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Transcriptional Regulation of the Copper Transporter Mfc1 in Meiotic Cells

ABSTRACT Mfc1 is a meiosis-specific protein that mediates copper transport during the meiotic program in Schizosaccharomyces pombe. Although the mfc1+ gene is induced at the transcriptional level in response to copper deprivation, the molecular determinants that are required for its copper starvation-dependent induction are unknown. Promoter deletion and site-directed mutagenesis have allowed identification of a new cis-regulatory element in the promoter region of the mfc1+ gene. This cis-acting regulatory sequence containing the sequence TCGGCG is responsible for transcriptional activation of mfc1+ under low-copper conditions. The TCGGCG sequence contains a CGG triplet known to serve as a binding site for members of the Zn(2)Cys(6) binuclear cluster transcriptional regulator family. In agreement with this fact, one member of this group of regulators, denoted Mca1, was found to be required for maximum induction of mfc1+ gene expression. Analysis of Mca1 cellular distribution during meiosis revealed that it colocalizes with both chromosomes and sister chromatids during early, middle, and late phases of the meiotic program. Cells lacking Mca1 exhibited a meiotic arrest at metaphase I under low-copper conditions. Binding studies revealed that the N-terminal 150-residue segment of Mca1 expressed as a fusion protein in Escherichia coli specifically interacts with the TCGGCG sequence of the mfc1+ promoter. Taken together, these results identify the cis-regulatory TCGGCG sequence and the transcription factor Mca1 as critical components for activation of the meiotic copper transport mfc1+ gene in response to copper starvation.

M eiosis is a specialized facet of the cell cycle whereby precursor diploid cells generate haploid gametes (1,2). Following a mitotic cell cycle block at the G 1 phase, diploid cells can undergo meiosis by replicating their chromosomal DNA, thereby producing pairs of homologous chromosomes. After DNA replication, recombination takes place between each pair of homologous chromosomes during the meiotic prophase. Homologous chromosomes and sister chromatids are then successively separated through two rounds of division (called meiosis I [MI] and MII) to generate four haploid sets of chromosomes. Once MI and MII divisions have been completed, a differentiation process is induced to produce four gametes ready for fertilization or germination. Recent studies have shown that micronutrients such as copper and zinc are required for normal progression of meiosis because insufficient amounts of these metal ions lead to meiotic blocks and defective gamete formation (3,4).
The relatively short life expectancy of mammalian meiotic cells in coculture represents a hurdle to the use of these cells for studies of the whole meiotic program (5). One compounded difficulty stems from the fact that animal models and tissue cocultures are difficult to synchronize with respect to their entry into meiosis. Consequently, the use of model organisms has become an attractive avenue for the study of the molecular mechanisms that initiate and regulate meiosis (6,7). Among these models, the fission yeast Schizosaccharomyces pombe is one of the best genetically amenable organisms to use in deciphering molecular aspects of meiotic division from initiation to generation of mature haploid cells (8). This organism essentially undergoes meiosis in a manner analogous to that of germ line cells of higher eukaryotes, except for some differences. First, a fusion of two haploid cells of opposite mating types precedes the premeiotic S phase. Second, in the terminal stage of meiosis, the resulting gametes differentiate into spores that are enclosed in an ascus. This membranous oval struc-ture offers strong protection for the spores because it is highly resistant to unfavorable environmental conditions. During the S. pombe cell cycle, the G 1 phase represents a critical point at which a diploid cell becomes committed to the mitotic cell cycle or to the meiotic program. Diploid S. pombe cells undergo meiosis when nitrogen levels are low. In contrast, under conditions of nitrogen availability, cells grow mitotically because they express an active Pat1 kinase that inhibits cells from entering meiosis by phosphorylating the transcription factor Ste11 and the meiotic inducer Mei2 (9). Under conditions of nitrogen starvation, the mating type loci are induced by Ste11, which itself becomes active. Consequently, cells of the opposite mating type conjugate, forming diploid zygotes. Activation of the mating pheromone signaling pathway fosters the expression of mei3 ϩ , encoding a negative regulator of Pat1 kinase (10,11), preventing phosphorylation of target protein substrates, including Ste11 and Mei2. As an active Ste11 induces mei2 ϩ gene expression, unphosphorylated Mei2 accumulates and triggers the initiation of zygotic meiosis (9). However, if the resulting zygotes are returned to a nitrogen-rich medium before definitive commitment to meiosis, they can resume vegetative growth and form colonies of diploid cells. Conveniently, these cells undergo azygotic meiosis in response to a nitrogen starvation shock in a more synchronous manner than zygotic meiosis (12). A mutant strain harboring the pat1-114 temperature-sensitive mutation produces a thermosensitive Pat1 kinase. Upon a heat shock at 34°C, Pat1 is readily in-hibited, therefore bypassing the Mei3-dependent inactivation pathway of Pat1. This temperature-sensitive mutant confers a marked advantage, since it is more synchronous than azygotic meiosis. Nitrogen starvation response, initiation, and progression throughout meiosis are characterized by the expression of numerous genes that are modulated in four successive waves (13). The first wave of genes encodes proteins that are involved in nitrogen starvation and pheromone responses. Early-phase genes (wave 2) encode proteins that participate in premeiotic S phase and recombination. Middle-phase genes (wave 3) produce cellular components that are responsible for meiotic divisions and early steps of spore formation. Late-phase genes (wave 4) generate the cellular products required for spore maturation (13,14).
Recent studies have shown that metal ions such as copper and zinc are required for normal progression of meiosis in S. pombe and mice, respectively (3,4,15). In S. pombe, copper-insufficient zygotes undergo a meiotic block at metaphase I (3). With respect to copper transport into S. pombe meiotic cells, low copper levels induce expression of the copper transport genes ctr4 ϩ and ctr5 ϩ within the first hour of meiosis, followed by their repression 3 h after meiotic induction. Consistent with ctr4 ϩ and ctr5 ϩ gene expression profiles, the two-component copper-transporting complex that is composed of Ctr4 and Ctr5 (16)(17)(18)(19) is observed at the plasma membrane within 1 h and remains at the cell surface until the 3-h meiotic time point is reached (3). This step is followed by a quick increase in mfc1 ϩ mRNA levels at the 3-h meiotic time point, with mfc1 ϩ transcript levels remaining sustained throughout the meiotic program. mfc1 ϩ encodes a major facilitator superfamily (MFS)-type copper transporter (3,20). During middle to late meiosis, Mfc1 localizes at the forespore membrane, where it potentially mediates copper uptake into the forespore. Studies of the full-scale meiotic transcriptional program have revealed that ctr4 ϩ and mfc1 ϩ transcript levels are induced at distinct times after meiotic induction, in response to copper starvation (3). Whereas deletion of the gene encoding the copper-sensing transcription factor Cuf1 (cuf1⌬) impairs the induction of ctr4 ϩ and ctr5 ϩ , the activation of mfc1 ϩ is unaffected, suggesting the existence of a distinct transcriptional regulator for the induction of mfc1 ϩ in response to copper starvation (3). Among transcription factors that are known to be required for successful meiosis and sporulation, global transcriptome and deletome profile analyses have shown that the mfc1 ϩ gene is not regulated by Rep1, Mei4, Cuf2, Rsv1, Rsv2, Atf21, and Atf31 (14,21). These observations add further support to the hypothesis that a new transcription factor is required for regulation of the mfc1 ϩ gene.
