Strains and media

S. pombe strains undergoing vegetative growth were isogenic derivatives of the parental strain FY435 (h⁺ his7-366 leu1-32 ura4-∆18 ade6-M210) [16] and their genotypes are described as follows: cuf1∆ (h⁺ his7-366 leu1-32 ura4-∆18 ade6-M210 cuf1∆::KAN^r) and mca1∆ (h⁺ his7-366 leu1-32 ura4-∆18 ade6-M210 mca1∆::KAN^r). In the case of strains that entered into meiosis, we used isogenic strains in which the pat1-114 temperature-sensitive mutation allowed synchronization of cells in terms of their entry into the meiotic program. Genotypes of strains harboring a pat1-114 mutation are summarized as follows: h⁺ pat1-114 ade6-M210 (JB484) [23], h⁺ pat1-114 ade6-M216 (JB485) [23], h⁺ pat1-114 ade6-M210 mca1∆::KAN^r (JSY26), h⁺ pat1-114 ade6-M216 mca1∆::KAN^r (JSY27), and h⁺/h⁺ pat1-114/pat1-114 ade6-M210/ade6-M216 mca1∆::KAN^r/mca1∆::KAN^r (JSY30). Standard methods were used for growth, sexual conjugation and sporulation of cells [4]. Non-selective yeast extract plus supplements (YES) medium for S. pombe contained 0.5% yeast extract, 3% glucose, and 225 mg/l of adenine, histidine, leucine, uracil and lysine. Strains used for DNA plasmid integration were grown in Edinburgh minimal medium (EMM), in which specific nutrients were lacking as required to foster chromosomal integration events. h⁺/h⁺ pat1-114/pat1-114 and h⁺/h⁺ pat1-114/pat1-114 mca1∆/mca1∆ diploid strains were generated by incubating mid-logarithmic phase cultures of haploid cells with 20 μg/ml carbendazim (Sigma-Aldrich) as described previously [24]. Preparation and synchronization of pat1-114/pat1-114 diploid cells for their entry into meiosis was performed as described previously [6].

Plasmids

The mca1⁺ promoter containing 500 bp of the 5’ noncoding region and the first 150 codons (450 bp) of the mca1⁺ gene was isolated by PCR, using primers designed to generate ApaI and XmaI restriction sites at the termini of the PCR product. The PCR product obtained was digested with ApaI and XmaI and cloned into the corresponding sites of pBPade6 [25]. The resulting plasmid was designated pBPade6prom-mca1DBD. To create a plasmid containing full-length Mca1, an XmaI-SacII PCR-amplified fragment containing the last 547 codons of the mca1⁺ ORF was isolated. The XmaI-SacII DNA fragment was inserted in-frame into the corresponding sites of pBPade6prom-mca1DBD, generating a plasmid that was denoted pBPade6prom-mca1⁺. To construct a TAP epitope-tagged Mca1, the TAP coding sequence derived from Cuf2-TAP [26] was isolated by PCR using primers designed to generate SacII and SacI sites at the 5’ and 3’ termini of the TAP ORF. The resulting DNA fragment was used to clone the TAP coding sequence into pBPade6prom-mca1⁺ plasmid to which SacII and SacI restriction sites had previously been introduced by PCR and placed immediately before the mca1⁺ stop codon. For this particular construct, named pBPade6prom-mca1⁺-TAP, the SacII-SacI TAP-encoded fragment was inserted in-frame with the C-terminal region of Mca1.

Two distinct ApaI-SacII PCR-amplified fragments containing the mca1⁺ locus starting at -500 bp relative to the A of the ATG codon up to +1710 and +1800 bp after the initiator codon were isolated. These two DNA fragments were swapped with a corresponding ApaI-SacII DNA fragment into pBPade6prom-mca1⁺ and pBPade6prom-mca1⁺-TAP plasmids. The resulting plasmids were named pBPade6prom-mca11-570, pBPade6prom-mca11-600, pBPade6prom-mca11-570-TAP, and pBPade6prom-mca11-600-TAP. Two C-terminal coding regions of Mca1 containing the last 398 (residues 299–697) and 371 (residues 326–697) codons were amplified from pBPade6prom-mca1⁺ and pBPade6prom-mca1⁺-TAP plasmids using two sets of primers designed to introduce XmaI and SacII or XmaI and SacI at the termini of DNA fragments. Each PCR product was purified, digested with XmaI and SacII or SacI, and then inserted into pBPade6prom-mca1DBD at the XmaI and SacII or XmaI and SacI sites. The resulting plasmids were named pBPade6prom-mca1DBD-299-697, pBPade6prom-mca1DBD-299-697-TAP, pBPade6prom-mca1DBD-326-697, and pBPade6prom-mca1DBD-326-697-TAP. A DNA fragment containing the mca1⁺ promoter up to -500 bp upstream from the start codon and the first 298 codons of the mca1+ gene was amplified from pBPade6prom-mca1⁺ using primers designed to introduce ApaI and XmaI at the termini of the DNA fragment. The PCR product was purified, digested with ApaI and XmaI, and used to replace an ApaI-XmaI DNA fragment in pBPade6prom-mca1DBD-326-697 or pBPade6prom-mca1DBD-326-697-TAP. The resulting plasmids were denoted pBPade6prom-mca1-1-298+326–697 and pBPade6prom-mca1-1-298+326-697-TAP. The mca1⁺ coding region corresponding to amino acid residues 601–697 was isolated by PCR using primers that contained XmaI and SacII or XmaI and SacI at their ends. Subsequently, purified XmaI-SacII and XmaI-SacI DNA fragments were exchanged with the XmaI-SacII and XmaI-SacI DNA regions in plasmids pBPade6prom-mca1-1-298+326–697 and pBPade6prom-mca1-1-298+326-697-TAP to generate pBPade6prom-mca1-1-298+601–697 and pBPade6prom-mca1-1-298+601-697-TAP, respectively.

