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Originally published In Press as doi:10.1074/jbc.M005946200 on August 1, 2000

J. Biol. Chem., Vol. 275, Issue 41, 32310-32316, October 13, 2000
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Identification of the Copper Regulon in Saccharomyces cerevisiae by DNA Microarrays*

Claudia GrossDagger , Mark Kelleher§, Vishwanath R. Iyer, Patrick O. Brown, and Dennis R. WingeDagger ||

From the Dagger  University of Utah Health Sciences Center, Departments of Medicine and Biochemistry, Salt Lake City, Utah 84132 and the § Department of Biochemistry, Stanford University School of Medicine, Howard Hughes Medical Institute, Stanford, California 94305-5428

Received for publication, July 6, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates copper-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiae genes, including CTR1, CTR3, FRE1, FRE7, YFR055w, and YJL217w. Two of the last three newly identified Mac1 target genes have no known function; the third, YFR055w, is homologous to cystathionine gamma -lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1up1, are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells because of constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1 and CRS5 and two genes, FET3 and FTR1, in the iron uptake system. Copper-induced FET3 and FTR1 expression arises from an indirect copper effect on cellular iron pools.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Copper ions are required for at least three key enzymes in the yeast Saccharomyces cerevisiae. The ability of cells to grow on nonfermentable carbon sources is dependent on having an active cytochrome oxidase complex, which requires copper ions as cofactors. Oxidative growth requires defense molecules against reactive oxygen intermediates. Superoxide dismutase is a copper-metalloenzyme that functions to disproportionate superoxide anions. A third, key copper-metalloenzyme is Fet3, which is a ferro-oxidase critical for uptake of Fe(II) (1). A myriad of other oxidases and oxygenases require Cu(II) as a functional cofactor in other species, so additional copper-metalloenzymes may exist in S. cerevisiae.

S. cerevisiae possesses mechanisms to maintain cellular copper levels at adequate but not excessive cellular copper levels. Copper ion homeostasis in yeast is maintained, in part, through copper-regulated expression of genes involved in copper ion uptake and Cu(I) sequestration. Positive and negative copper ion regulation of gene expression is observed, and both effects occur at the level of transcription (2). Distinct mechanisms mediate activation and inhibition of transcription in response to copper.

Conditions of copper deficiency result in the derepression of a family of genes whose products are involved in cellular uptake of copper ions (3-6). Two of these genes encode high affinity plasma membrane copper ion permeases, Ctr1 and Ctr3 (5, 7). A third gene encodes the Fre1 metalloreductase, which mobilizes copper ions from oxidized copper complexes (4, 8, 9). The expression of these genes is a cellular response to inadequate intracellular copper levels. In copper-replete cells, expression of CTR1, CTR3, and FRE1 is inhibited (3-5), but copper ion uptake can still occur through low affinity transporters, such as Ctr2, Fet4, and Smf1 (10-12).

The expression of high affinity copper uptake genes is regulated at the level of transcription through the Mac1 transcriptional activator (13). Mac1 is a functional transcriptional activator in copper-deficient cells but is inhibited in copper-replete cells by a copper-induced, intramolecular interaction that represses both DNA binding and transactivation activities (14-16). MAC1 was originally identified as a partially dominant mutation, designated MAC1up1 (13). Cells harboring the MAC1up1 allele were found to be incapable of repression of copper-uptake genes (4, 6). As a result of constitutive expression of the transporters, MAC1up1 cells are hypersensitive to elevated levels of copper salts in the growth medium (13). In contrast, cells lacking a functional Mac1 show reduced copper transport and exhibit copper-deficient phenotypes (13). Mac1 binds to duplicated TTTGCTCA promoter sequences in CTR1, CTR3, FRE1, and the FRE1 homolog FRE7 (5, 6, 17, 18). Two repeats of this element are necessary for in vivo activity, although neither the orientation nor spacing between repeats appears critical (5, 6, 17).

A different subset of genes is transcriptionally activated when the extracellular copper concentration exceeds 1 µM. This is a protective response to counteract the cytotoxicity of copper ions. The copper-activated genes include CUP1, CRS5, and SOD1 (19-21). CUP1 and CRS5 encode cysteinyl-rich polypeptides in the metallothionein family (19, 22). CUP1 is the dominant locus that confers the ability of yeast cells to propagate in medium containing copper salts (22-24). Cells highly resistant to copper salts contain a CUP1 locus with tandem arrays of genes encoding the Cup1 metallothionein (23). In addition to its role in dismuting superoxide anions, Sod1 has a secondary role of contributing to copper buffering in S. cerevisiae (25).