In the present report, we show that mfc1 ϩ transcriptional induction is exclusively detected after treatment with a copper chelator and not by iron or zinc chelators. Analysis of regions in the promoter of mfc1 ϩ reveal that two TCGGCG regulatory elements containing CGG triplets are required for the induction of mfc1 ϩ in response to copper starvation. We consistently find that Mca1, a putative member of the Zn (2) Cys (6) binuclear cluster class of regulators which are known to bind repeated cis-acting elements containing CGG triplets, is necessary to mount a maximal transcriptional response of mfc1 ϩ . Microscopic analyses reveal that a functional Mca1-Cherry protein localizes to the nucleus during the course of vegetative growth of diploid cells and colocalizes with chromosomes during the meiotic process of differentiation. Although mca1⌬/mca1⌬ cells exhibit normal progression under basal and copper-replete conditions, these mutant cells undergo a meiotic block at metaphase I under conditions of copper starvation. Binding studies reveal that the N-terminal 150-residue segment of Mca1 expressed as a fusion protein in Escherichia coli specifically binds to the TCGGCG sequences of the mfc1 ϩ promoter region. Taken together, these results have identified cis-and trans-acting elements involved in molecular control of the meiosis-specific copper transporter Mfc1.

MATERIALS AND METHODS
Strains and media. The S. pombe strains used in this study are listed in Table 1. Standard methods were used for growth, mating, and sporulation of fission yeast cells (22). Under nonselective conditions, S. pombe cells were grown on yeast extract plus supplements (YES) containing 225 mg/ liter of adenine, histidine, leucine, uracil, and lysine. When plasmid transformation was required, cells were grown in Edinburgh minimal medium (EMM) lacking specific nutrients to select and purify cells expressing the transformed plasmid. The h ϩ /h Ϫ diploid strains used for azygotic meiosis were isolated as follows. Haploid cells of the opposite mating types were conjugated on a solid malt extract (ME) medium, and the resulting zygotes were then returned to rich media (YES) prior to commitment to meiosis. After this step, diploid cells can undergo azygotic meiosis following a synchronized nitrogen-starvation shock. Azygotic meiosis was induced using EMM lacking nitrogen (EMM-N) and supplemented with 10 mg/liter of adenine or 10 mg/liter of adenine, histidine, leucine, uracil, and lysine. Diploid strains homozygous for the mating type (h ϩ /h ϩ ) were generated by protoplast fusion, as described previously (23).
To synchronize pat1-114/pat1-114 diploid cells for their entry into meiosis, cells were precultured in EMM supplemented with adenine (225 mg/liter) at 25°C. Liquid cultures were seeded to an A 600 of 0.2 and grown to mid-log phase (A 600 of 0.5). The cells were harvested, washed twice, and transferred to EMM-N supplemented with 10 mg/liter of adenine. After incubation for 16 h at 25°C, NH 4 Cl (0.5 mg/liter) was added and cells were separated into different lots which were treated with ammonium tetrathiomolybdate (TTM), 2,2=-dipyridyl (Dip), N,N,N=,N=-tetrakis(2-pyridylmethyl)-1,2-ethanediamine (TPEN), and CuSO 4 or were left untreated. At this point, the temperature was shifted to 34°C to induce meiosis. Meiosis progression was monitored with Hoechst 33342 stain (5 g/ml) added at various times following meiotic induction.
Plasmids. The mfc1 ϩ promoter containing 800, 600, 200, 109, or 79 bp of the 5= noncoding region and the first 10 codons of the mfc1 ϩ gene was isolated by PCR. The first set of primers were designed to generate BamHI and ApaI restriction sites at the 5= termini of the PCR products, whereas the second primer was engineered to generate Bsu36I and EcoRI restriction sites at the 3= end of the PCR-amplified DNA fragments. Each PCR product was purified, digested with BamHI and EcoRI, and then introduced into the BamHI-EcoRI-digested Yep357R vector (24). The indicated mfc1 ϩ promoter region was isolated from Yep357Rmfc1 ϩ -800lacZ, Yep357Rmfc1 ϩ -600lacZ, Yep357Rmfc1 ϩ -200lacZ, Yep357Rmfc1 ϩ -109lacZ, and Yep357Rmfc1 ϩ -79lacZ after digestion with ApaI and Bsu36I. Each of these promoter regions was then swapped for the equivalent DNA restriction fragment in pBPade6str1 ϩ -296lacZ, creating pBPade6mfc1 ϩ -800lacZ, pBPade6mfc1 ϩ -600lacZ, pBPade6mfc1 ϩ -200lacZ, pBPade6mfc1 ϩ -109lacZ, and pBPade6mfc1 ϩ -79lacZ. To generate the pBPade6str1 ϩ -296lacZ vector, the pBPade plasmid (17) was digested with SalI, filled in using Klenow polymerase, and digested with PstI. Subsequently, a SpeI (filled in with Klenow)-PstI PCRamplified DNA segment containing the str1 ϩ regulatory region (up to Ϫ296 from the initiator codon) in addition to the E. coli lacZ gene was isolated from plasmid pSP1str1 ϩ -296lacZ (25). This DNA fragment was inserted into the SalI (filled in with Klenow)-PstI-digested pBPade6 plasmid.