To create the pSKcuf1⁺ plasmid, an ApaI-XmaI PCR-amplified DNA segment containing the cuf1⁺ locus starting at -977 bp from the translational start codon up to the stop codon was inserted into the corresponding sites of pBluescript SK (Stratagene). Subsequently, the myc₁₂ epitope coding region was isolated from pctr4⁺-X-myc₁₂ plasmid [27] using XmaI and XbaI and then inserted into the corresponding sites of pSKcuf1⁺ plasmid, resulting in an in-frame fusion of the cuf1⁺ gene with twelve copies of the Myc epitope. Once created, the cuf1⁺-myc₁₂ fusion gene and its promoter region were isolated using ApaI and SacI and inserted into the corresponding sites of pBPade6. The resulting integrative plasmid was designated pBPade6cuf1⁺-myc₁₂. In the case of the untagged cuf1⁺ allele, the integrative plasmid pBPade6cuf1⁺ was constructed by subcloning the ApaI-XmaI fragment from pSKcuf1⁺ into the corresponding sites of pBPade6.

RNA isolation and analysis

Total RNA was extracted using a hot phenol method as described previously [28]. Gene expression profiles were analyzed using RNase protection assays as described previously [29]. Plasmids pSKmfc1⁺ [6], pSKmca1⁺ [16], pSKctr4⁺ [30] and pSKact1⁺ [31] were used to produce antisense RNA probes that served to determine mfc1⁺, mca1⁺, ctr4⁺, and act1⁺ steady-state mRNA levels, respectively. ³²P-labeled antisense RNA probes were produced using the above-mentioned BamHI-linearized plasmids and with the use of [α-³²P]UTP and T7 RNA polymerase. The act1⁺ riboprobe was used to detect act1⁺ transcript as an internal control for normalization during quantification of the RNase protection products.

Protein extraction and analysis

To determine the steady-state protein levels of Mca1-TAP or its mutant derivatives and α-tubulin, whole cell extracts were prepared using a trichloroacetic acid (TCA) extraction method [32]. Equal amounts of each sample preparation were resuspended in sodium dodecyl sulfate loading buffer and proteins were resolved by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels. Proteins were then electroblotted onto nitrocellulose membranes for 1 h at 4°C. Membranes were blocked by treatment with 5% powdered skim milk (Difco) in TBS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% bovine serum albumin) containing 0.1% Tween 20 (TBST). Following washings with TBST, membranes were incubated with primary antibodies dissolved in 1% powdered skim milk in TBST for 16 h at 4°C. The following antibodies were used for immunodetection of Mca1-TAP (or its mutant derivatives) and α-tubulin: polyclonal anti-mouse IgG antibody (ICN Biomedicals) and monoclonal anti-α-tubulin antibody (clone B-5-1-2; Sigma-Aldrich), respectively. Following incubation, the membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), developed with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences), and visualized by chemiluminescence using an ImageQuant LAS 4000 instrument (GE Healthcare) equipped with a Fujifilm High Sensitivity F0.85 43 mm camera.

ChIP assays

In vivo cross-linking of proteins was performed by incubating cell cultures with 1% formaldehyde for 20 min at the indicated time after meiotic induction. After formaldehyde-mediated cross-links and neutralization with glycine, cell lysates were prepared by glass bead disruption in lysis buffer containing 100 mM HEPES-KOH pH 7.5, 1% Triton X-100, 0.1% Na-deoxycholate, 1 mM EDTA, 140 mM NaCl, 2X cOmplete ULTRA Tablets (protease inhibitors, Roche), 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF and 0.2 mM Na₃VO₄, as described previously [33]. Samples were then sonicated using a Branson 450 sonicator to shear chromatin DNA into fragments of ~500 to 1000 bp. Immunoprecipitation of Mca1-TAP and its mutant derivatives bound to chromatin was performed using immunoglobin G (IgG)-Sepharose beads. In the case of the RNA polymerase II, immunoprecipitation was carried out using protein G Sepharose beads that were coupled to a monoclonal anti-Rpb1 antibody (clone 8WG16, Covance), which specifically recognizes a repeated seven-residue motif (YSPTSTS) located at the C-terminus of Rpb1, termed the C-terminal domain (CTD). In the case of Cuf1-Myc₁₂, immunoprecipitation was performed using protein G Sepharose beads that were coupled to a monoclonal anti-myc antibody (9E10, Roche). Handling of beads, including washings and elution, reversed cross-linking, and DNA precipitation were performed as described previously [34, 35]. Quantification of immunoprecipitated DNA was carried out by real-time PCR (qPCR) using different sets of primers that spanned mfc1⁺, cuf2⁺, and ctr4⁺ promoter or coding regions. Primers for ChIP assays were designated by the name of the gene, followed by the position of their 5’ ends relative to that of the translational start codon: mfc1-183 (5’-GTATCGCTAAACTCCGAGGATATAAGTG-3’), mfc1-32 (5’- GAAGATTGAGAATGATGAGAATATATATATCATTTGC-3’), mfc1+875 (5’-TCGTTGGCACTGTTTATGGA-3’), mfc1+960 (5’-TCCCGAAGTGAAGTGGTATTG-3’), cuf2-1139 (5’-TCAGCATCTAGCAGCAAATATACA-3’), cuf2-999 (5’-CGAAGTGATTTCATCAAATTAAGGAACG-3’), ctr4-737 (5’-GAAATGTCACTGTTTGTGATGCTG-3’), and ctr4-584 (5’-CAGCTCTTTTACACGGCAATAATAC-3’). Mca1-TAP, Cuf1-Myc₁₂ or RNA polymerase II density at mfc1⁺, cuf2⁺, and ctr4⁺ genes was calculated as the enrichment of specific genomic mfc1⁺, cuf2⁺, and ctr4⁺ DNA regions relative to a 18S ribosomal DNA coding region in which no functional TCGGCG or CuSE element was present. The primers (denoted 18S-a and 18S-b) that were used to amplify a 18S ribosomal DNA region were described previously [7]. Each qPCR was run in triplicate using Perfecta SYBR Green Fast mix (Quanta) on a LightCycler 96 Real-Time PCR instrument (Roche). All ChIP experiments were repeated at least three times using independent chromatin preparations.

