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Genetics, Vol. 174, 191-201, September 2006, Copyright © 2006
doi:10.1534/genetics.106.059964
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Department of Molecular and Cellular Biology, Division of Genetics, Genomics, and Development, University of California, Berkeley, California 94701-3202
2 Corresponding author: Department of Molecular and Cellular Biology, Division of Genetics, Genomics, and Development, 522 Barker Hall, University of California, Berkeley, CA 94701-3202.
E-mail: jrine{at}berkeley.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Upc2p and Ecm22p, two transcription factors with similar sequences, bind a sequence motif known as the sterol regulatory element (SRE) and regulate the ergosterol biosynthetic genes ERG1, ERG2, ERG3, ERG7, ERG25, ERG26, and ERG27 (VIK and RINE 2001; GERMANN et al. 2005). Although binding of Upc2p and Ecm22p at other ERG genes has not been tested directly, the SRE binding site is found in many of these genes, suggesting that these two proteins are major regulators of ergosterol biosynthesis.
In addition to binding the promoters of ERG genes, Upc2p binds a similar sequence in the regulatory region of hypoxically induced mannoprotein-encoding genes known as the DAN/TIR genes (ABRAMOVA et al. 2001a,b). These genes are responsible for changes in the cell wall of yeast grown hypoxically. Upc2p has also been implicated in the uptake of sterols under hypoxic conditions (LEWIS et al. 1988; CROWLEY et al. 1998; WILCOX et al. 2002; ALIMARDANI et al. 2004). Normally, yeast cells take up sterols from their environment only under hypoxic conditions (ANDREASEN and STIER 1953), but an overactive allele of UPC2, UPC2-1, allows aerobic uptake of sterols (LEWIS et al. 1988). Moreover, nearly one-third of hypoxically induced genes contain at least one potential Upc2p/Ecm22p binding site (KWAST et al. 2002). These observations suggest that Upc2p, and perhaps Ecm22p, are major factors in the adaptation to hypoxia.
Upc2p and Ecm22p share similar binuclear cluster DNA binding domains (70% identical in sequence) and similar activation/regulatory domains (76% identical in sequence), but Upc2p and Ecm22p function at different times. Ecm22p is more abundant at ERG promoters under normal laboratory growth conditions. However, when sterols are depleted, Ecm22p levels drop and Upc2p replaces Ecm22p at the promoters of ERG genes (DAVIES et al. 2005). Such an activator switch implies that there must be other factors that regulate the activation, abundance, and localization of Upc2p and Ecm22p, as well as conditions that favor the use of one activator over the other.
Other transcription factors also participate in the regulation of ergosterol biosynthesis genes. Hap1p, a heme-activated transcriptional regulator, regulates HMG1 (THORSNESS et al. 1989), the structural gene for 3-hydroxy-3-methylglutaryl CoA reductase. A HAP1 mutant allele can cause lower expression in several ergosterol biosynthetic genes with a corresponding reduction in ergosterol levels (TAMURA et al. 2004). Yer064Cp, a nuclear protein of unknown function, is important for the activation of some ERG genes (KENNEDY et al. 1999; GERMANN et al. 2005). Finally, deletion of MOT3, a zinc-finger protein that can act as both a transcriptional activator and a repressor, leads to increased expression of ERG2, ERG6, and ERG9 (HONGAY et al. 2002).
In this study, we established the functions of Hap1p and Mot3p in regulating the gene targets of Upc2p and Ecm22p. In addition we tested the hypothesis that sterol, not heme, depletion was the signal responsible for hypoxic activation of Upc2p and Ecm22p and uncovered unanticipated complexity in how oxygen levels affect the expression of Upc2 and Ecm22p gene targets.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Genotyping of HAP1 allele:
Yeast spores were subjected to colony PCR with the primers bd194 (5'-GGAGCTGGAACTGCGAATAC-3'), bd195 (5'-CATTTGCGTCATCTTCTAACACCG-3'), and bd196 (5'-CTCCCATATTGGAAAATCTGCTC-3'). In the presence of wild-type HAP1, bd194 and bd196 amplify a 250-bp product. In the presence of the S288C HAP1 mutation, bd194 and bd195 amplify a 350-bp product.
ß-Galactosidase assays:
ß-Galactosidase assays were performed essentially as previously described (BURKE et al. 2000).