Cu(I) activation of CUP1 expression is mediated by Ace1 in S. cerevisiae (26, 27). Cu(I) ion binding within the copper-regulatory domain of Ace1 stabilizes a specific tertiary fold capable of high affinity interaction with specific DNA promoter sequences. Cu(I) triggering involves formation of a tetracopper thiolate cluster within the regulatory domain (28). CUP1, CRS5, and SOD1 contain CuAce1 binding sites within 5' promoter sequences (20, 29).

Thus, the known genes regulated by Mac1 and Ace1 in S. cerevisiae are functionally important in copper homeostasis. To identify other yeast genes that may likewise be important in copper homeostasis, we used DNA microarray technology to evaluate differential expression of genes in cells varying in copper ion status. DNA microarrays containing approximately 6120 genes from S. cerevisiae printed on glass slides were used to identify genes up- or down-regulated by activated Mac1 and Ace1.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Culture Conditions-- The yeast strains used, CM66J (MATalpha , ino1-13, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2) and CM66J-up (MATalpha , trp1-63, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2, MAC1up1), were derived from strains 66 (MATalpha , trp1-63, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2) and CM3262 (MATa, ino1-13, gcn4-101, his3-609, ura3-52, leu2-3, 112, ino1-13), gifts from A. Dancis (30). CM66mac1Delta , was constructed by integrating URA3 at the MAC1 locus in strain CM66E (MATa, trp1-63, gcn4-101, his3-609, ura3-52, FRE1-HIS3::LEU2) by one-step gene replacement as described in Ref. 31. CM3260aft1Delta (MATalpha , trp1-63, leu2-3, 112, gcn4-101, his3-609, ura3-52, aft1::LEU2) was constructed by integrating LEU2 at the AFT1 locus by one-step gene replacement (31). In both cases, allele status was verified by diagnostic polymerase chain reaction and sequencing. The ace1Delta strain was constructed by the one-step gene replacement method mentioned integrating kanMX in the ACE1 locus in strain CM66G. The pho4 strain used was W303 containing a pho4::TRP1 locus transformed with pRSMAC1 and pRSMAC1up1. The vector pRSMAC1 was the YCp-based pRS316 (32) containing a 1.7-kilobase Sau3A genomic fragment of MAC1 or MAC1up1. Yeast strains were cultured in either CM with YNB (Difco) or low copper complete medium (LCCM)1 using copper and iron limiting yeast nitrogen base (Bio101) and lacking the appropriate amino acids to ensure maintenance of plasmid and reporters.

RNA Isolation and Microarray Analysis-- Cells were harvested from 300 ml of culture at an A600 nm of 0.4 or following 60 min of treatment with 100 µM BCS or 30 min of treatment with 100 µM CuSO4. Total RNA was isolated by the hot acid phenol method. Quantitation of RNA was carried out by UV spectroscopy. mRNA was isolated from total RNA using the Poly(A)T tract mRNA Isolation System IV kit from Promega following the manufacturer's instructions.

Three types of microarray experiments were conducted. First, wild-type and MAC1up1 cells were cultured in LCCM. Second, wild-type cells pregrown in LCCM medium to an A600 nm of 0.4 were incubated in the presence and absence of 100 µM BCS for 30 or 60 min prior to harvesting. Third, wild-type cells pregrown in CM medium to an A600 nm of 0.4 were incubated in the presence and absence of 100 µM CuSO4 for 30 min. Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech) was incorporated during reverse transcription of the polyadenylated RNA. The fluorescently labeled product was recovered and hybridized to microarrays, washed, and scanned as described previously (33).