Mca1 binds to the mfc1⁺ promoter in vivo in a copper-independent manner

We have previously reported that the transcription factor Mca1 was required for maximal induction of mfc1⁺ mRNA levels in response to low concentrations of copper (Fig 1). Despite its transcriptional regulatory role, mfc1⁺ promoter occupancy in vivo by Mca1 remained uncharacterized. To understand the extent by which Mca1 occupies the promoter of mfc1⁺ in vivo under copper starvation compared to its occupancy in the presence of copper, we used a pat1-114/pat1-114 mca1∆/mca1∆ mutant strain that had been transformed with a control integrative plasmid or a plasmid harboring an untagged or a TAP-tagged version of Mca1 under the control of its own promoter. pat1-114/pat1-114 mca1⁺/mca1⁺ (control) and pat1-114/pat1-114 mca1∆/mca1∆ diploid cells expressing the indicated integrative plasmid (empty, mca1⁺ or mca1⁺-TAP) were induced to undergo synchronous meiosis in the presence of the copper chelator TTM (150 μM) or CuSO₄ (25 μM). Subsequently, total RNA was isolated from culture aliquots taken at different time points. In the case of mca1∆/mca1∆ cells expressing Mca1 or Mca1-TAP, mfc1⁺ transcripts were initially detected after 3 h of meiotic induction under low copper. These observations contrasted with the absence of significant induction of transcription of mfc1⁺ mRNA in cells harboring an empty plasmid at the 3-h time point under similar conditions (Fig 1). Results showed that, under copper-starved conditions, mfc1⁺ mRNA levels reached high levels after 7 h of meiotic induction in mca1∆/mca1∆ cells expressing an untagged or a TAP-tagged version of Mca1 (7.2-fold) in comparison with cells transformed with the vector alone (Fig 1). The expression profile of mfc1⁺ mRNA in mca1∆/mca1∆ cells expressing Mca1 or Mca1-TAP was similar to that observed in the case of mca1⁺/mca1⁺ (control) cells, except that the magnitude of induction of mfc1⁺ mRNA was modestly higher (9%, 3%, and 23% at 3-, 5- and 7-h time points, respectively) (Fig 1). Under copper-replete conditions, transcript levels of mfc1⁺ were expressed to near background levels in mca1⁺/mca1⁺ or mca1∆/mca1∆ cells whether the cells contained an empty vector or an integrative vector expressing Mca1 or Mca1-TAP (Fig 1).

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Fig 1. Mca1 is required for maximal copper-starvation-induced expression of mfc1⁺.

A, pat1-114/pat1-114 mca1∆/mca1∆ cells containing an empty integrative vector (vector alone) and re-integrated untagged mca1⁺/mca1⁺ and mca1⁺-TAP/mca1⁺-TAP alleles were synchronously induced to undergo meiosis under copper-starved (TTM, 150 μM) and copper-replete (Cu, 25 μM) conditions. Total RNA was isolated from culture aliquots taken at the indicated time points after meiotic induction. Following RNA isolation, mfc1⁺ and act1⁺ steady-state mRNA levels were analyzed by RNase protection assays. pat1-114/pat1-114 mca1⁺/mca1⁺ is the isogenic parental strain that was used as a control. B, Graphic representation of the quantification of the results of three independent RNase protection assays, including assays shown in panel A. Values are represented as averages ± S.D. The asterisks correspond to p<0.0001 (****) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g001

Results showed that the in-frame TAP insertion did not interfere with Mca1 function. For example, when Mca1-TAP was expressed in mca1∆/mca1∆ cells, it conferred copper starvation-dependent regulation of mfc1⁺ expression in a fashion similar to that of the untagged Mca1 protein. This observation was taken as evidence that Mca1-TAP retained wild-type function. Using mca1∆/mca1∆ cells expressing Mca1-TAP, we first determined the steady-state protein levels of Mca1-TAP over a time period of 1 to 7 h after meiotic induction. Results showed that Mca1-TAP levels were constitutively present irrespective of the cellular copper status (Fig 2). We took advantage of the constitutive expression of Mca1-TAP to test whether Mca1-TAP could be detected at the mfc1⁺ and cuf2⁺ promoters in vivo using a ChIP approach. pat1-114/pat1-114 mca1∆/mca1∆ diploid cells expressing either an untagged or a TAP-tagged Mca1 were synchronized to initiate and proceed through meiosis under copper-deficient or copper-replete conditions. After 1, 3, 5, and 7 h of meiotic induction, results showed that Mca1-TAP constitutively occupied the mfc1⁺ promoter (Fig 2). Although results showed that Mca1-TAP chromatin occupancy of mfc1⁺ promoter was slightly higher in copper-starved cells compared to copper-replete cells, Mca1-TAP clearly occupied the mfc1⁺ promoter under both low and high copper conditions (Fig 2). At the 1-h time point, Mca1-TAP level occupancy was lower in comparison to level observed after 3, 5, and 7 h of meiotic induction. This reduced association of Mca1-TAP with the mfc1⁺ promoter may contribute to the markedly reduced transcription of mfc1⁺ in cells at this meiotic time point under low and high copper concentrations (Fig 1). Promoter occupancy by Mca1-TAP was detected using primers amplifying a DNA region located between positions -183 and -32 relative to the initiator codon of mfc1⁺. This amplified promoter region is known to contain two functional TCGGCG elements that are bound by Mca1 in vitro [16]. Results from ChIP analysis showed that Mca1-TAP did not associate with the cuf2⁺ promoter (used as a negative control). Only very weak levels of cuf2⁺ promoter fragments were immunoprecipitated (Fig 2). These low levels of immunoprecipitated chromatin were similar to the background signals observed when ChIP assays were performed in mca1∆/mca1∆ cells expressing untagged mca1⁺, which had been re-integrated (Fig 2). Taken together, these results showed that Mca1 is recruited to the promoter of mfc1⁺ in a constitutive manner, with a slight increased ability to bind mfc1⁺ promoter under conditions of low levels of copper.