Analysis of protein levels:
Strains were grown to midlog phase and whole-cell extracts were prepared as described previously (FOIANI et al. 1994). Extracts were subjected to SDS–PAGE and immunoblotting. FLAG-tagged proteins were detected by immunoblotting with anti-flag (rabbit or mouse; Sigma). TAP-tagged proteins were immunoblotted with an anti-flag antibody (rabbit; Sigma) to detect the TAP tag. All immunoblots were also blotted with anti-3-phosphoglycerate kinase antibodies (Molecular Probes) as a loading control. Immunoblots were scanned using the Li-Cor Odyssey imaging system.
Chromatin immunoprecipitation:
Chromatin immunoprecipitations were performed as described previously (DAVIES et al. 2005). For TAP-tagged proteins (Figures 2 and 4), immunoprecipitations were performed using 30 µl IgG sepharose (Amersham Biosciences). For Hap1-flag (Figure 2), immunoprecipitations were performed using 25 µl anti-flag M2-agarose (Sigma). Typically, IPs were performed overnight at 4°. DNA enrichment was analyzed by real-time PCR and Syber-Green fluorescence on a Stratagene MX3000 real-time PCR instrument. Real-time PCR was performed using primers to the ERG3 promoter (5'-GACGCCTTTTGTTGCGATTGTCG-3' and 5'-CAGCAACAACAATACCCGATCGC-3') and to ACT1 (5'-GGCATCATACCTTCTACAACGAATTG-3' and 5'-CTACCGGAAGAGTACAAGGACAAAAC-3') with DNA derived from whole-cell extracts as a standard.
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Analysis of gene expression:
Gene expression was measured using quantitative RT–PCR. RNA was prepared as described previously (SCHMITT et al. 1990). RNA preps were digested with RNase-free DNase I (Roche and QIAGEN) and cDNA was synthesized using SuperScript III first-strand synthesis system for RT–PCR (Invitrogen) and oligo(dT). cDNA was analyzed by real-time PCR and Syber-Green fluorescence on a Stratagene MX3000 real-time PCR instrument. Samples were analyzed in triplicate for each of two independent RNA preparations. cDNA from ACT1 was used as a loading control. Primer sequences are listed in supplemental Table 1 at http://www.genetics.org/supplemental/.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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HAP1—the genetic basis of strain differences in ERG2 expression:
To find proteins that, in conjunction with Upc2p and Ecm22p, contribute to the regulation of ERG genes, a genetic screen was designed using a pERG2::lacZ reporter plasmid (pJR2316) and the yeast deletion collection (Research Genetics). The plan was to transform the knockout collection with the reporter plasmid and screen for either increased or decreased ERG2 expression compared with the parent strain. However, when ERG2 expression was tested in BY4742, the parent strain of the MAT
deletion collection, we found that expression was fivefold lower than what had been observed previously in W303-derived strains (VIK and RINE 2001; DAVIES et al. 2005). A hybrid diploid formed between the two strains exhibited an intermediate level of ERG2::lacZ expression (Figure 1A).
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Prior to initiating mapping of the locus responsible for this difference, we tested whether known differences between these two strains could account for the difference in ERG2 expression. Strains derived from S288c (such as BY4742) have mutant forms of several genes including a mutant allele of HAP1, which encodes a heme-responsive transcriptional regulator (GAISNE et al. 1999). This mutation, caused by an insertion near the 3' end of the HAP1 ORF, has a detrimental effect on some, but not all, Hap1p targets (GAISNE et al. 1999). Several observations suggested that HAP1 may be the basis of the difference in ERG2 expression in these two strains: Deletion of both UPC2 and ECM22 results in lethality in S288C (SHIANNA et al. 2001) but not in W303 (VIK and RINE 2001). The lethality of the upc2 ecm22 double null mutation in S288c appears to result from the hap1 mutant allele, as restoration of the wild-type HAP1 restores viability (M. VALACHOVIC, personal communication). Moreover, deletion of UPC2, ECM22, and HAP1 results in lethality in W303 (data not shown). The haploid progeny from the W303/BY4742 diploid were therefore tested for the S288c allele of HAP1. In every case tested, spores with low ERG2 expression had the S288c-derived mutant allele of HAP1 and spores with high expression had the W303-derived allele, indicating that low ERG2 expression was a result of the mutant allele of HAP1 (data not shown).