Plasmid pBPade6mfc1 ϩ -109lacZ was used to introduce mutations to each or both of the TCGGCG elements (positions Ϫ80 to Ϫ85 and positions Ϫ99 to Ϫ104 with respect to the A of the ATG codon of mfc1 ϩ ). PCR amplification reactions of the mfc1 ϩ promoter were carried out using primers designed to generate 5=-GATTAT-3= instead of 5=-TCGGCG-3= in each or both of the regulatory cis-acting elements. Each PCR product was purified, digested with ApaI and Bsu36I, and then used to replace the equivalent wild-type DNA restriction fragment in pBPade6mfc1 ϩ -109lacZ. The resulting plasmids were named pBPade6mfc1 ϩ -109lacZ-mut1, pBPade6mfc1 ϩ -109lacZmut2, and pBPade6mfc1 ϩ -109lacZ-mut1-2. To generate wild-type and mutant pCF83mfc1 ϩ -125/-65lacZ fusion plasmids, a series of purified oligonucleotides (with upper and lower strands that are complementary to each other) were annealed pairwise to form wild-type (TCGGCG elements) and mutant (GATTAT elements) double-stranded DNA matrices. Once annealed, each doublestranded DNA oligomer derived from the mfc1 ϩ promoter (position Ϫ125 to Ϫ65) was ligated at the XmaI and XhoI sites of CYC1-lacZ fusion plasmid pCF83 (19).
PCR amplification of the mca1 ϩ gene was carried out with primers designed to generate XmaI and SacII restriction sites at upstream and downstream termini of the open reading frame, respectively. The fulllength gene was isolated from S. pombe strain FY435 genomic DNA. The PCR product was digested with XmaI and SacII and cloned into the corresponding sites of pBPade6 plasmid, creating plasmid pBPade6mca1 ϩ . Subsequently, the mca1 ϩ promoter region from position Ϫ500 upstream of the start codon of the mca1 ϩ gene was isolated by PCR amplification and was then inserted into pBPade6mca1 ϩ at the ApaI and XmaI sites. This pBPade6mca1 ϩ derivative was named pBPade6prom-mca1 ϩ . The Cherry coding sequence derived from pBM46SIT1mCherry (26) was isolated by PCR using primers designed to generate SacII and SacI sites at the 5= and 3= termini of the Cherry gene. The resulting DNA fragment was used to clone the Cherry gene into pBPade6prom-mca1 ϩ plasmid to which SacII and SacI restriction sites had previously been introduced by PCR and placed immediately before the mca1 ϩ stop codon. For this particular construct, named pBPade6prom-mca1 ϩ -Cherry, the SacII-SacI Cherry-encoded fragment was inserted in frame with the C-terminal region of Mca1. An identical strategy was used to clone the TAP coding sequence into pBPade6prom-mca1 ϩ plasmid to generate pBPade6prom-mca1 ϩ -TAP.
RNA analysis. Total RNA was extracted using the hot phenol method as described previously (27). RNA samples were quantified spectrophotometrically, and 15 g of RNA per sample was used for RNase protection assays, which were performed as described previously (28). Plasmid pSK-mca1 ϩ was constructed by inserting a 214-bp BamHI-EcoRI fragment from the mca1 ϩ gene into the same sites of pBluescript SK (Stratagene, La Jolla, CA). The antisense RNA hybridizes to the region between ϩ1001 and ϩ1215, downstream of the initiator codon of mca1 ϩ . Riboprobes derived from plasmids pSKmfc1 ϩ (3) and pKSlacZ (29) were used to detect mfc1 ϩ and lacZ transcripts, respectively. The act1 ϩ riboprobe (30) was used to detect act1 ϩ mRNA as an internal control for normalization during quantification of RNase protection products.
Fluorescence microscopy. h ϩ mca1⌬ and h Ϫ mca1⌬ haploid cells expressing the mca1 ϩ -Cherry allele were grown under conditions of low nitrogen and then crossed in order to produce diploid zygotes. After mating, the cells were quickly transferred to rich YES medium to stabilize their diploid state. The azygotic meiosis of diploid cells was synchronously induced by transferring the cells to nitrogen-poor EMM, as described previously (21). After the cells had just entered meiosis, culture aliquots were sampled every hour and Hoechst 33342 stain (5 g/ml) was added to analyze the progression of meiosis of individual cells. At the indicated meiotic phase, the cells were analyzed by microscopy using ϫ1,000 magnification with the following filters: 510 to 560 nm (Cherry) and 340 to 380 nm (Hoechst 33342 stain). Fluorescence and differential interference contrast images (Nomarski) were obtained using a Nikon Eclipse E800 epifluorescence microscope (Nikon, Melville, NY) equipped with a Hamamatsu ORCA-ER digital cooled camera (Hamamatsu, Bridgewater, NJ). Fields of cells shown in this study correspond to a minimum of five independent experiments. Merged images were obtained using Simple PCI software version 5.3.0.1102 (Compix, Sewickly, PA).
Protein extraction and Western blot analysis. Whole-cell extracts were prepared using a trichloroacetic acid extraction method (31). Equal amounts of proteins of each sample were subjected to electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose Hybond-ECL membranes (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Immunoblots were analyzed for the steady-state levels of Mca1-TAP and the ␣-tubulin protein using a polyclonal anti-mouse IgG antibody (ICN Biomedicals, Aurora, OH) and a monoclonal anti-␣-tubulin antibody (clone B-5-1-2; Sigma-Aldrich Canada, Oakville, Ontario, Canada), respectively. After 1 h of incubation with the primary antibodies in 1% powdered skimmed milk-PBST (10.1 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 138 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20, pH 7.4,), the membranes were washed three times with PBST, incubated with the appropriate horseradish peroxidase-conjugated anti-mouse secondary antibodies (GE Healthcare), and visualized by chemiluminescence detection on X-ray films.

Expression of the MBP-Mca1 fusion protein.
The DNA containing mca1 ϩ codons 1 to 150 was amplified by PCR, purified, and inserted in frame in pMAL-c2X vector (New England BioLabs, Ipswich, MA) at EcoRI and SalI restriction sites. Plasmid pMAL-1 mca1 ϩ150 was transformed in E. coli BL21(DE3). Fresh transformants of BL21 cells containing plasmid pMAL-c2X or pMAL-1 mca1 ϩ150 were grown to an A 600 of 0.5. At this growth phase, the cells were induced with 0.4 mM isopropyl-␤-Dthiogalactopyranoside for 18 h at 18°C in the presence of 2% ethanol. Harvested cells were washed once with ice-cold water and resuspended in G200 buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 M ZnCl 2 , pH 7.5) and a cocktail of protease inhibitors (P8340; Sigma-Aldrich). The mixture was incubated for 20 min at 4°C. Cell lysis was achieved using a FastPrep-24 instrument (MP Biomedicals, Solon, OH) for 45 s at the maximum speed setting. Insoluble material was removed by centrifugation (15,000 rpm, 20 min). The supernatant was applied to a 1-ml column of amylose resin (New England BioLabs) that had been equilibrated with G200 buffer. Beads were washed with 10 ml of buffer G200 and then were eluted stepwise with G200 buffer containing 10 and 20 mM maltose. SDSpolyacrylamide gel electrophoresis analysis showed that the affinity-purified maltose-binding protein (MBP)-1 Mca1 150 protein was recovered predominantly in the 10 mM maltose eluate fractions.