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Fig 2. Mca1 constitutively binds the mfc1⁺ promoter in vivo.

A, pat1-114/pat1-114 mca1∆/mca1∆ cells expressing Mca1-TAP were synchronously induced into meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM). The upper panel shows Western blots of Mca1-TAP and α-tubulin protein levels at different time points after meiotic induction. Positions of the molecular weight standards (M) are indicated to the left. B, Schematic representation of mfc1⁺ and cuf2⁺ promoter regions. Nucleotide numbers refer to the position relative to the A of the initiator codon of each gene (mfc1⁺ or cuf2⁺). Numbers in parentheses indicate promoter regions that were used for qPCR analysis in ChIP assays. Black boxes depict wild-type TCGGCG sequences known to serve as a binding site for Mca1. C–D, pat1-114/pat1-114 mca1∆/mca1∆ cells expressing Mca1-TAP or untagged Mca1 were synchronously induced to undergo meiosis. Following induction of meiosis, chromatin was immunoprecipitated using Sepharose-bound anti-mouse IgG antibodies at the indicated time points. Specific regions of mfc1⁺ and cuf2⁺ promoters were analyzed by qPCR to determine Mca1-TAP occupancy in the presence of TTM or copper. Binding of Mca1-TAP to promoters was calculated as the enrichment of specific mfc1⁺ and cuf2⁺ promoter regions relative to a 18S ribosomal DNA coding region. The cuf2⁺ promoter served as a negative control. ChIP data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments.

https://doi.org/10.1371/journal.pone.0201861.g002

The RNA polymerase II is recruited to mfc1⁺ locus in vivo in response to copper starvation

The RNA polymerase II (Pol II) is a fundamental multi-subunit enzyme that transcribes numerous genes into mRNA in eukaryotic cells [36]. General, signal- and stress-responsive transcription factors control either positively or negatively gene expression through multiple mechanisms that affect Pol II occupancy at specific coding regions of target genes [37]. Taking into account the result that Mca1 constitutively occupied the mfc1⁺ promoter, we used a Pol II ChIP assay to determine whether the presence of Mca1 was required for Pol II chromatin occupancy and whether Pol II was primarily recruited in response to low copper availability. Analysis of Pol II chromatin occupancy was performed using an anti-Rpb1 antibody that recognizes the heptapeptide sequence YSPTSPS, which is found in multiple copies at the C-terminal domain (CTD) of Pol II [38]. pat1-114/pat1-114 mca1∆/mca1Δ cells harboring an empty integrative plasmid or expressing untagged mca1⁺ were synchronously induced into meiosis. Immediately before their passage to meiosis, cell cultures were treated with TTM (150 μM) or CuSO₄ (25 μM). Aliquots of cell cultures were taken 1, 5, and 7 h after meiotic induction and cells were fixed by formaldehyde treatment for ChIP assays. Primers that hybridized immediately upstream (positions -183 to -32) and at the 3’ end of mfc1⁺ (positions +875 to +960) coding region were used for qPCR analysis (Fig 3A). Results of ChIP analysis showed that in cells expressing Mca1, association of Pol II with the mfc1⁺ locus was highly enriched in response to copper starvation conditions and was negligible or absent under copper-replete conditions (Fig 3B). In copper-starved cells expressing Mca1, Pol II exhibited 14.0-, 40.1-, and 45.0-fold increases in its binding to mfc1⁺ (positions -183 to -32) relative to a control 18S ribosomal DNA coding region after 1, 5, and 7 h of meiotic induction (Fig 3B). Under the same conditions, levels of Pol II enrichment were higher (17.6-, 92.4-, and 100.0-fold increases) in its binding at the 3’ end of mfc1⁺ (positions +875 to +960) coding region relative to a 18S ribosomal DNA region reference (Fig 3B). Taken together, these results revealed that association of Pol II with an mfc1⁺ transcribed region in vivo requires the presence of the transcription factor Mca1 in response to low concentrations of copper.

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Fig 3. Mca1 is required for copper starvation-dependent RNA polymerase II chromatin occupancy at the mfc1⁺ locus after 5 and 7 h of meiotic induction.