Vectors-- Plasmids Dg5-7b, generously supplied by D. Eide, contains a 29-bp insert of the ZRE from the ZRT1 promoter (34) in the Delta UAS vector pNB404, respectively (35). Plasmid pFC-W, generously provided by A. Dancis, contains a 30-bp cassette of the FET3 UAS (-263 to -234) fused to lacZ (36). Plasmid pFC-LM2 is a mutant version of pFC-W containing a mutation in the Aft1 binding site (36).

mRNA Quantitation by S1 Nuclease Analysis-- Total RNA isolated from mid-logarithmic cells was hybridized with a 32P-labeled, single-stranded DNA oligonucleotide and digested with S1 nuclease. The samples were electrophoresed through a 8% polyacrylamide, 5 M urea gel, and data were quantified by using a Bio-Rad FX Imager and Quantity one software (Bio-Rad Inc.). S1 probes used included 57-75 nucleotides of the 5' open reading frame sequences of candidate genes, 40 nucleotides of the calmodulin (CDM1) 5' open reading frame, and 60 nucleotides from the lacZ open reading frame.

Metal Ion Analysis of Cells-- Cells were harvested, washed twice, and digested for 60 min with 200 µl nitric/perchloric acid mixture (5:2) at 110 °C. Following digestion, the sample volume was adjusted to 1 ml and clarified, prior to analysis, using a Perkin-Elmer Optima 3100 XL ICP. For each element two absorption lines were used, and readings from four independent measurements were taken, averaged, and normalized to cell number.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our strategy to catalog genes activated by Mac1 and Ace1 was to use duplicated DNA microarray experiments as a screen to identify genes consistently up-regulated under activating conditions. In each case, candidate genes were evaluated by secondary tests to verify the differential expression and to determine whether the induction was a direct or indirect effect of Mac1 or Ace1 activation.

The strategy used to screen for Mac1-induced genes was 4-fold. First, DNA microarray experiments were conducted with a yeast strain containing a gain-of-function MAC1up1 allele compared with its isogenic control. MAC1up1 cells are constitutively active in expressing CTR1, CTR3, FRE1, and FRE7 (5, 13, 17). Many laboratory strains do not express CTR3 because of a transposon insertion (37). This DNA microarray experiment was carried out in triplicate. The second strategy was to identify genes induced in copper-deficient cells as Mac1 is activated under these conditions. Efficient Mac1 activation is dependent on culturing cells in low copper medium and adding the Cu(I)-specific chelator BCS to further lower the available copper ion pools (5). Thus, DNA microarray experiments were carried out on cells treated with BCS for short periods of time to focus on primary effects of copper deficiency. Third, genes differentially expressed both in MAC1up1 cells and by the addition of BCS were tested for differential mRNA expression by the S1 nuclease protection assay to quantify mRNA levels. Fourth, these genes were evaluated for Mac1 DNA-binding sites in 5' promoter sequences. The consensus Mac1 binding site is TTTGC(T/G)C(A/G) (17). Genes with elevated expression in MAC1up1 cells and induced by the addition of BCS as determined by both DNA microarray and S1 nuclease analyses and containing at least one Mac1 DNA-binding site were considered candidates for primary Mac1 induced genes. Genes up-regulated in MAC1up1 cells but not induced by short BCS treatment and lacking a candidate Mac1 binding site were considered candidates for secondary responses.

Three DNA microarray experiments were conducted comparing wild-type and MAC1up1 cells. Duplicate experiments were conducted on mRNA isolated from exponentially growing wild-type and MAC1up1 cells of S. cerevisiae strain CM66J. Fluorescently labeled cDNA was prepared from mRNA isolated from wild-type cells by reverse transcriptase in the presence of Cy3 (green)-labeled dUTP and from MAC1up1 cells by reverse transcription in the presence of Cy5 (red)-labeled dUTP. The labeled cDNAs were mixed and hybridized to the microarrays. The fluorescence intensities of the Cy3 and Cy5 fluorophores were quantified to provide a quantitative measure of the relative abundance of mRNA levels in the two cell populations. A third microarray experiment was conducted with wild-type and MAC1up1 cells of strain BR10 grown to late log phase. Table I shows fluorescence ratios of genes differentially expressed in these three experiments. The known Mac1 regulated genes (CTR1, FRE1, and FRE7) were clearly up-regulated in each experiment. Ratios of mRNA levels of CTR1, FRE1, and FRE7 in MAC1up1 versus wild-type cells exhibited mean values of 4, 7, and 6, respectively. Because strain CM66J contained a FRE1/HIS3 fusion gene, differential expression of HIS3 from the FRE1 promoter was also observed. CTR3 was induced 8-fold in MAC1up1 cells of strain BR10 but was not expressed in strain CM66J. In addition to known Mac1-regulated genes, >50 genes exhibited a moderate (>1.5-fold) induction in MAC1up1 cells in any given experiment. However, only five new genes were moderately induced in all three experiments. These genes included CUP1, PHO5, and YJL217w. CUP1 expression is likely elevated in MAC1up1 cells as expression of the high affinity copper transport system is constitutive, resulting in elevated uptake of copper ions (4). Metal analysis revealed that cellular copper levels were elevated 6-fold in MAC1up1 cells compared with wild-type cells.

