An organism's ability to respond to changes in oxygen tension depends in large part on alterations in gene expression. The oxygen sensing and signaling mechanisms in eukaryotic cells are not fully understood. To further define these processes, we have studied the ⌬9 fatty acid desaturase gene OLE1 in Saccharomyces cerevisiae. We have confirmed previous data showing that the expression of OLE1 mRNA is increased in hypoxia and in the presence of certain transition metals. OLE1 expression was also increased in the presence of the iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3 to the previously identified fatty acid response element was identified as critical for the induction of OLE1 in response to these stimuli using OLE1 promoter-lacZ reporter constructs. Electromobility shift assays confirmed the presence of an inducible band shift in response to hypoxia and cobalt. Mutational analysis defined the nonameric sequence ACTCAACAA as necessary for transactivation. A 20-base pair oligonucleotide containing this nonamer confers up-regulation by hypoxia and inhibition by unsaturated fatty acids when placed upstream of a heterologous promoter in a lacZ reporter construct. Additional yeast genes were identified which respond to hypoxia and cobalt in a manner similar to OLE1. A number of mammalian genes are also up-regulated by hypoxia, cobalt, nickel, and iron chelators. Hence, the identification of a family of yeast genes regulated in a similar manner has implications for understanding oxygen sensing and signaling in eukaryotes.

Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expression under normoxia, hypoxia, and cobalt treatment conditions in ⌬mga2 or ⌬spt23 deletion strains. The results of OLE1 promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia-and cobalt-induced OLE1 expression was dramatically decreased in a ⌬mga2 strain but not in a ⌬spt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae ⌬mga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the ⌬mga2 strain. These results indicate that MGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.Humans and other eukaryotes have developed sophisticated mechanisms to respond to decreased oxygen tension. These mechanisms are fundamentally important for developmental, physiological, and pathophysiological processes. Because of the importance of the response to hypoxic conditions, many mammalian cell types share a common mechanism of oxygen sensing and signal transduction (reviewed in references 3 and 22). One of the best-studied signal transduction pathways involved in the response to decreased oxygen tension contains the transcription factor, hypoxia-inducible factor 1 (HIF-1). Many hypoxia-inducible genes, such as erythropoietin and vascular endothelial growth factor, are upregulated by hypoxia via the activation of HIF-1 (reviewed in references 2 and 27). HIF-1 is a heterodimer consisting of an ␣ subunit and a ␤ subunit, two basic helix-loop-helix proteins in the PAS family of transcription factors. The ␤ subunit is also known as the aryl hydrocarbon receptor nuclear translocator ARNT. ARNT mRNA and protein levels are not significantly affected by ambient oxygen tension. In contrast, though HIF-1␣ mRNA levels are not appreciably affected by oxygen tension, HIF-1␣

In Saccharomyces cerevisiae, OLE1 encodes a ⌬9 fatty acid desaturase, an enzyme that plays a critical role in maintaining the correct ratio of saturated to monounsaturated fatty acids in the cell membrane. Previous studies have demonstrated that (i) OLE1 expression is repressed by unsaturated fatty acids (UFAs) and induced by low oxygen tension, (ii) a component of this regulation is mediated through the same low oxygen response element (LORE) in the OLE1 promoter, and (iii) Mga2p is involved in LORE-dependent hypoxic induction of OLE1. We now report that LORE-CYC1 basal promoter-lacZ fusion reporter assays demonstrate that UFAs repress the reporter expression under hypoxic conditions in a dose-dependent manner via LORE. Electrophoretic mobility shift assays show that UFAs repress the hypoxia-induced complex formation with LORE. Studies with a construct encoding a truncated form of Mga2p support the hypothesis that both hypoxia and UFA signals affect the processing of Mga2p and the UFA repression of OLE1 hypoxic induction is mediated through Mga2p. Data from Western blot assays provide evidence that under normoxic conditions, Mga2p processing produces approximately equimolar levels of the membrane-bound and processed forms and is unaffected by UFAs. Hypoxic induction of OLE1, however, is associated with increased processing of the protein, resulting in an approximately fivefold increase in the soluble active form that is counteracted by exposure of the cells to unsaturated fatty acids. Data from this study suggest that the Mga2p-LORE interaction plays an important role in OLE1 expression under both normoxic and hypoxic conditions. Saccharomyces cerevisiae is a facultative aerobe that can utilize oxygen for energy production and therefore has developed a number of mechanisms by which to sense and respond to changes in oxygen availability. The regulatory pathways that are involved with the response to hypoxia (low oxygen) have been studied for decades, and there has been significant progress in this field (3,23,43).In mammalian cells, the best-studied hypoxia signal transduction pathway is mediated by hypoxia-inducible factor 1 (HIF-1) (3, 32). HIF-1 is a heterodimer comprised of HIF-1␣ and ARNT, two basic helix-loop-helix proteins in the PAS family (39). ARNT mRNA and protein levels are not significantly affected by ambient oxygen tension. HIF-1␣ mRNA levels are also not appreciably affected by oxygen tension, but HIF-1␣ protein is only minimally present in normoxia and is rapidly degraded by the ubiquitin-proteasome pathway under this condition (15,16,21,30). This process is dependent on the von Hippel-Lindau tumor suppressor protein (pVHL) (24), which serves as the recognition component of a ubiquitin ligase that promotes ubiquitin-dependent proteolysis of HIF-1␣ and directly interacts with a highly conserved region within the HIF-1␣ oxygen-dependent degradation domain (7,22,28,35).Recent studies have indicated that a proline hydroxylation within that conserved region is responsible for targeting HIF-1␣ for pVH...