A, Schematic representation of the mfc1⁺ gene in which ChIP1 and ChIP2 regions indicate locations of the primers that were used for qPCR analysis. Arrows indicate the direction of transcription. Nucleotide numbers refer to the position relative to the A of the initiator codon of mfc1⁺. B, Cultures of pat1-114/pat1-114 mca1∆/mca1∆ cells expressing untagged Mca1 or harboring an empty integrative vector were induced to initiate and proceed through meiosis. Cells underwent meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM). At the indicated times after meiotic induction, chromatin was immunoprecipitated using antibodies against RNA Pol II CTD. ChIP results (RNA Pol II occupancy) are presented as enrichments of specific regions (ChIP1 and ChIP2) relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments. The asterisks correspond to p<0.05 (*) and p<0.001 (**) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g003

The mfc1⁺ promoter is constitutively bound by Mca1-TAP during mitosis but there is absence of expression of mfc1⁺ regardless of changes in copper concentrations

Previous studies have shown that the copper transporter ctr4⁺ mRNA levels are induced in a Cuf1-dependent manner in cells proliferating in mitosis under conditions of copper starvation [30, 39, 40]. After treatment with copper, Cuf1 is inactivated [39, 41, 42] and then ctr4⁺ mRNA is not detected because its expression is dependent of Cuf1 [40]. Here, we used ctr4⁺ as a control gene known to be induced under copper-limiting conditions (Fig 4A). Furthermore, we performed ChIP assays using Cuf1-Myc₁₂ as a control transcription factor known to interact with the ctr4⁺ promoter in vivo under low copper conditions (Fig 4B and 4C) [43]. In the case of the mfc1⁺ transcription profile, there was no detection of the mfc1⁺ transcript in mitotically growing cells in the presence or absence of TTM or CuSO₄ (Fig 4A) [6]. However, the ability of Mca1 to associate with mfc1⁺ promoter in vivo in cells proliferating in mitosis remains unknown. To examine whether there was absence or presence of Mca1 in the promoter region of mfc1⁺ in mitotic cells, ChIP assays were performed in vegetatively growing mca1∆ cells expressing a functional mca1⁺ or mac1⁺-TAP allele under copper-starved or copper-replete conditions. ChIP analysis showed that in the case of cells treated with TTM for 1 and 3 h, Mca1-TAP immunoprecipitated 3.8- and 4.3-fold more chromatin corresponding to mfc1⁺ promoter compared to an 18S ribosomal region reference (Fig 4B and 4C). In the case of cells exposed to copper for the same times of treatments, chromatin enrichment levels by Mca1-TAP were 3.2- and 3.6-fold, respectively (Fig 4C). These results were consistent with the interpretation that in mca1∆ cells expressing Mca1-TAP, the mfc1⁺ promoter was occupied by Mca1-TAP in the presence of TTM in a manner similar to that observed for mca1∆ cells expressing Mca1-TAP in the presence of copper (Fig 4C; Figure A in S1 File). Results showed that untagged Mca1 (expressed in mca1∆ cells) immunoprecipitated only background levels of mfc1⁺ promoter region (Fig 4C). As control experiments, results from ChIP analysis showed that in the case of cuf1∆ cells expressing Cuf1-Myc₁₂ that were treated with TTM for 1 and 3 h, Cuf1-Myc₁₂ occupied the ctr4⁺ promoter with 3.7- and 3.8-fold enrichments, respectively, relative to a control 18S ribosomal DNA region reference (Fig 4C). In these cells, steady-state levels of ctr4⁺ mRNA consistently increased 11- and 9-fold above the levels observed in copper-replete cells (Fig 4A). In contrast, when cells expressing Cuf1-Myc₁₂ were incubated in the presence of copper for 1 and 3 h, only very weak levels of ctr4⁺ promoter fragments were immunoprecipitated (Fig 4C). These low levels of immunoprecipitated chromatin were similar to the background signals observed when ChIP assays were performed in a cuf1∆ strain expressing an untagged cuf1⁺ allele, which had been re-integrated (Fig 4C). The association of Cuf1-Myc₁₂ with the ctr4⁺ promoter was detected using primers amplifying a DNA region located between positions -737 and -584 relative to the initiator codon of ctr4⁺ (Fig 4B). This amplified promoter region is known to contain functional CuSE elements that are specifically recognized by Cuf1 [30].

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Fig 4. Mca1 interacts with the mfc1⁺ promoter when expressed in mitotically growing cells but fails to activate mfc1⁺ transcription in response to copper deficiency.

A, The indicated strains were proliferated in mitosis in the presence of TTM (150 μM) or copper (Cu, 25 μM) for 1 and 3 h. In the cases of cuf1∆ cells expressing an integrated untagged or a Myc-tagged cuf1⁺ allele and mca1∆ cells expressing an integrated untagged or a TAP-tagged mca1⁺ allele, aliquots of these cultures were fixed by formaldehyde treatment for ChIP assays and at the same time collected to isolate RNA for assessment of transcript levels. Shown are representative RNase protection assays of ctr4⁺, mfc1⁺, and act1⁺ mRNA steady-state levels at the indicated time points. B, Schematic representation of ctr4⁺ and mfc1⁺ promoter regions. Nucleotide numbers refer to the position relative to the A of the initiator codon of each gene. Numbers in parentheses indicate promoter regions that were used for qPCR analysis in ChIP assays. White and black boxes depict wild-type CuSE and TCGGCG sequences known to serve as binding sites for Cuf1 and Mca1, respectively. C, Specific regions of ctr4⁺ (left) and mfc1⁺ (right) promoters were analyzed by qPCR to determine chromatin occupancy by Cuf1-Myc₁₂ and Mca1-TAP, respectively. Association of Cuf1-Myc₁₂ and Mca1-TAP to promoters was calculated as the enrichment of specific ctr4⁺ and mfc1⁺ promoter regions relative to a 18S ribosomal DNA coding region. ChIP data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments. D, Schematic representation of ctr4⁺ and mfc1⁺ coding regions. Numbers in parentheses indicate coding regions that were used for qPCR analysis in ChIP assays. E, ChIP analysis was performed to determine RNA Pol II chromatin occupancy at ctr4⁺ and mfc1⁺ genes. Chromatin was immunoprecipitated using RNA Pol II CTD-specific antibodies. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments. The asterisks correspond to p<0.001 (**) and p<0.0001 (****) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g004

