlling expression of other genes, or in responses to other stresses, another possibility is that subtle regulatory effects matter to the cell. Stress responses typically involve induction of large numbers of genes, and large energetic costs are associated with such a gene expression program. The contribution of Nrg1/Nrg2 to fine-tuning the regulation of stress-responsive genes may serve to minimize unnecessary energy expenditures and enhance competitiveness in natural environments.