mfc1 ؉ mRNA levels are induced in response to low concentrations of copper but not regulated by iron or zinc deprivation.
Our previous studies identified a novel meiotic copper transporter that we named Mfc1 (3). As previously observed and as shown in Fig. 1, when diploid strain pat1-114/pat1-114 mfc1 ϩ/ϩ was synchronized through meiosis in the presence of the copper chelator TTM (150 M), mfc1 ϩ mRNA levels were induced ϳ4-fold compared to basal levels observed in untreated cells after 3 h of meiotic induction (Fig. 1). Results showed that, under low-copper conditions, the transcript levels of mfc1 ϩ remained upregulated (ϳ4-to 12-fold) throughout the remainder of the meiotic program, being detected even after 9 h of meiotic induction (Fig. 1). To further examine whether mfc1 ϩ transcription was induced by the presence of other metal ion chelators, synchronized pat1-114/pat1-114 mfc1 ϩ/ϩ cells were incubated in the presence of the iron chelator Dip (150 M) or the zinc chelator TPEN (150 M). Results showed no induction of transcription of mfc1 ϩ mRNA in response to these chelators ( Fig. 1 and data not shown). We therefore concluded that induction of mfc1 ϩ is specific to copper deprivation and not to regulation by iron or zinc deficiency.
Analysis of mfc1 ؉ promoter sequences required to induce gene expression under copper-limiting conditions. mfc1 ϩ is induced at the transcriptional level in response to copper starvation in a manner similar to that seen with the ctr4 ϩ and ctr5 ϩ genes that encode the high-affinity copper heteromeric transport complex located at the plasma membrane. In contrast to ctr4 ϩ and ctr5 ϩ , for which the transcription factor Cuf1 is required for their induction under copper-limiting conditions, the inactivation of the cuf1 ϩ gene does not affect the transcription of mfc1 ϩ (3). This result prompted us to search for the presence of cis-acting elements within the promoter region of mfc1 ϩ that were required for its induction in response to copper starvation. A series of mfc1 ϩ promoter fragments were fused upstream of and in frame to the lacZ gene in a pBPade6lacZ derivative vector, creating pBPade6mfc1 ϩ - 600lacZ, pBPade6mfc1 ϩ -200lacZ, pBPade6mfc1 ϩ -109lacZ, and pBPade6mfc1 ϩ -79lacZ. Gene expression of these plasmids was analyzed by RNase protection experiments. Results showed that removal of DNA sequences between Ϫ600 and Ϫ109 of the mfc1 ϩ promoter had no significant effect on the copper starvation-dependent activation of mfc1 ϩ -lacZ fusions (Fig. 2). In the presence of TTM, plasmids pBPade6mfc1 ϩ -600lacZ, pBPade6mfc1 ϩ -200lacZ, and pBPade6mfc1 ϩ -109lacZ were still induced ϳ27-, 29-, and 25-fold, respectively, compared to their levels of expression in untreated (basal) or copper-treated cells (7-h time point). In the case of all three of these mfc1 ϩ -lacZ promoter derivatives, basal and elevated copper concentrations resulted in a loss of mfc1 ϩ -lacZ mRNA expression. When the mfc1 ϩ promoter was deleted to position Ϫ79, TTM-dependent expression was completely abolished, lowering its expression to a minimal threshold that was similar to that observed in the case of basal and copper-replete conditions (Fig. 2).
The finding that the presence of the promoter region between Ϫ109 and Ϫ79 was sufficient to drive copper deprivation-dependent expression of the mfc1 ϩ -lacZ fusion gene prompted us to examine whether a mfc1 ϩ promoter segment that includes this region could contain a cis-acting element responsible for TTM- mediated responsiveness. Interestingly, the mfc1 ϩ promoter region between Ϫ109 and Ϫ79 contains two copies of the sequence TCGGCG. These repeats are separated by a sequence of 13 bp and are oriented in the opposite direction with respect to the A of the ATG codon of the mfc1 ϩ gene. CGG triplets are found within these sequences, and these triplets are quite often bound by transcription factors of the family of zinc binuclear cluster proteins (32). To determine whether these TCGGCG repeats played a role in mfc1 ϩ gene induction in response to low copper availability, we inserted multiple point mutations in each or both of the TCGGCG elements to mimic changes known to abolish binding of the zinc cluster transcription factors to CGG triplet sequences. Mutation of the base pairs within the Ϫ99 TCGGCG Ϫ104 element (GATTAT instead of TCGGCG; denoted Mut1) abolished copper starvation-dependent induction of the mfc1 ϩ -109lacZ fusion (Fig. 3). When the second TCGGCG element, Ϫ80 TCGGCG Ϫ85 , was mutated (G ATTAT instead of TCGGCG; denoted Mut2), lacZ transcript levels were very low in response to copper-limiting conditions; the overall magnitude of the response was decreased by ϳ95% (5-and 7-h time points) compared to cells containing wild-type reporter plasmid. When both TCGGCG elements were mutated, there was a complete lack of TTM responsiveness of the reporter gene (Fig. 3).