Despite a constitutive mfc1⁺ promoter occupancy by Mca1-TAP, mitotic cells grown under low and high concentrations of copper revealed that the target gene mfc1⁺ was silent (Fig 4A). To gain insight into this observation, we analyzed Pol II chromatin occupancy using a pair of primers located between positions +875 and +960 relative to the initiator codon of mfc1⁺. Results showed no enrichment of immunoprecipitated chromatin corresponding to the mfc1⁺ coding region relative to an 18S ribosomal region reference. The absence of association of Pol II with the mfc1⁺ locus was observed under low and high concentrations of copper (Fig 4D and 4E). In the case of ctr4⁺, ChIP experiments showed that Pol II occupied the ctr4⁺ coding region upstream of the initiator codon of ctr4⁺ (positions +718 and +817) under copper-limiting conditions. In contrast, in cells undergoing a transition from low to high copper, Pol II was absent from the ctr4⁺ locus (Fig 4D and 4E). Taken together, these results suggested that cells proliferating in mitosis lacked a factor (perhaps meiosis-specific) or possessed a mechanism that prevented Pol II-dependent transcriptional activation of mfc1⁺ despite the fact that Mca1 constitutively occupied the mfc1⁺ promoter in vivo.

In vivo mapping of a middle region of Mca1 that is required for induction of mfc1⁺ expression

In cells undergoing synchronous meiosis, Mca1-TAP was constitutively bound to the mfc1⁺ promoter but recruitment of Pol II and, therefore, mfc1⁺ expression occurred only under conditions of copper starvation. We thus sought to investigate whether a domain of Mca1 was required for copper starvation-dependent induction of mfc1⁺. Previous studies had shown that the N-terminal 150-residue segment of Mca1 constituted its DNA-binding domain (DBD) [16]. Because this domain binds DNA constitutively, we kept it intact and generated two internal in-frame Mca1 deletions, starting from Ile¹⁵¹ and extending to residues Leu²⁹⁹ and Gly³²⁶, respectively. The rationale for investigating these 2 mutant forms of Mca1 was the fact that the first deleted region (residues 151–298) contains 2 potential metal-binding motifs (Cys¹⁶³-X-Cys-X-His¹⁶⁵ and Met²³⁴-X₃-Met²³⁸). The resulting C-terminal portions of Mca1 (residues 299–697 and 326–697) were fused in-frame to the DNA binding domain of Mca1 (residues 1–150) (Fig 5A). Because a Mca1-TAP protein triggers activation of mfc1⁺ gene expression in a manner similar to that of wild-type untagged protein, each mutant derivative was tagged with TAP. The mca1⁺-TAP allele and its mutant derivatives were expressed in pat1-114/pat1-114 mca1∆/mca1∆ cells that underwent synchronous meiosis under copper-starved or copper-replete conditions. Maximal activation of mfc1⁺ expression was observed in copper-starved cells expressing mca1⁺-TAP and mca1DBD-299-697-TAP alleles after 7 h of meiotic induction (Fig 5B). When the middle region of Mca1 was further deleted to residue 326 to generate the mca1DBD-326-697-TAP allele, TTM-dependent expression of mfc1⁺ was strikingly decreased by 85% to a minimal level of activation that was similar to that observed in the case of cells harboring an empty vector (TTM, 7-h time point) (Fig 5B). In the case of all three alleles (mca1⁺-TAP, mca1DBD-299-697-TAP, and mca1DBD-326-697-TAP) expressed in mca1∆/mca1∆ cells, elevated copper concentrations resulted in a strong repression of mfc1⁺ mRNA expression (Fig 5B).

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Fig 5. Deletion of the middle region 151–326 of Mca1 inactivates its ability to induce mfc1⁺ gene expression in response to low copper.

A, Schematic diagram of truncated versions of the Mca1-TAP protein. The amino acid sequence of Mca1-TAP is numbered relative to its initiator codon. The black arrow indicates the position of the in-frame deletion within Mca1. B, Representative expression profiles of mfc1⁺ and act1⁺ transcripts in synchronous meiosis experiments. Mca1-TAP and the indicated mutant proteins were expressed in pat1-114/pat1-114 mca1∆/mca1∆ cells and assessed for their ability to activate mfc1⁺ expression in the presence of TTM (150 μM) or copper (Cu, 25 μM). At the indicated times after meiotic induction, total RNA was isolated and steady-state mRNA levels of mfc1⁺ and act1⁺ were analyzed by RNase protection assays. C–E, ChIP analysis of the mfc1⁺ promoter to determine Mca1-TAP, Mca1DBD-299-697-TAP, and Mca1DBD-326-697-TAP chromatin occupancy. TAP-tagged protein density at the mfc1⁺ promoter was determined as the enrichment of the specific mfc1⁺ promoter region (positions -183 to -32) relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments using distinct chromatin preparations. F, Schematic representation of the mfc1⁺ gene in which ChIP1 region indicates the location of the primers that were used for qPCR analysis. Nucleotide numbers refer to the position relative to the A of the initiator codon of mfc1⁺. Black boxes depict wild-type TCGGCG sequences known to serve as a binding site for Mca1. G–H, Protein extracts were analyzed by Western blots for steady-state protein levels of Mca1-TAP and its mutant derivatives and α-tubulin after 1 and 7 h of meiotic induction.