Upc2p and Ecm22p had different requirements for Hap1p:
To examine the role of Hap1p in the regulation of ERG genes, especially with respect to Upc2p and Ecm22p, hap1
(JRY8038), hap1upc2 (JRY8040), and hap1ecm22 (JRY8042) derivatives of W303 were tested for ERG2 expression in both the presence and the absence of lovastatin, an inhibitor of HMG-CoA reductase, which catalyzes an early step in sterol biosynthesis. Thus the presence or absence of lovastatin corresponds to inducing and noninducing conditions, respectively. Under noninducing conditions, ERG2 expression was profoundly reduced in all strains lacking HAP1 (Figure 1B, shaded bars). Thus, Hap1p was required for the basal expression of ERG2, regardless of whether that expression was activated by Ecm22p or Upc2p.
Unlike the upc2ecm22 double mutant strain, however, a strain without HAP1 was able to induce ERG2 expression when sterols were depleted by growth in the presence of lovastatin, albeit to a lower level (Figure 1B, solid bars). Induction in the absence of HAP1 depended mostly on Upc2p, as very little induction was observed in a strain when both UPC2 and HAP1 were deleted. Thus, upon inducing conditions, Upc2p could activate ERG2 expression in a Hap1-independent manner, whereas Ecm22p was a Hap1p-dependent activator of ERG2 expression.
Hap1p acted at ERG promoters:
There are several possible ways that Hap1p could regulate Ecm22p and, to a lesser extent, Upc2p at ERG promoters. Hap1p might modulate ERG gene expression indirectly either by regulating transcription of UPC2 and ECM22 or by regulating the stability of Upc2p and Ecm22p. Alternatively, Hap1p might act more directly to influence either the binding of Upc2p and Ecm22p at ERG promoters or the activity of these two proteins once bound. To distinguish among these possibilities, the levels of Upc2p and Ecm22p were measured in a hap1 strain grown in the presence or absence of lovastatin. Neither Upc2p nor Ecm22p levels were affected by the deletion of HAP1, under inducing or noninducing conditions (Figure 2A). Similarly, Hap1p levels were unaffected by lovastatin treatment (data not shown). The increase in Upc2p and decrease in Ecm22p evident upon induction were described previously (DAVIES et al. 2005). These data argued for a direct mechanism of Hap1 function.
To determine if Hap1p acted directly on ERG gene promoters, a flag-tagged version of Hap1p was used for chromatin-immunoprecipitation experiments. The tagged version of Hap1p complemented the hap1 null mutation with respect to ERG2 expression (data not shown). As chromatin immunoprecipitation of ERG2, with its single SRE, has proven difficult (data not shown) and regulation of ERG3 parallels regulation of ERG2 (VIK and RINE 2001), Hap1p and Ecm22p were tested for their enrichment at the ERG3 promoter, which contains five SREs. Hap1p was enriched at the ERG3 promoter and to approximately the same extent under inducing and noninducing conditions (Figure 2B). Although Hap1p was at the ERG3 promoter, deletion of HAP1 had no effect on the presence of Ecm22p at the same promoter in cells grown in either the presence or the absence of lovastatin (Figure 2C). Thus, Hap1p influenced the activity, but not the presence, of Ecm22p at ERG promoters.
Mot3p inhibited Ecm22p:
Mot3p, a dose-dependent repressor/activator, represses expression of several ERG genes including ERG2 (HONGAY et al. 2002), although the mechanism was unknown. To determine whether Mot3p acted through Upc2p, Ecm22p, or by some independent mechanism, mot3 (JRY8044), mot3upc2 (JRY8048), mot3ecm22 (JRY8050), and mot3upc2ecm22 (JRY8056) deletion strains were constructed and tested for ERG2 expression. As reported by HONGAY et al. (2002), the deletion of MOT3 led to an increase in basal ERG2 expression (Figure 3, first two shaded bars). The increase in ERG2 expression depended primarily on Ecm22p, as the deletion of MOT3 had no significant effect on ERG2 activation by Upc2p (Figure 3). The activation of ERG2 by Ecm22p was increased in a mot3 strain in both inducing and noninducing conditions (Figure 3, compare upc2 to upc2mot3). Thus, Mot3p repressed Ecm22p-dependent expression of ERG2 but not Upc2-dependent expression. It is important to note that induction of ERG2 by Ecm22p in response to sterol depletion was not due simply to relief of Mot3p repression, as considerable ERG2 induction was still evident in a mot3 mutant.
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strain: strain. Deletion of MOT3 had little, if any, effect on Upc2p levels. However, upon sterol depletion, the level of Ecm22p was approximately twofold higher in mot3 strains (Figure 4A), which corresponded quantitatively to the twofold greater ERG2 induction by Ecm22p in mot3 strains (Figure 3). A similar pattern was observed when the enrichment of Ecm22p at the ERG3 promoter was tested using chromatin immunoprecipitation in a mot3 strain (Figure 4B). Thus, Mot3p caused at least part of the reduction in overall Ecm22p levels under inducing conditions, and at least part of the reduction in SRE occupancy at the ERG3 promoter.
