MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces 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 α subunit and a β subunit, two basic helix-loop-helix proteins in the PAS family of transcription factors. The β 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-1α mRNA levels are not appreciably affected by oxygen tension, HIF-1α protein is rapidly degraded by the ubiquitin-proteosome pathway in normoxia (15, 26). However, HIF-1α protein accumulates in response to hypoxia, certain transition metals (e.g., cobalt and nickel), and iron chelators such as desferrioxamine. Once the heterodimer is formed under these conditions, it can interact with other DNA-binding proteins which function in part to provide tissue and developmental specificity (reviewed in references 3 and 7). HIF-1α has also been shown to associate with the coactivator protein p300 (also called CBP) (1), which interacts with the basal transcription machinery (19, 32). The yeast Saccharomyces cerevisiae is a facultative anaerobic eukaryote, which differentially expresses a large number of genes in response to changes in oxygen availability (reviewed in references 3, 17, and 35). The oxygen-sensing and signal transduction pathways involved in the regulation of these genes have been the focus of several studies. Many yeast genes, such as ANB1, have been shown to be upregulated by complete anaerobiosis and are mediated in large part through the Rox1p protein, a DNA-binding protein which functions as a repressor (reviewed in reference 35). Recently, it has been shown that some genes in S. cerevisiae exhibit increased expression in response to hypoxia, cobalt, and iron chelation, mimicking hypoxia-regulated genes in higher organisms (18, 30). We have studied one such gene, OLE1, which encodes a Δ9 fatty acid desaturase gene in S. cerevisiae. A cis transactivation element, the low-oxygen response element (LORE), was identified and characterized in the promoter region of OLE1. The LORE sequence is also found in a family of yeast genes which may also be regulated via LORE (30). A similar but longer element has also been reported by Nakagawa et al. (23). The results of these studies strongly indicate that there is another hypoxia signal transduction pathway in yeast, in addition to the Rox1p-dependent repression mechanism. MGA2 and SPT23 are two functionally and genetically related genes. MGA2 was identified as a multicopy suppressor of a transcription defect caused by a null mutation in the SNF2 gene in S. cerevisiae (33). SPT23 is also functionally related to SNF2 (4, 33). It has been shown that Snf2p is a key component of the SWI-SNF nucleosome remodeling complex, which plays an important role in activating the transcription of many genes (16, 28). Sequence analysis shows that MGA2 and SPT23 have considerable homology, with 43% of the amino acids overall being identical and 60% being similar (33). Deletion of either one of these genes has only a modest effect on cell growth. However, cells with a MGA2 SPT23 double mutation are not viable (33). Studies have also shown that both MGA2 and SPT23 can activate transcription when fused to a Gal4 DNA-binding domain (33). A subsequent search for genes which are functionally related to or controlled by MGA2 and SPT23 led to the identification of OLE1 as a gene which is positively influenced by MGA2 and SPT23 at the transcription level (34). It is possible that MGA2 and SPT23 control cell viability by stimulating OLE1 transcription (34). In eukaryotes, regulated intracellular turnover of many proteins, such as the α subunit of HIF-1, is primarily mediated by the ubiquitin/proteasome pathway, which normally results in the complete proteolysis of a targeted protein (12). In a few cases, however, this pathway is involved in partial, rather than complete, proteolysis. Examples include the proteasome-dependent processing of the p105 precursor of the transcription factor NF-κB from mammalian cells and the processing of the precursors of certain yeast transcription factors (20). Recently, Hoppe et al. (14) identified a novel processing pathway in S. cerevisiae involving SPT23 and MGA2, which they have termed regulated ubiquitin/proteasome-dependent processing. Spt23p and Mga2p are initially made as dormant precursors that are firmly anchored in the endoplasmic reticulum or nuclear envelope membranes by their C-terminal tails. The shortage of unsaturated fatty acid leads to the RSP5-mediated ubiquitination of Spt23p and Mga2p, which leads to the release of the N-terminal transcription factor domain into the cytosol and finally to the enhanced expression of OLE1 mRNA. Addition of unsaturated fatty acids that contain more than one double bond almost completely blocks Spt23p precursor processing. Data suggest that this pathway controls the level of unsaturated fatty acids in S. cerevisiae by regulating OLE1 expression (14). Previously, OLE1 expression has also been shown to be increased by oxygen deprivation (18, 23, 30). Oxygen is critical for Ole1p function. Given the critical need for unsaturated fatty acid production in S. cerevisiae to maintain membrane integrity, we hypothesized that MGA2 and/or SPT2 may also mediate OLE1 expression by hypoxia. Here, we demonstrate that MGA2, but not SPT23, is required for LORE-dependent hypoxic induction of gene expression in S. cerevisiae.