https://doi.org/10.1371/journal.pone.0201861.g005

To further verify that Mca1-TAP and its two mutant derivatives were associated with the mfc1⁺ promoter in vivo, ChIP assays were performed in pat1-114/pat1-114 mca1∆/mca1∆ cells expressing either an untagged or a TAP-tagged version of Mca1 (wild-type or the indicated mutant version). Enrichment levels of Mca1, Mca1-TAP, Mca1DBD-299-697-TAP, and Mca1DBD-326-697-TAP proteins were assessed at the mfc1⁺ promoter after 1 and 7 h of meiotic induction and compared to enrichment levels of a 18S ribosomal region reference. Results of ChIP assays showed that chromatin enrichment levels by Mca1-TAP and Mca1DBD-299-697-TAP were similar (with values ranging between 2.5- and 2.8-fold), regardless of the copper status (Fig 5, C–D). In the case of Mca1DBD-326-697-TAP, chromatin enrichment levels were lower by 14% (under TTM conditions) and 30% (under copper conditions) compared to enrichment levels of Mca1-TAP and Mca1DBD-299-697-TAP. However, the mfc1⁺ promoter was still clearly bound by Mca1DBD-326-697-TAP compared to an untagged Mca1, which immunoprecipitated only background levels of mfc1⁺ promoter region (Fig 5C and 5E). The association of Mca1-TAP, Mca1DBD-299-697-TAP, and Mca1DBD-326-697-TAP with the mfc1⁺ promoter was detected using primers amplifying a DNA region located between positions -183 and -32 relative to the initiator codon of mfc1⁺ (Fig 5F). To assess that Mca1-TAP and its mutant derivatives were produced, total protein extracts were analyzed by immunoblotting at the indicated meiotic time points. Results showed that detectable levels of Mca1-TAP, Mca1DBD-299-697-TAP, and Mca1DBD-326-697-TAP proteins were present in mca1∆/mca1∆ cells (Fig 5G and 5H).

We next sought to examine whether Mca1, Mca1DBD-299-697, and Mca1DBD-326-697 enhanced Pol II occupancy at the mfc1⁺ locus. To carry out these experiments, we used untagged Mca1 proteins to avoid any interference of the TAP tag (which can bind to the Fc region of IgG) with the monoclonal anti-RNA Pol II antibody (isotype IgG) that was used for immunoprecipitation of Pol II bound to chromatin. mca1∆/mca1∆ cells expressing untagged Mca1, Mca1DBD-299-697, and Mca1DBD-326-697 were synchronously induced into meiosis and treated with either TTM (150 μM) or CuSO₄ (25 μM). Aliquots of cultures were analyzed for their ability to activate mfc1⁺ transcript levels in response to low concentrations of copper. Results showed that mca1∆/mca1∆ cells expressing untagged Mca1 and Mca1DBD-299-697 proteins conferred copper starvation-dependent activation of mfc1⁺ expression in a manner similar to that of TAP-tagged Mca1 and Mca1DBD-299-697 proteins (Figs 5 and 6). mfc1⁺ mRNA levels were induced within 5 h, exhibiting high levels of expression after 7 h of meiotic induction (Fig 6A). In the case of cells expressing an untagged Mca1DBD-326-697 protein, TTM-dependent transcription of mfc1⁺ was strongly reduced by 95% to a minimal level of expression that was similar to that observed in the case of cells containing an empty vector (TTM, 7-h time point) (Fig 6A). In the case of all three untagged alleles (mca1⁺, mca1DBD-299-697, and mca1DBD-326-697) expressed in mca1∆/mca1∆ cells, mRNA levels of mfc1⁺ remained very low under copper-replete conditions (Fig 6A). Given the fact that Mca1 and its mutant derivatives were untagged, we assessed the mRNA levels of the mca1⁺, mca1DBD-299-697, and mca1DBD-326-697 alleles using aliquots of cultures that served to analyze the expression profile of mfc1⁺. Results showed that mca1⁺, mca1DBD-299-697, and mca1DBD-326-697 genes were constitutively expressed as their corresponding transcripts were detected throughout the meiotic program under copper-starved and copper-replete conditions (Fig 6B).

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Fig 6. The central deletion 151–326 of Mca1 affects RNA polymerase II chromatin occupancy at the mfc1⁺ gene under low copper conditions.

A–B, pat1-114/pat1-114 mca1∆/mca1∆ cells harboring either pBPade6 (vector alone), mca1⁺/mca1⁺, mca1DBD-299-697/mca1DBD-299-697 or mca1DBD-326-697/mca1DBD-326-697 alleles were synchronously induced into meiosis. Following induction of meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM), total RNA was isolated at the indicated time points. After RNA preparation, mfc1⁺ (panel A) and mca1⁺ (panel B) steady-state mRNA levels were analyzed by RNase protection assays using actin (act1⁺) as an internal control. Results shown are representative of three independent experiments. C, Schematic representation of the mfc1⁺ gene in which the ChIP2 region indicates locations of the primers that were used for qPCR analysis. The arrow indicates the direction of transcription. Nucleotide numbers refer to the position relative to the A of the initiator codon of mfc1⁺. D–F, At the 1 and 7-h meiotic time points, ChIP analysis was performed to determine RNA Pol II chromatin occupancy at the mfc1⁺ gene. Chromatin was immunoprecipitated using RNA Pol II CTD-specific antibodies. ChIP results are presented as enrichments of mfc1⁺ (+875/+960) coding region relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments. The asterisks correspond to p<0.001 (**) and p<0.0001 (****) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g006

In copper-starved mca1∆/mca1∆ cells expressing Mca1 and Mca1DBD-299-697, association of Pol II with the mfc1⁺ locus was enriched 43.0- and 38.2-fold, respectively, relative to a 18S ribosomal region reference (Fig 6C–6E). However, these levels of Pol II enrichment due to the presence of Mca1 and Mca1DBD-299-697 were 33.2- and 29.5-fold when we took into account that Pol II chromatin occupancy was slightly enriched in TTM-treated cells lacking Mca1 (containing an empty vector) after 7 h of meiotic induction (9.8- and 8.7-fold) (Fig 6D and 6E). In the case of copper-starved mca1∆/mca1∆ cells expressing Mca1DBD-326-697, results showed that Pol II enrichment levels (9.3-fold) were similar to those in mca1∆/mca1∆ cells containing an empty vector (8.9-fold) (Fig 6F). We therefore concluded that Mca1DBD-326-697 interacted with the mfc1⁺ promoter in vivo (Fig 5E). However, this mutant was unable to foster a significant recruitment of Pol II at the mfc1⁺ locus, resulting in low levels of mfc1⁺ mRNA expression even under low concentrations of copper (Fig 6A and 6F). Under copper-replete conditions, results showed that Pol II immunoprecipitated very low or background levels of mfc1⁺ DNA coding region when mca1∆/mca1∆ cells expressed mca1⁺, mca1DBD-299-697 or mca1DBD-326-697 alleles (Fig 6D–6F).