Based on the findings that the integrity of the TCGGCG elements located between positions Ϫ99 and Ϫ104 as well as Ϫ80 and Ϫ85 was essential to trigger TTM-dependent induction of the mfc1 ϩ -lacZ fusion, we examined whether these two elements could regulate a heterologous reporter gene in a TTM-dependent , is shown. Black boxes depict wild-type TCGGCG sequences, whereas empty boxes represent their mutant counterpart, GATTAT. pat1-114/pat1-114 cells were transformed with pBPade6mfc1 ϩ -109lacZ, pBPade6mfc1 ϩ -109lacZmut1, pBPade6mfc1 ϩ -109lacZmut2, or pBPade6mfc1 ϩ -109lacZmut1-2. Cultures were presynchronized by nitrogen starvation at 25°C and then induced to undergo synchronous meiosis at 34°C. Following induction of meiosis, total RNA was isolated at the indicated time points. After RNA preparation, the lacZ steady-state mRNA levels were analyzed by RNase protection assays using actin (act1 ϩ ) as an internal control. Data are representative of the results of three independent experiments. manner. A short DNA fragment derived from the mfc1 ϩ promoter (positions Ϫ65 to Ϫ125) was inserted in its natural orientation upstream of the minimal promoter of the CYC1 gene fused to lacZ in pCF83 (19). The fact that the upstream region of lacZ in pCF83 contains the CYC1 minimal promoter may explain the very weak levels of lacZ transcript that were detected in cells transformed with the plasmid alone (data not shown). However, the very weak level of lacZ mRNA from the plasmid alone was mostly observed during the early and middle phases of meiosis (after 1 to 5 h of meiotic induction) (data not shown). When a wild-type Ϫ65 mfc1 ϩϪ125 -CYC1-lacZ fusion reporter was expressed in pat1-114/pat1-114 cells undergoing meiosis in the presence of the cop-per chelator TTM (150 M), lacZ mRNA expression was induced ϳ17-fold compared to transcript levels detected in control (basal) or copper-exposed cells (Fig. 4). When the first Ϫ99 TCGGCG Ϫ104 element was mutated and the second one ( Ϫ80 TCGGCG Ϫ85 ) left unchanged, the steady-state levels of lacZ mRNA were decreased by 92% under copper-starved (TTM) conditions compared to transcript levels of wild-type Ϫ65 mfc1 ϩϪ125 -CYC1-lacZ fusion under the same experimental conditions (3-h time point). Furthermore, the very low levels of lacZ transcript showed an expression profile similar to that observed in the case of cells transformed with plasmid alone (data not shown). When the first Ϫ99 TCGGCG Ϫ104 element was left unaltered and the second one Cultures of a pat1-114/pat1-114 strain transformed with wild-type (WT) and mutant TCGGCG (mut1, mut2, and mut-2) fusions were presynchronized by nitrogen starvation and were then induced to undergo synchronous meiosis. At the indicated times following meiotic induction, total RNA was isolated and analyzed by RNase protection assays (right side). Steady-state levels of lacZ and act1 ϩ (as internal control) mRNAs were determined in the absence (basal) or the presence of TTM (150 M) or CuSO 4 (50 M). Data are representative of the results of three independent experiments. mutated, TTM-dependent induction of lacZ mRNA was compromised in a manner similar to that observed in the case of the Ϫ65 mfc1mut1 Ϫ125 -CYC1-lacZ mutant (Fig. 4). When both TC GGCG elements were mutated, a lack of TTM response of the reporter gene was observed (Fig. 4). In the case of the wild-type Ϫ65 mfc1 ϩϪ125 -CYC1-lacZ fusion and its mutant derivatives, there was a lack of significant down-or upregulation of lacZ mRNA levels under basal or copper-replete conditions. Taken together, the results were consistent with the interpretation that both TC GGCG elements in the mfc1 ϩ promoter are required to confer copper limitation-dependent induction of expression of mfc1 ϩ .
Mca1 plays a major role in activation of mfc1 ؉ expression in response to copper starvation. We next sought to identify a transacting factor that recognized the cis-acting element 5=-TCGGC G-3= DNA binding motif required for appropriate induction of mfc1 ϩ gene expression under low-copper conditions. Analysis of genomic DNA sequences of the S. pombe database revealed the existence of 31 genes that encode known or putative members of the Cys (6) Zn (2) binuclear cluster protein family. Among them, 30 of these genes are expressed during the entire course of meiosis or at a precise step during meiotic development. Transcriptional profiles of these 30 genes revealed that 26 genes are expressed during the middle meiotic phase, which corresponds to the meiotic period where Mfc1 is expressed as a function of copper availability. Of interest, among the 26 genes that were expressed during the middle meiotic phase where Mfc1 is expressed as a function of copper availability, we identified 4 genes (SPAPB1A11.04c, SPCC777.02, SPAPB24D3.01, and SPAC11D3.07c) containing coding sequences rich in Cys, Met, and His residues. Because several Cys, Met, and His residues were scattered throughout these proteins and may represent potential metal-binding ligands, we further investigated a potential role for SPAPB1A11.04c, SPCC777.02, SPAPB24D3.01, and SPAC11D3.07c in the regulation of Mfc1 expression. Using isogenic strains harboring insertionally inactivated SPAPB1A11.04c⌬, SPCC777.02⌬, SPAPB24D3.01⌬, and SPAC11D3.07c⌬ genes, we assessed the potential role of the predicted SPAPB1A11.04c-, SPCC777.02-, SPAPB24D3.01-, and SPAC11D3.07c-encoded zinc binuclear cluster transcription factors in the regulation of mfc1 ϩ . Among the four different disruption genes tested, only SPAPB1A11.04c altered the activation of Mfc1 expression. The SPAPB1A11.04c gene was found to be expressed on the same chromosome as the mfc1 ϩ gene and was located 7,038 bp downstream of mfc1 ϩ . As is the case with most of the zinc binuclear cluster transcription factors, SPAPB1A11.04c protein contains a DNA-binding domain, a middle homology regulatory region, and a transactivation domain (Fig. 5A). When pat1-114/pat1-114 SPAPB1A11.04c ϩ / ϩ and pat1-114/pat1-114 SPAPB1A11.04c⌬/⌬ diploid strains were presynchronized in the G 1 phase by nitrogen starvation and then incubated at 34°C to inactivate Pat1 kinase, cells initiated and  (2) Cys (6) binuclear cluster motif (amino acids 24 to 51) that is followed by a linker region (amino acids 52 to 116) and one heptad repeat of leucine residues (amino acids 117 to 138). A regulatory domain (amino acids 336 to 412) is located on the C-terminal side of the DNA-binding domain, and it is termed the middle homology region (MHR). The C-terminal 44 amino acids of Mca1 comprise an overall majority of acidic amino acid residues. This region is predicted to act as an activation domain. Amino acid numbers refer to the position relative to the first amino acid residue of the protein. Consensus amino acid sequences that represent a Zn (2) Cys (6) -type finger and a heptad repeat of leucine residues are shown. (B) pat1-114/pat1-114 (mca1 ϩ /mca1 ϩ ) and pat1-114/pat1-114 mca1⌬/mca1⌬ strains were presynchronized by nitrogen starvation at 25°C (t ϭ 0, basal) and then induced to undergo synchronous meiosis at 34°C under basal, copper-replete, and copper-depleted conditions. At the indicated time points, mfc1 ϩ and act1 ϩ (internal control) mRNA levels were analyzed in the control strain (mca1 ϩ /mca1 ϩ ) and an isogenic strain lacking the mca1 ϩ alleles. Data are representative of the results of three independent experiments. proceeded through a synchronous meiosis. In the presence of TTM, inactivation of SPAPB1A11.04c resulted in strongly (ϳ8fold) reduced mfc1 ϩ mRNA levels in comparison to those seen with wild-type cells grown under the same conditions (Fig. 5B). Under basal and copper-replete conditions, transcript levels of mfc1 ϩ remained very low (background threshold) in wild-type and SPAPB1A11.04c⌬/⌬ strains. We named the SPAPB1A11.04c gene product Mca1 (meiosis copper starvation-dependent activator) based on the fact that its presence was required to fully activate mfc1 ϩ gene expression under conditions of copper starvation.