                              
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Table I
Genes up-regulated >1.5 fold in MAC1up1 arrays
Three experiments were conducted comparing expression levels of wild-type cells versus cells containing a MAC1up1 allele. The ratio shown is of expression levels in MAC1up1 cells over that in wild-type cells. In the experiments in which Mac1 was activated by copper-deficient conditions, exponentially growing wild-type cells were incubated in the presence and absence of 100 µM BCS for either 30 or 60 min prior to cell harvest.

To determine whether these genes were directly Mac1-induced, we carried out microarray experiments in which wild-type cells cultured in low copper medium were treated with the Cu(I)-specific chelator for either 30 or 60 min. These conditions are known to induce CTR1 expression (3-5). mRNA was isolated from cells cultured in the presence and absence of BCS, and labeled cDNA was prepared as before for DNA microarray hybridization. As expected, CTR1, FRE1, and FRE7 were up-regulated by short treatments with BCS (Table I). In contrast, neither CUP1 nor PHO5 were BCS-induced.

Thirty genes showed moderately elevated expression in MAC1up1 exponentially growing cells. Only 17 of these were also induced by BCS (60-min treatment) (Table I). Two of these genes, YFR055w and YJL217w, showed marked enhancements in expression in MAC1up1 cells and BCS-treated cells (Fig. 1A). S1 analysis was performed as an independent verification of differential expression using the same RNA samples used to prepare mRNA for the array experiments. Transcripts of YFR055w and YJL217w were markedly elevated in MAC1up1 cells and by BCS treatment (60 min BCS) (Fig. 1A). YFR055w expression was elevated 8-fold in MAC1up1 cells and 3-fold by 60 min BCS treatment. YJL217w expression was enhanced 6-fold in MAC1up1 cells (same RNA as in experiment 2) and 2-fold by the 60 min BCS treatment. Maximal expression of FRE1, FRE7, CTR1, YFR055w, and YJL217w occurred by 60 min of treatment with BCS (data not shown). Neither YFR055w nor YJL217w were induced by BCS in mac1-1 cells, suggesting that copper metalloregulation of these genes is mediated by Mac1 (Fig. 1B).


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  		Fig. 1.   S1 nuclease protection assays to quantify mRNA levels of three genes up-regulated by Mac1. The upper band for each sample is the specified gene. The lower band in each case, indicated by the arrow, is the calmodulin (CMD1) loading control. fp lanes show undigested, free probes. A, RNA was isolated from wild-type (wt) CM66J cells cultured in LCCM in the presence and absence of 100 µM BCS or MAC1up1 (UP) cells. B, RNA was isolated from wild-type cells or mac1Delta cells cultured in LCCM in the presence or absence of 100 µM BCS.

The four known Mac1-regulated genes contain at least two binding sites for Mac1 activation. However, YFR055w and YJL217w each have one perfect Mac1 consensus DNA-binding site, although additional nonconsensus sites exist that may be functional. Thus, YFR055w and YJL217w satisfy the criteria of being direct target genes of Mac1.

A gene divergently expressed from FRE1, YLR213c, was up-regulated in MAC1up1 cells and induced by BCS in strain BR10. S1 analysis of wild-type BR10 cells treated with 100 µM BCS for 60 min resulted in a 15-fold induction in FRE1 and a weaker 4-fold induction of YLR213c. In contrast, expression of YLR213c in strain CM66J was very low under all conditions.

A number of genes in the PHO regulon (PHO5, PHO11, PHO12, and PHO84) were up-regulated in MAC1up1 cells but not induced by BCS treatment. PHO5 expression was enhanced 7- and 4-fold in MAC1up1 cells in two array experiments but unaffected by BCS treatment. Evaluation of PHO5 mRNA levels by S1 analysis confirmed the induction in MAC1up1 cells (13-fold enhancement) and failed to show any induction by BCS treatment. The lack of induction by BCS suggested that PHO5 induction may be a secondary effect of phosphate depletion in MAC1up1 cells. PHO5 expression is regulated by the Pho4 transcription factor through, in part, cellular phosphate levels. MAC1up1 cells containing a pho4 mutation failed to show a Mac1up1-dependent enhanced expression of PHO5, consistent with the postulate that elevation of the PHO regulon arises from selective depletion of the cellular phosphate pool in MAC1up1 cells (data not shown).