Mot3p interacted directly with Ecm22p:
To test whether Mot3p affects Ecm22p by direct physical interaction, TAP-tagged Ecm22p was immunoprecipitated from cells with FLAG-tagged Hap1p, Mot3p, or Upc2p and then immunoblotted with an anti-FLAG antibody. These experiments revealed a strong and specific interaction between Ecm22p and Mot3p (Figure 5A). Adding a DNAse digestion prior to precipitation had no effect on this interaction, thus precluding a DNA bridge between two proteins binding at the same promoter as an explanation (Figure 5B). It should be noted that the observed band did not correspond to full-length Mot3p, but did correspond to the major MOT3-flag-specific band observed in whole-cell extracts (data not shown). Thus a major mechanism for the repression of ERG gene expression by Mot3p was by direct binding to the Ecm22p activator.
Testing the role of heme A in ERG gene expression:
As lovastatin inhibits an early step in ergosterol biosynthesis, it reduces not only the synthesis of ergosterol, but also the synthesis of certain nonsterol products, such as heme A. Heme A, a farnesylated version of heme, is an essential prosthetic group of cytochrome C oxidase (MORAES et al. 2004). Because cytochrome C oxidase is thought to play a role in oxygen sensing and the activation of some hypoxically expressed genes (KWAST et al. 1999), in principle, depletion of heme A by lovastatin might result in a hypoxic response. Thus, depletion of heme A, rather than sterol depletion per se, might be responsible for the switch from Ecm22p to Upc2p at ERG promoters under inducing conditions. Such a model would be consistent with Upc2p's role in activating the hypoxically expressed DAN/TIR genes if they too are keyed into hypoxia through a cytochrome C oxidase-mediated signal (ABRAMOVA et al. 2001a,b). The importance of Hap1p, a heme-activated transcription factor, in the activation of ERG genes again suggested that heme levels may have a prominent role in the regulation of Upc2p and Ecm22p.
If the activation of either Upc2p or Ecm22p were the result of reduced heme A, rather than the result of sterol depletion, depleting sterols without lowering heme A levels would not be expected to induce ERG gene expression through that transcription factor. To test this hypothesis directly, ketoconazole was used to deplete sterols. Ketoconazole inhibits Erg11p, an ergosterol biosynthetic enzyme that acts in the sterol-specific part of the pathway, downstream of the point from which nonsterol products of the pathway, such as heme A, are derived. By treating with ketoconazole, sterols can be depleted without depleting the nonsterol products of the pathway. If changes in Upc2p and Ecm22p activity or levels were indeed the result of heme depletion and not sterol depletion, following ketoconazole treatment neither ERG2 expression nor Upc2p or Ecm22p levels would be expected to change as they do following lovastatin treatment. In wild-type, upc2 (JRY7179), ecm22 (JRY7180), and upc2ecm22 (JRY7181) strains treated with ketoconazole, induction of ERG2 was similar to that seen after lovastatin treatment (Figure 6A). More tellingly, Upc2p and Ecm22p levels following ketoconazole treatment changed just as with lovastatin treatment (Figure 6B). Thus, induction of ERG genes and changes in Upc2p and Ecm22p levels resulted from sterol depletion and not from depletion of heme A or any other early product from the sterol pathway.
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To test whether hypoxic targets of Upc2p were induced in response to sterol depletion or heme depletion, the expression of several Upc2p/Ecm22p target genes was measured by quantitative RT–PCR in wild-type, upc2, ecm22, and upc2ecm22 strains grown aerobically with or without ketoconazole or hypoxically with or without hemin (Table 2). The logic behind this experiment is as follows: genes that are induced by sterol depletion should be induced both by ketoconazole treatment and by hypoxic growth, and addition of hemin should not reduce hypoxic induction. Genes induced only by low heme should not be induced by ketoconazole, but should be induced under hypoxic growth. When hemin is added to hypoxically growing strains, hypoxic induction should be reduced or eliminated, as heme is no longer limiting. If a gene is activated by low heme and low sterols in combination, ketoconazole might induce the gene to some degree, while hypoxia will result in even greater induction. Adding hemin should reduce, but not eliminate, hypoxic induction of such genes, as sterols will still be limiting.