To provide additional data to support the notion of an important role of Mca1 in mfc1 ϩ gene activation, we used the mfc1 ϩ promoter region between positions Ϫ109 and Ϫ1, which was sufficient to drive TTM-dependent induction of the mfc1 ϩ -lacZ gene (Fig. 3). In the case of pat1-114/pat1-114 mca1 ϩ / ϩ diploid cells synchronously induced into meiosis, mfc1 ϩ -109lacZ promoter fusion strongly upregulated lacZ mRNA expression under conditions of copper deficiency (Fig. 6). In contrast, pat1-114/pat1-114 mca1⌬/⌬ mutant cells containing the mfc1 ϩ -109lacZ promoter fusion plasmid showed poor induction of lacZ mRNA which was decreased by ϳ85% compared to the level seen with wild-type strain (mca1 ϩ / ϩ ) under conditions of low levels of copper (Fig. 6). Furthermore, results showed that the integrity of the TCGGCG sequence was essential because a TCGGCG box mutant (with a mutation in each or both of the TCGGCG elements) abrogated TTM-dependent induction of the mfc1 ϩ -109lacZ reporter gene (Fig. 6). Taken together, these results indicate that, under conditions of low concentrations of copper, the TCGGCG promoter sequence and the Mca1 zinc binuclear cluster protein play critical roles in the activation of mfc1 ϩ gene expression.
Mca1 is constitutively expressed during mitosis and meiosis. We next analyzed steady-state mRNA levels of mca1 ϩ as a function of copper availability during mitosis and meiosis. Experiments using cells proliferating in mitosis showed that mca1 ϩ steady-state mRNA levels were constitutive and unresponsive to cellular copper status (Fig. 7A). In contrast, ctr4 ϩ copper transporter mRNA levels (assayed as a control) were induced or repressed, depending on the presence of TTM or copper, with respect to basal conditions (Fig. 7A). To further investigate the expression profile of mca1 ϩ during meiosis, a pat1-114/pat1-114 diploid strain was synchronized to initiate and proceed through the meiotic program. Immediately prior their entry into the meiotic program, the cells were either left untreated or exposed to TTM (150 M) or CuSO 4 (50 M). Aliquots of cultures were retrieved at distinct time intervals following meiotic induction, and steady-state levels of mca1 ϩ mRNA were analyzed by RNase protection assays. Results showed that mca1 ϩ transcripts were detected under basal (untreated), copper-starved, or copper-replete conditions (Fig. 7B). Interestingly, in the presence of TTM, mca1 ϩ mRNA levels were slightly increased after 5 and 7 h of meiotic induction (ϳ1.5-fold compared to levels observed after 3 h of meiotic induction). Similarly, a slight increase of mca1 ϩ expression was observed in untreated cells after 5 h of meiotic induction (ϳ1.4-fold compared with levels observed after 1 and 3 h of meiotic induction) (Fig. 7B). To ascertain whether the steady-state levels of Mca1 followed those of mca1 ϩ mRNA, we used a pat1-114/pat1-114 mca1⌬/⌬ strain in which a functional mca1 ϩ -TAP fusion allele was returned into the genome by integration. Results showed that, under basal or low-or high-copper conditions, Mca1-TAP levels were constitutively present. In addition, we ob-served that steady-state levels of Mca1 remained slightly higher 7 h and 9 h after meiotic induction (Fig. 7C). To ensure that cell incubation conditions (TTM, basal, or CuSO 4 ) had no major negative effect on meiotic progression and sporulation, a series of microscopic analyses were performed. pat1-114/pat1-114 diploid cells were synchronously induced into meiosis, and Hoescht 33342 stain (0.5 g/l) was added every hour to cell culture aliquots to visualize DNA and to monitor meiotic progression. Under basal conditions, meiosis I occurred primarily between 3 h and 6 h after meiotic induction, meiosis II between 5 h and 8 h, and sporulation after 8 to 10 h (Fig. 7D). Although meiotic progression under conditions of copper starvation (TTM, 150 M) was reduced by approximately 2 h compared to untreated (basal) cell results, spore formation was clearly observed at the end of meiosis (Fig. 7D). Similarly, although copper-replete (CuSO 4 , 50 M) zygotes exhibited a meiotic progression that was prolonged by ϳ1 h compared to control cells, the resulting meiotic products were tetranucleated asci (Fig. 7D). Taken together, these results indicate that, under basal, copper-depleted, and copper-replete conditions, the mca1 ϩ gene is constantly expressed during meiosis, although the process of meiotic maturation varies slightly as a function of time.
Subcellular localization of Mca1 during meiosis. We next determined the subcellular location of Mca1 during the meiotic program. The Cherry fluorescence-coding sequence was fused in frame with the 3=-terminal end of the mca1 ϩ gene. When the Mca1-Cherry fusion protein was expressed in meiosis, it triggered the induction of mfc1 ϩ mRNA to levels similar to that of wild-type Mca1 (untagged) or TAP epitope-tagged Mca1 (data not shown). A functional mca1 ϩ -Cherry allele was integrated into h Ϫ mca1⌬ and h ϩ mca1⌬ cells to visualize the Mca1-Cherry in living zygotes and asci. Following mating, h Ϫ /h ϩ mca1⌬/mca1⌬ mca1 ϩ -Cherry/ mca1 ϩ -Cherry diploid cells were cultured under conditions chosen to induce them to undergo azygotic synchronous meiosis. Following induction of meiosis under basal or low-copper conditions (TTM, 50 M), fluorescent Mca1-Cherry was readily detected (at the 0-h time point) in the nucleus of vegetative azygotic meiotic cells (Fig. 8). During meiotic prophase (3-h time point), S. pombe cells displayed Mca1-Cherry fluorescence as an elongated spot that appeared to correspond to the elongated nucleus that is generally called the "horse-tail" nucleus (33,34). At metaphase I (4-h time point), Mca1-Cherry fluorescence in meiotic cells was observed as a single spot per cell that colocalized with chromosomal material (as marked by Hoechst 33342 staining). After 5 h of meiotic induction (late anaphase I), Mca1-Cherry fluorescence was observed as a pair of spots in each cell and their locations appeared to correspond to homologous chromosomes that had undergone the first meiotic division (Fig. 8). At early and late anaphase II (6-and 7-h time points), meiotic cells displayed Mca1-Cherry fluorescence as two pairs of spots per cell and this result was interpreted to correspond to chromosomal material that had undergone the second meiotic division, in which case sister chromatids segregate (Fig. 8). Cells displayed Mca1-Cherry fluorescence as four distinct spots in the zygote during forespore membrane formation and sporulation (Fig. 8). Collectively, data from microscopic analysis of meiotic cells reveal that a functional Mca1-Cherry protein colocalizes with the chromosomes that had undergone every step of the process of meiosis. These observations add further support for the notion of a regulatory role of Mca1 at the DNA level.