Additional studies were carried out on the remaining marginal candidates listed in Table I. A yeast DNA microarray experiment was repeated at the University of Utah microarray facility comparing wild-type cells to MAC1up1 cells. None of the marginal candidates were up-regulated in this experiment. Secondly, most of these genes lacked a consensus Mac1 binding site in 5' DNA sequences. Those containing a candidate Mac1 binding site were tested by S1 analysis. Quantitation of transcripts of YBR105c, YHR088w, and YKL082c revealed no enhancement in MAC1up1 cells and no induction by BCS.

DNA microarrays were also used to screen for genes with diminished expression in MAC1up1 cells. Two genes, ZRT1 and ZRT2, encoding zinc plasma membrane transporters, were markedly repressed in MAC1up1 cells. S1 analysis confirmed that ZRT1 is markedly inhibited (25-fold) in MAC1up1 cells, and ZRT2 was down 2-fold (Fig. 2A). In addition to ZRT1 and ZRT2, five other genes were slightly down-regulated in MAC1up1 cells as well as in BCS-treated cells by microarray experiments. However, S1 analysis did not corroborate the down-regulation (data not shown).


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  		Fig. 2.   S1 nuclease protection assays to quantify mRNA levels of genes down-regulated by Mac1. The upper band for each sample is the specified gene. The lower band in each case, indicated by an arrow, is the calmodulin (CMD1) loading control. fp lanes show undigested, free probes. A, RNA was isolated from cells cultured in LCCM in the absence or presence of 100 µM BCS. B, RNA was isolated from wild-type (wt) cells or MAC1up1 (UP) cells. without or harboring a ZRE/lacZ fusion gene. C, RNA was isolated from wild-type CM66J cells (wt) or MAC1up1 (UP) cells in the absence or presence of 10 µM ZnSO4.

Because ZRT1 and ZRT2 are transcriptionally activated by the Zap1 transcription factor in zinc-deficient cells, the down-regulation of ZRT1 may arise from enhanced zinc uptake in MAC1up1 cells and zinc inhibition of Zap1. Alternatively, ZRT1 may be repressed by a direct effect of Mac1. The sequence upstream of ZRT1 contains a candidate Mac1 binding site. To determine whether the Mac1up1 effect was primary or secondary, the zinc-responsive promoter element (ZRE) from ZRT1 was cloned into a UAS-less vector (pNB404) with lacZ as the reporter gene. ZRT1 and ZRE/lacZ expression were both markedly diminished in MAC1up1 cells relative to wild-type cells, whereas CTR1 showed the expected elevated expression in Mac1up1 cells (Fig. 2B). However, ZRT1 and ZRE/lacZ expression was unaffected in wild-type cells treated with BCS for 60 or 90 min, suggesting that the down-regulation was not a direct effect of Mac1. Furthermore, no enhanced ZRT1 expression was observed in mac1Delta cells, which may occur if Mac1 was an active repressor of ZRT1 expression. The MAC1up1-dependent inhibition of ZRT1 expression was observed in synthetic growth medium, but not in low zinc medium in which ZRT1 expression is fully derepressed (Fig. 2C). One prediction from these experiments is that the cellular zinc pool sensed by Zap1 is increased in MAC1up1 cells resulting in zinc inhibition of ZRT1 expression. To evaluate whether cellular Zn(II) levels were increased in MAC1up1 cells, metal analysis was carried out in cells cultured in medium of varying zinc levels. As expected, ZRT1 expression was fully derepressed in wild-type cells cultured in low zinc medium in which the cellular zinc content was 0.3 ng of zinc/106 cells. In contrast, cells cultured with 10 µM Zn(II) containing 12.7 ng of zinc/106 cells showed only minimal ZRT1 expression. Wild-type cells and MAC1up1 cells cultured in CM medium contained 2.2 ± 0.03 (n = 4) and 2.9 ± 0.3 (n = 4) ng of zinc/106 cells, respectively. The slight but reproducibly elevated level of Zn(II) in MAC1up1 cells may occur near some subtle, and hence difficult to detect, threshold level of cellular zinc important in homeostatic regulation of ZRT1 expression. Alternatively, copper ions may compete with Zn(II) for an intracellular site, thereby increasing the Zap1-responsive nonsequestered zinc pool more than total cellular Zn(II). The combined evidence is consistent with an indirect effect of Mac1 on ZRT1 expression.