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ERG2, ERG3, ERG10, and IDI1 all encode enzymes in the sterol biosynthesis pathway. ERG2 and ERG3 encode late enzymes in the sterol-specific branch of the pathway and are known targets of Upc2p and Ecm22p (VIK and RINE 2001; DAVIES et al. 2005). ERG10 encodes the enzyme that catalyzes the first step in the pathway and contains a potential SRE in its promoter. IDI1 also encodes an early gene in the sterol pathway, but does not contain an SRE in its promoter. Expression of ERG2, ERG3, and ERG10 was induced by ketoconazole, but this induction required either Upc2p or Ecm22p. Hypoxia also induced these three genes, and when either Upc2p or Ecm22p is present hypoxic induction is not affected by the addition of heme. Interestingly, in the absence of both Upc2p and Ecm22p, hypoxic induction of ERG2, ERG3, and ERG10 is observed. This Upc2/Ecm22p-independent induction appeared to be a response to low heme, as the addition of heme greatly reduced the hypoxic response. In contrast, IDI1 was not induced by sterol depletion, but, as with the other three ERG genes, was induced by hypoxia in a Upc2p- and Ecm22p-independent manner. Thus it appeared that hypoxia created two induction signals, one mimicked by ketoconazole that could not be suppressed by hemin and a second that could be. The hemin suppression was robust only in the upc2ecm22 strain, which lacked the proteins that respond to sterol depletion.
The DAN/TIR genes encode hypoxically expressed mannoproteins and are known targets of Upc2p induction (ABRAMOVA et al. 2001a,b). DAN1 and TIR1 appeared to be induced both by low sterols and by low heme. Ketoconazole treatment resulted in some induction, but hypoxic induction was 10- to 100-fold higher than ketoconazole induction. Much, but not all, of the hypoxic induction could be abated by adding back heme. This result is consistent with previous reports that DAN1 and TIR1 expression are regulated both by Upc2p and by Rox1p, a transcriptional repressor that is expressed when heme levels are high (ABRAMOVA et al. 2001b; KWAST et al. 2002; LAI et al. 2005). DAN2 and DAN4, which are not regulated by Rox1p (ABRAMOVA et al. 2001b), appeared to be induced primarily by low sterols. Ketoconazole and hypoxia (with or without hemin) induced both genes in a Upc2p/Ecm22p-dependent manner. Interestingly, unlike previous reports, under hypoxic conditions Ecm22p was quite capable of inducing all of the DAN/TIR genes tested.
UPC2 itself was induced by ketoconazole and by hypoxia, consistent with previous reports (ABRAMOVA et al. 2001b; AGARWAL et al. 2003; DAVIES et al. 2005). The addition of heme to hypoxically growing cells did not reduce induction of UPC2, indicating that this induction was a result of low sterols rather than of low heme. Thus, as reported previously, Upc2p appears to be autoregulated, most likely through the SRE elements in its promoter (ABRAMOVA et al. 2001b).
ECM22 expression was modestly induced by hypoxia. This induction could be suppressed by additional heme, indicating that this induction was the result of heme depletion. As reported above, activation of target genes by Ecm22p was the result of low sterols, suggesting a certain disconnect between ECM22 expression and Ecm22p activity.
To control for the possibility that sterol depletion might create an unanticipated hypoxic response through heme levels, ERG2 expression was monitored in the presence and absence of lovastatin and in the presence and absence of exogenous heme. No effects of heme levels on induction by sterol depletion were evident under these conditions (Figure 7). Thus, although hypoxic conditions created depletion of both heme and sterol levels, the responses to heme and sterol depletion were distinct and resolvable.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Upc2p and Ecm22p regulate the expression of many of the ergosterol biosynthesis (ERG) genes (VIK and RINE 2001; GERMANN et al. 2005) as well as the transcription of several hypoxically expressed genes, including genes involved in anaerobic cell wall reorganization and anaerobic sterol uptake (ABRAMOVA et al. 2001a,b; WILCOX et al. 2002). As expression of these genes responds to oxygen availability, it makes sense that the activity of Upc2p and Ecm22p must also be regulated by oxygen. Because low oxygen limits biosynthesis of both heme and sterols, regulation of Upc2p and Ecm22p activity may respond to either or to both of these signals.