Inactivation of the mca1 ؉ gene alters the process of meiosis under copper-limited conditions. In addition to the Mca1 requirement for expression of the mfc1 ϩ gene under conditions of copper deficiency, we hypothesized that Mca1 was also important for normal progression of meiosis under copper-starved conditions. To test this hypothesis, h ϩ /h Ϫ mca1⌬/mca1⌬ diploid cells were used and results compared to h ϩ /h Ϫ mca1 ϩ /mca1 ϩ control cell results. Diploid strains were synchronously induced by transferring the cells at the same time to nitrogen-poor medium, thus allowing cells to undergo azygotic meiosis. Following induction of meiosis (zero time point), cells were left untreated or were treated with TTM (50 M). In the case of wild-type cells (mca1 ϩ /mca1 ϩ ), meiosis I occurred mainly between 5 h and 7 h, meiosis II between 8 h and 10 h, and spore formation after 11 h of meiotic induction under basal (untreated) conditions (Fig. 9A). Furthermore, in the case of control cells, where meiosis was induced in the presence of TTM (50 M), the first meiotic division was postponed by ϳ2 h (Fig. 9B). Despite this delay, control cells proceeded through meiosis and formed asci containing four spores after 12 h of meiotic induction. In the case of mca1⌬/mca1⌬ mutant cells, where azygotic meiosis was induced under basal conditions, the first meiotic division was delayed by ϳ3 h compared to the results seen with control cells (Fig. 9A). Despite this delay, mca1⌬/mca1⌬ cells underwent meiosis II (12-h time point), spore maturation, and formation (14-h time point) to produce 4-spore asci similarly to the wild-type strain (Fig. 9A). In contrast, TTM (50 M)-treated mutant cells lacking Mca1 (mca1⌬/mca1⌬) proceeded through metaphase I but then stopped their progression, exhibiting meiotic arrest (Fig. 9B). Taken together, the data suggest that, under basal conditions, Mca1 plays an important role for normal meiotic progression based on the fact that cells carrying inactivated mca1⌬/⌬ alleles display delayed and prolonged meiosis. Under conditions of copper deprivation, the presence of Mca1 becomes even more critical since its absence results in arrested meiosis at metaphase I.
Mca1 interacts with two mfc1 ؉ promoter elements containing CGG triplets. The bulk of the results were consistent with the notion that the mca1 ϩ gene was required for maximal activation of mfc1 ϩ gene expression under conditions of copper starvation. We thus investigated the possibility that Mca1 interacted directly with the sequences Ϫ99 TCGGCG Ϫ104 and Ϫ80 TCGGCG Ϫ85 located in the promoter region of mfc1 ϩ (Fig. 10A). To test this hypothesis, we made a construct where the N-terminal 150 amino acids of Mca1 were fused to MBP and expressed the plasmid in E. coli. The purified recombinant Mca1 fusion protein was used for binding studies. Results showed that a wild-type 32 P-end-labeled 73-bp mfc1 ϩ promoter fragment, which contained the TCGGCG sequences, formed a DNA-protein complex in the presence of Mca1 (Fig. 10B). The presence of Mca1 in the complex was assessed in supershift experiments using an anti-MBP antibody. Results showed the formation of a complex of slightly lower electrophoretic mobility, consistent with the interpretation of the presence of a DNA-MBP-1 Mca1 150 complex (Fig. 10B). The specificity of the DNA-protein complex was confirmed by competition assays using unlabeled oligomers containing either wild-type TCGGCG elements or mutated elements (GATTAT instead of TCGGCG) (Fig. 10B). Formation of the DNA-protein complexes was inhibited by incubation with excess wild-type oligomer but not by mutant competitor (Fig. 10B). These results indicate that the N-terminal 150 amino acids of Mca1 associate with TCGGCG promoter elements of the mfc1 ϩ gene.

DISCUSSION
A finely tuned regulation of copper uptake is required to maintain copper homeostasis in S. pombe. On the one hand, it provides copper-dependent protein activity with sufficient amounts of copper and, on the other hand, it protects the cells against the toxic effects of copper overload. Efficient copper transport in S. pombe cells that grow mitotically requires the copper ion to be in the Cu 1ϩ state of oxidation to be transported by a heteroprotein complex of Ctr4 and Ctr5 proteins (16,18,35). Transcriptional regulation of the ctr4 ϩ and ctr5 ϩ genes is under the control of the copper-sensing transcription factor Cuf1 which induces their expression under copper-deficient conditions (36,37). Under high-copper conditions, Cuf1 becomes inactive and is subsequently exported outside the nucleus (38,39).