The copper-activated CUP1 gene is induced within 30 min of the addition of 100 µM CuSO4 to yeast cells. Elevated copper levels are known to induce expression of CUP1, CRS5, and SOD1 through the copper-activated Ace1 transcription factor. To determine whether other genes are modulated by elevated copper in the growth medium, mRNA samples from wild-type cells growing exponentially in the presence or absence of a 30-min exposure to 100 µM CuSO4 were compared by differential hybridization to DNA microarrays. The experiment was carried out with two different pairs of cultures. CUP1 was enhanced 8- and 4.5-fold in cells treated with 100 µM Cu(II) (Table II). In contrast, CRS5 was elevated 1.8- and 2.6-fold in the two experiments. Curiously, SOD1 expression was elevated in only one of the two experiments. In addition to the known targets of Ace1, only two other genes, FET3 and FTR1, showed elevated expression (>1.5-fold) in copper-treated cells (Table II). These two genes encode proteins involved in the high affinity iron uptake system of yeast. FET3 expression was elevated 5- and 3-fold in the two experiments, whereas FTR1 was elevated 5- and 2-fold. S1 analysis of the same RNA used in the microarray experiments confirmed the copper-induced expression of FET3 and FTR1 (Fig. 3A).

                              
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Table II
Genes induced in copper-treated wild-type cells
Wild-type cells were cultured in the presence or absence of 100 µM CuSO4 for 30 min prior to harvest. The data shown represent the ratio of expression in the presence or absence of added CuSO4.


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  		Fig. 3.   S1 nuclease protection assay of genes up-regulated in copper-treated cells. A, independent RNA isolates 1 and 2 from cells cultured in the absence and presence of 100 µM CuSO4 for 30 min (used in microarray experiments 1 and 2; Table II). B, RNA isolated from CM66J wild-type cells cultured in low iron medium (-Fe), with 100 µM bathophenantroline sulfonate (BPS), or supplemented with 1.5 µM ferric chloride. In each case, mid-log phase cells were incubated in the presence and absence of 100 µM Cu(II) for 30 min. C, RNA isolated from from CM3260 wild-type cells (wt) or aft1Delta cells incubated in the presence and absence of Cu(II) for 30 min.

FET3 and FTR1 are known to be highly expressed in iron-deficient yeast cells (36)). Expression of these genes is induced in iron-deficient cells by the Aft1 transcriptional activator (36). At least three models may explain the copper induction of FET3. These include: 1) copper-induced diminution in cellular iron pools, activating Aft1, 2) direct activation of FET3 expression by CuAce1, and 3) copper-dependent loss of potential repression activity of Mac1. To determine whether FET3 induction was dependent on Aft1, we investigated whether copper induction of FET3 was related to the iron status of cells. Copper-induced expression of FET3 was observed in iron-replete cells, but not Fe-deficient cells, in which FET3 expression was fully derepressed (Fig. 3B). Furthermore, no copper-stimulated FET3 and FTR1 expression was observed in aft1Delta cells (Fig. 3C).

Both FET3 and FTR1 contain candidate Ace1 DNA-binding sites in their 5' sequences, suggesting that the observed copper induction may in part be mediated by Ace1. Consistent with this postulate, copper induction of FET3 and CUP1 was abrogated in ace1Delta cells (Fig. 4A). This result is consistent with Ace1 being important in copper induction of FET3. However, insertion of two candidate Ace1 binding sites from FET3 into a UAS-less vector with a lacZ reporter gene failed to show copper induction of lacZ (data not shown). In contrast, a lacZ gene with the Aft1 binding site from FET3 cloned into a UAS-less vector showed copper-induced expression of lacZ, suggesting that the effect is mediated by Aft1 (Fig. 4B). No copper induction was observed with a mutant FET3/lacZ fusion gene containing a mutation in the Aft1 binding site (Fig. 4B). The observed copper-induced expression of FET3 may arise from a transient copper-induced diminution in cellular iron pools resulting in Aft1 activation. Quantitation of cellular copper and iron levels in wild-type cells revealed a minimal (5%) copper-induced diminution in total cellular Fe levels.