One important result of this study was the clear identification of sterol levels as the primary regulator of Upc2p (see Figure 8). The activation of target genes by Upc2p occurred in response to low sterols, whether caused by early blocks in ergosterol biosynthesis (lovastatin), by late blocks (ketoconazole), or by hypoxia. ERG2, ERG3, ERG10, DAN2, and DAN4 were activated by Upc2p solely in response to sterol depletion rather than to heme depletion, whereas DAN1 and TIR1 responded to both sterols and heme (Table 2), consistent with previously reported repression of these genes by Rox1p and Mot3p and activation by Upc2p (ABRAMOVA et al. 2001b).
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Clearly, both heme levels and sterol levels contribute to oxygen sensing in yeast. Although the role of heme in oxygen sensing has been well studied (ZHANG and HACH 1999; HON et al. 2003), the broader role of Upc2p and its activation in response to oxygen through sterol depletion is just beginning to emerge. Several targets have been identified (ABRAMOVA et al. 2001b; VIK and RINE 2001; AGARWAL et al. 2003; TER LINDE et al. 2003; GERMANN et al. 2005), but if, as indicated by KWAST et al. (2002), Upc2p regulates nearly one-third of all anaerobically expressed genes, activation of Upc2p targets following sterol depletion represents a significant contribution to the adaptation to hypoxia.
Certainly, response to low heme and response to low sterols are not completely separable. Even within a specific pathway, such as ergosterol biosynthesis or the DAN/TIR gene family, mechanisms responding to both low heme and low sterols are at work. The involvement of Hap1p in ergosterol biosynthesis serves as one such example. Both Upc2p and Ecm22p required a functional version of Hap1p for basal expression of ERG2 (Figure 1). In contrast, when sterols were depleted with lovastatin, Upc2p's dependence on Hap1p was largely abolished, as robust induction of ERG2 was observed, whereas Ecm22p still depended upon Hap1p for ERG gene activation. Upc2p's ability to induce ERG2 expression in the absence of Hap1p may be the consequence of several factors. Both Upc2p levels and the amount of Upc2p at ERG promoters increase upon sterol depletion (DAVIES et al. 2005), whereas Ecm22p levels and their occupancy at SREs decreased. The derepression of Upc2p activity upon sterol depletion (DAVIES et al. 2005) and the resulting increases in Upc2p levels may enable Upc2p to induce ERG2 expression in a HAP1-independent manner.
It is unlikely that the need for both Hap1p and Upc2p or Hap1p and Ecm22p for basal gene expression is limited to ERG2. Full expression of HMG1 requires both the Hap1p binding site and an additional 55-bp region in its promoter (TAMURA et al. 2004). This region contains the SRE binding site for Upc2p and Ecm22p (VIK and RINE 2001). Indeed, conserved core consensus binding sites are found for both Hap1p and Upc2p/Ecm22p in several ERG genes (CHIANG et al. 2003).
The involvement of Mot3p in ERG gene expression revealed that ERG gene expression involved both induction and repression. Mot3p modulates the transcription of a diverse set of genes in a dosage-dependent manner (GRISHIN et al. 1998; ABRAMOVA et al. 2001a). Mot3p acts in combination with Rox1p to recruit the Tup1-Ssn6 repressor complex to anaerobically expressed genes under aerobic conditions (KASTANIOTIS and ZITOMER 2000; MENNELLA et al. 2003; SERTIL et al. 2003; KLINKENBERG et al. 2005). Mot3p inhibited Ecm22p, but not Upc2p (Figure 3), and the direct physical interaction of Mot3p with Ecm22p (Figure 5) was likely an important part of the inhibitory mechanism. Mot3p inhibited Ecm22p-mediated ERG2 activation both before and after sterol depletion, so it remains unclear what signal or influence Mot3p responds to. Mot3p seemed to play some role in the decreased Ecm22p levels under inducing conditions, but not under noninducing conditions (Figure 4A).
One unexpected result from this study was the component of the hypoxic induction of ERG genes that was Upc2p/Ecm22p independent. In the absence of Upc2p and Ecm22p, all the ERG genes tested, including IDI1, were hypoxically induced (Table 2). Unlike Upc2p- or Ecm22p-dependent induction, this induction responded to heme levels rather than to sterol levels. None of the DAN/TIR genes exhibited this type of induction, suggesting that this type of gene activation was specific to the ergosterol pathway. It is unlikely that this activation results from depression by Rox1p or Mot3p, as deletion of ROX1 does not change expression of any ERG genes apart from ERG26 and HMG2 (TER LINDE and STEENSMA 2002), and, in the absence of Upc2p or Ecm22p, deletion of MOT3 does not result in induction of ERG2 (Figure 3).
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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