Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae

Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Δ or mot3Δ cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1–Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present—at much lower levels—demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.     

Multiple Elements and Auto-Repression Regulate Rox1, a Repressor of Hypoxic Genes in Saccharomyces Cerevisiae

The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function.     

Metabolic-state-dependent remodeling of the transcriptome in response to anoxia and subsequent reoxygenation in Saccharomyces cerevisiae

We conducted a comprehensive genomic analysis of the temporal response of yeast to anaerobiosis (six generations) and subsequent aerobic recovery ( approximately 2 generations) to reveal metabolic-state (galactose versus glucose)-dependent differences in gene network activity and function. Analysis of variance showed that far fewer genes responded (raw P value of or=2 generations) with the activation of Upc2- and Mot3-regulated networks involved in sterol and cell wall homeostasis. The response to reoxygenation was rapid (<5 min) and similar in both media, dominated by Yap1 networks involved in oxidative stress/redox regulation and the concomitant activation of heme-regulated ones. Our analyses revealed extensive networks of genes subject to combinatorial regulation by both heme-dependent (e.g., Hap1, Hap2/3/4/5, Rox1, Mot3, and Upc2) and heme-independent (e.g., Yap1, Skn7, and Puf3) factors under these conditions. We also uncover novel functions for several cis-regulatory sites and trans-acting factors and define functional regulons involved in the physiological acclimatization to changes in oxygen availability.     

The anatomy of a hypoxic operator in Saccharomyces cerevisiae.

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.