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  		Fig. 4.   S1 nuclease protection assay of FET3 in cells treated with CuSO4. A, RNA was isolated from wild-type and ace1Delta cells treated in the absence and presence of 100 µM CuSO4. fp lanes show undigested, free probe. B, RNA was isolated from wild-type cells harboring either plasmid pFC-W or pFL-LM2. Plasmid pFC-W contains the Aft1-responsive, 30-bp portion of the FET3 promoter (-263 to -234) fused to lacZ. Plasmid pFC-LM2 contains a triple mutant in the FET3 promoter element, upstream of lacZ. The cells were cultured in LCCM and either untreated or treated with 100 µM CuSO4 for 30, 60, or 90 min.

The third candidate mechanism involves copper inhibition of Mac1 function. Mac1 was recently reported to be an active repressor of FET3 in S. cerevisiae (38). Copper-induced expression of FET3 may therefore arise from the known copper-inactivation of Mac1 function. This clearly is not the mechanism of copper-induced expression of FET3 and FTR1 for two reasons. First, no reproducible diminution in FET3 was observed in the various MAC1up1 array experiments or S1 analyses of MAC1up1 cells. Second, the reported derepression of FET3 levels in mac1 null cells was not reproduced in two distinct strains of S. cerevisiae, including the strain used in the published experiment.

Expression of a limited number of other genes was inhibited in cells treated with 100 µM Cu(II) for 30 min. Genes activated by Mac1 were, as expected, inhibited in their expression by added copper. Both newly identified Mac1-regulated genes (YFR055w and YJL217w) were inhibited in their expression in copper-treated cells (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNA microarray hybridization experiments identified a specific set of yeast genes differentially expressed under growth conditions of copper deficiency or copper excess. Two transcription factors, Mac1 and Ace1, were previously shown to mediate transcriptional regulation of S. cerevisiae genes by copper. In addition to the known targets of Mac1 and Ace1, a limited number of new genes were identified. A single DNA microarray experiment has the potential to reveal candidate genes differentially expressed under two different conditions. However, a more systematic strategy, using DNA microarrays to compare multiple related conditions, combined with secondary tests successfully restricted the candidate list to a common set of genes.

Mac1 mediates activation of genes under conditions of copper deficiency. Of the four known targets of Mac1, FRE1, FRE7, CTR1, and CTR3, three encode proteins important in high affinity uptake of copper ions. Thus, the genomic screens have the potential of identifying other genes whose products are important in copper homeostasis. Two new Mac1 regulated genes are YFR055w and YJL217w. The clearest evidence that these genes are Mac1-regulated is that they are induced by the copper chelator BCS in a wild-type strain but not in a mac1Delta strain. YFR055w belongs to a family of transsulfuration enzymes that includes yeast and rat cystathionine gamma -lyase, yeast homocysteine synthase, and E. coli cystathionine gamma  synthase. The predicted product encoded by YFR055w is 28% identical to the CYS3 product, which catalyzes production of cysteine from cystathionine and 26% identical to Met17, which converts O-acetylhomoserine into homocysteine. YFR055w may encode one of several cystathionine gamma -lyase isozymes in S. cerevisiae that generate cysteine from cystathionine. Cys3 is the major cystathionine gamma -lyase important in cysteine biosynthesis in yeast (39). The observation that YFR055w is Mac1-induced may suggest that one cellular response to copper deficiency is an expansion of Cys pools. YJL217w is predicted to encode an unknown 198-residue polypeptide. Ongoing studies of cells lacking a functional YJL217w may provide insight into the physiological role of this molecule.

YFR055w and YJL217w each have one perfect Mac1 consensus binding site in their 5'-flanking sequences. The four known Mac1-regulated genes require two binding sites for Mac1 activation. Although YFR055w and YJL217w contain only one consensus site, additional nonconsensus sites exist that may be functional.

A third candidate Mac1-regulated gene, YLR213c, is adjacent to, and transcribed divergently from FRE1. A 644-bp interval separates the two open reading frames. Two direct repeats of the Mac1 binding site are found halfway between the two open reading frames. These promoter elements may therefore mediate copper regulation of expression of both divergent genes. The significance of this response is uncertain, however, because YLR213c was markedly induced by copper deficiency in only one of the two strains used in the present studies.