There are relatively few data concerning the intrinsic role of copper during meiosis. It has recently been reported that S. pombe undergoes meiotic arrest at metaphase I under strong copper starvation conditions, suggesting a critical role for copper in meiotic maturation and progression (3). During early meiosis, copper uptake is most likely ensured by the heteromeric Ctr4-Ctr5 protein complex, since Ctr4 localizes to the cell surface of developing zygotic cells (3). Once middle-phase meiosis has been initiated, Ctr4 expression is abolished and Mfc1, a meiosis-specific major facilitator superfamily (MFS)-type transporter, is induced. Mfc1 is first detected in precursor vesicles and then at the forespore membrane of ascospores (3). In late-phase meiosis, Mfc1 is found at the forespore membrane, and the use of the coppersensor-1 tracker suggests that it transports copper into the forespore (3,40). In a manner similar to that seen with the ctr4 ϩ and ctr5 ϩ genes, mfc1 ϩ is induced at the level of gene transcription in response to copper starvation. However, in contrast to ctr4 ϩ and ctr5 ϩ , for which the transcription factor Cuf1 is required for their induction under copper-limiting conditions, the inactivation of the cuf1 ϩ gene does not affect the transcriptional activation of mfc1 ϩ (3). This observation prompted us to look for a transcription factor that was required for copper starvation-dependent induction of mfc1 ϩ during meiosis. One potential candidate was the meiosis-specific Cuf2 transcription factor, which exhibits high homology of its N terminus with the N terminus of Cuf1 (21). Total RNA isolated from an isogenic pair of strains, one carrying an inactivated allele of cuf2 ϩ (cuf2⌬) and one containing a wild-type copy of the gene (cuf2 ϩ ), showed no effect on mfc1 ϩ transcript levels when the availability of copper was limited. mfc1 ϩ was strongly induced in response to copper deficiency in both strains (data not shown).
Functional dissection of the mfc1 ϩ promoter revealed the presence of two copies of a consensus TCGGCG element required for diploid (h ϩ /h Ϫ mca1 ϩ /mca1 ϩ ) and h ϩ /h Ϫ mca1⌬/mca1⌬ mutant cells expressing Sad1-Cherry were synchronously induced to undergo azygotic meiosis under basal conditions. For each strain, samples were taken every 60 min, and percentages of cells with 1 nucleus, 2 nuclei, or 3 or 4 nuclei as well as percentages of asci were calculated (graphics on left). Results are reported as the means of the results of three replicates Ϯ standard deviations. Although the entire meiotic program occurred in both strains, meiotic maturation of a strain lacking Mca1 (mca1⌬/mca1⌬) was significantly delayed compared to that of the control. Representative microscopic images of asci 12 h and 14 h after meiotic induction in the case of the wild-type (mca1 ϩ /mca1 ϩ ) and mca1⌬/mca1⌬ mutant cells, respectively, are shown (right side). (B) The entire meiotic program in wild-type zygotic cells (mca1 ϩ /mca1 ϩ ) incubated in the presence of TTM (50 M) was delayed compared to that seen with untreated mca1 ϩ /mca1 ϩ cells. TTM-treated mca1⌬/mca1⌬ mutant cells underwent a meiotic block at metaphase I. The graphics (left) indicate the meiotic profiles of these cells. The numbers of cells with 1 nucleus, 2 nuclei, or 3 or 4 nuclei were determined by counting Hoechst-stained nuclei after meiotic induction. Typical images of wild-type (mca1 ϩ /mca1 ϩ ) and mca1⌬/mca1⌬ mutant cells at the 12-h time point are shown in the right panels. FSM, forespore membrane. copper-starvation-induced expression of mfc1 ϩ . The two regulatory cis-acting elements contained CGG triplets that were disposed in a direct-repeat and symmetrical format, but they were oriented in the opposite direction from the A of the ATG of the mfc1 ϩ gene. The spacing between the two CGG triplets was 16 bp, which represents a long spacer compared to other known similar binding elements containing CGG trinucleotides (41). Multiple point mutations within mfc1 ϩ promoter TCGGCG sequences abolished copper starvation-dependent activation of mfc1 ϩ . We thus tested the possibility that a CGG-binding zinc binuclear cluster transcription factor played a role in the induction of mfc1 ϩ gene expression. We determined that cells bearing a disruption of mca1 ϩ , which encodes a Zn (2) Cys (6) cluster-type protein, were unable to fully induce mfc1 ϩ in response to copper deficiency. Consistent with a role for Mca1 as a positive regulator of mfc1 ϩ (a meiosis-specific gene), its expression and gene product were observed during meiosis. It should be noted that Mca1 was also constitutively expressed during mitosis, but its role during vegetative proliferation remains unknown. Mca1 possesses one zinc finger unit (residues 24 to 51) that includes 6 Cys residues which are predicted to coordinate 2 zinc ions. The N-terminal zinc-binding motif of Mca1 is followed by an unusual large linker region (residues 51 to 117) and a heptad repeat of Leu residues (residues 117 to 138). The fact that the Mca1 linker region is particularly large may explain the ability of Mca1 to bind CCG triplets which are separated by 16 bp in the mfc1 ϩ promoter. The heptad repeats of Leu residues represent the predicted dimerization region of Mca1.
Molecularly, the zinc finger unit, the linker, and the dimerization region form the putative N-terminal DNA-binding domain of Mca1 (residues 1 to 138).
We found that a direct palindrome, TCGGCGN 13 TCGGCG, was required for copper starvation-activation of mfc1 ϩ . Given the fact that two zinc finger units are required to bind a pair of CGG triplets, this implies that Mca1 may bind the TCGGCGN 13 TC GGCG sequence as a homodimer. Unexpectedly, a low level of mfc1 ϩ mRNA was still induced in a mca1⌬/mca1⌬ mutant strain under conditions of copper starvation. One interpretation of this result is that at least one other member of the family of zinc binuclear cluster proteins may be involved in the regulation of mfc1 ϩ . Data that showed that the TCGGCGN 13 TCGGCG sequence was necessary for copper-starvation-mediated gene expression are consistent with this interpretation. Thus, in the absence of Mca1 (mca1⌬/mca1⌬), a second zinc binuclear cluster transcription factor may act as a homodimer in regulating mfc1 ϩ . In the wild-type strain (mca1 ϩ /mca1 ϩ ), where many proteins within this class of transcription factors are found as heterodimers (32,42,43), the possibility exists that Mca1 may form a heterodimer complex with a second unidentified zinc binuclear cluster protein. Homodimeric or heterodimeric complexes of zinc cluster proteins may influence promoter recognition or transcriptional effects on target genes (44)(45)(46). Among the 31 putative members of the Cys (6) Zn (2) binuclear cluster protein family, 3 of these proteins (SPBPB8B6.04c, SPAC1486.10, and SPAC11D3.07c) have a large linker region similar to that seen with Mca1. If the length of the The anti-MBP antibody produced an additional DNA-protein complex (indicated with an asterisk) that showed electrophoretic mobility that was slower than that seen with the complex observed without the addition of ␣MBP antibody. Competition experiments were performed with double-stranded DNA unlabeled oligomers corresponding to wild-type (WT) and mutant elements. The amounts of competitor oligonucleotides used in each reaction are shown on top of the lanes. The probe concentration was 1 ng/reaction in each case. S, supershifted DNA-protein complex; B, bound probe DNA; F, free probe DNA.