Although a number of genes were observed to be slightly induced in Mac1up1 cells and in response to BCS treatment, we consider it unlikely that any of these are directly activated by Mac1, because their induction could not be confirmed by S1 analysis. Thus, the Mac1 regulon appears to consist of only seven genes activated under copper-deficient conditions.

A number of genes differentially expressed in MAC1up1 cells appear not to be direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of physiological changes in cells containing a constitutively active Mac1. Genes up-regulated in MAC1up1 cells include CUP1, PHO regulon genes, and a candidate hexose transporter HXT17. CUP1 expression is likely enhanced by virtue of constitutive expression of the high affinity copper transport system. Elevated copper transport will stimulate CUP1 expression through copper activation of Ace1. The PHO regulon is derepressed presumably by virtue of phosphate deprivation in MAC1up1 cells. The apparent phosphate deprivation occurring in MAC1up1 cells is not obviously explained based on the function of known Mac1-regulated genes. Microarray experiments are useful for evaluating changes in cellular physiology. Genes down-regulated in MAC1up1 cells include ZRT1 and ZRT2. These two zinc transporter genes are down-regulated in zinc-replete cells, suggesting that MAC1up1 cells may either preferentially accumulate Zn(II) or contain an elevated nonsequestered Zn(II) pool.

Ace1 mediates induction of gene expression under growth conditions of excess copper. The three known targets of Ace1 include the two metallothionein genes CUP1 and CRS5 and the SOD1 superoxide dismutase. The unexpected result was the copper-induced activation of FET3 and FTR1. Fet3 and Ftr1 function in high affinity iron uptake and are induced in iron-deficient cells through the Aft1 transcriptional activator (40).

Three lines of evidence suggest that the rapid copper-induced expression of FET3 arises from a transient diminution in a cellular iron pool resulting in Aft1 activation. First, copper induction of FET3 was abrogated in an aft1Delta strain. Second, copper induction was observed of a FET3/lacZ reporter gene containing a 30-bp segment of FET3 with the Aft1 binding site but not with a mutant FET3/lacZ fusion gene containing a mutation in the Aft1 binding site. Third, other genes in the Aft1 regulon are also induced by the short term copper treatment. YOR382w encodes a gene highly responsive to Aft1,2 and it is clearly copper-induced by S1 analyses (data not shown). The only suggestion that the copper effect was a direct effect of Ace1 was the lack of copper induction of FET3 and FTR1 in an ace1Delta strain. Because copper-mediated expression of FET3 is dependent on the cellular Fe status, it is conceivable that metal pools may be altered in cells lacking a functional Ace1. Thus, the available evidence is consistent with an indirect copper effect on iron pools.

Several interconnections between copper and iron metabolism are known. Copper is important for iron uptake through its role as a cofactor in the Fet3 trinuclear copper oxidase. Copper ions are provided for Fet3 biosynthesis by the copper-transporting, P-type ATPase Ccc2 (41). In addition, the Fre1 metalloreductase is co-regulated by copper and iron ions through Mac1 and Aft1 factors, respectively.

The observation that the cellular concentrations of copper ions can be coupled to activation and/or inhibition of gene expression in yeast implies that similar reactions may occur in other eukaryotes. Many examples exist of yeast being a Rosetta stone for the understanding of animal physiological processes, so a significant question arises of how applicable copper metalloregulation in yeast will be to animal cells. Clearly, microarray analysis using animal DNA clones may be useful in determining whether copper metalloregulation of transcription occurs in animal cells.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health Grants CA61286 (to D. R. W.) and HG00983 (to P. O. B.) and by funds from the Howard Hughes Medical Institute. The Biotechnology Core Facility for DNA synthesis at the University of Utah is supported by National Institutes of Health Grant 5P30-CA 42014.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Associate investigator of the Howard Hughes Medical Institute.

|| To whom correspondence should be addressed. Tel.: 801-585-5103; Fax: 801-585-5469; E-mail: dennis.winge@hsc.utah.edu.

Published, JBC Papers in Press, August 1, 2000, DOI 10.1074/jbc.M005946200

2 C. Philpott, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: LCCM, low copper complete medium; BCS, bathocuproine sulfonate; bp, base pair(s).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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