In vivo mapping of a C-terminal region of Mca1 that is required for induction of mfc1⁺ expression

To further delineate a region of Mca1 that was required for transcriptional activation of mfc1⁺ expression under low copper conditions, truncations were created from the C-terminal end of a functional Mca1-TAP protein (Fig 7A). We started with two small deletions due to the fact that the C-terminal end of Zn₂Cys₆ cluster transcription factors are known to be required for transcriptional activity of this group of regulators. The wild type mca1⁺-TAP and mutant mca1 1-600-TAP and mca1 1-570-TAP alleles were returned to pat1-114/pat1-114 mca1∆/mca1∆ cells. Subsequently, these cells were synchronously induced into meiosis and treated with TTM (150 μM) or CuSO₄ (25 μM). At the indicated times after meiotic induction, total RNA was isolated from aliquots of cultures and assayed for copper-dependent regulation of the mfc1⁺ copper-responsive transcript to determine whether the C-terminal end was important for Mca1 function in vivo. In the case of mca1∆/mca1∆ cells expressing Mca1 1-600-TAP under low copper conditions, mfc1⁺ mRNA levels were first detected at 3- and 5-h time points. RNA levels reached a maximum 7 h after meiotic induction, exhibiting a 15-fold up-regulation compared with mfc1⁺ transcript levels observed at the same time point (7 h) under copper-replete conditions (Fig 7B). As a control, a similar expression profile of mRNA levels of mfc1⁺ was observed in mca1∆/mca1∆ cells expressing Mca1-TAP (Fig 7B). In contrast, mca1∆/mca1∆ cells expressing the Mca1 1-570-TAP mutant were unable to mediate a robust activation of mfc1⁺ expression in response to copper starvation. The magnitude of the copper starvation-dependent induction was decreased by 86% and 92% at 5- and 7-h time points, respectively, compared to cells expressing wild-type Mca1-TAP (Fig 7B). In fact, copper-starved mca1∆/mca1∆ cells expressing Mca1 1-570-TAP exhibited a minimal level of mfc1⁺ expression that was similar to that observed in the case of cells containing an empty vector (Fig 7B).

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Fig 7. Deletion of the last 127 amino acid residues of Mca1 abolishes its capacity to activate mfc1⁺ expression under conditions of copper starvation.

A, Schematic representation of TAP-tagged Mca1, Mca1 1–600, and Mca1 1–570 proteins. The amino acid sequence of the indicated protein is numbered relative to its initiator codon. The black arrow indicates the position of the in-frame truncation at the C terminus of Mca1. B, pat1-114/pat1-114 mca1∆/mca1∆ cells expressing TAP-tagged mca1⁺/mca1⁺, mca1 1-600/mca1 1–600, and mca1 1-570/mca1 1–570 alleles or an empty vector (vector alone) were synchronously induced to undergo meiosis under copper-depleted (TTM, 150 μM) and copper-replete (Cu, 25 μM) conditions. Total RNA was isolated from culture aliquots taken at the indicated time points. Steady-state levels of mfc1⁺ and act1⁺ mRNAs were analyzed by RNase protection assays. Results shown are representative of three independent experiments. C–E, ChIP assays were performed from aliquots of cultures used in panel B after 1 and 7 h of meiotic induction. Binding of Mca1-TAP, Mca1 1-600-TAP or Mca1 1-570-TAP to the mfc1⁺ promoter was calculated as the enrichment of the specific mfc1⁺ proximal regulatory region (positions -183 to -32) relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments using separate chromatin preparations. F, Schematic representation of the mfc1⁺ gene in which ChIP1 region indicates the location of the primers that were used for qPCR analysis. Black boxes depict wild-type TCGGCG sequences. G–H, Whole-cell extracts were prepared from aliquots of cultures used in panel B after 1 and 7 h of meiotic induction. Samples were analyzed by immunoblot assays using anti-IgG and anti-α-tubulin antibodies to detect TAP-tagged proteins and α-tubulin steady-state protein levels, respectively.

https://doi.org/10.1371/journal.pone.0201861.g007

To test whether Mca1 1-600-TAP and Mca1 1-570-TAP occupied the mfc1⁺ promoter region, pat1-114/pat1-114 mca1∆/mca1∆ cells expressing wild-type Mca1-TAP and these mutant proteins were synchronized to initiate and proceed through the meiotic program. Immediately prior to their entry into meiosis, the cells were either exposed to TTM (150 μM) or CuSO₄ (25 μM). Aliquots of cultures were retrieved after 1 and 7 h of meiotic induction and ChIP assays were performed to assess the levels of mfc1⁺ promoter occupancy by Mca1-TAP, Mca1 1-600-TAP, and Mca1 1-570-TAP proteins. Results showed that the mfc1⁺ promoter was bound by Mca1 1-600-TAP (2.5- and 2.8-fold after 1 and 7 h in the presence of TTM, and 2.3- and 2.7-fold after 1 and 7 h in the presence of CuSO₄) in a manner similar to that observed for Mca1-TAP (2.5- and 3.0-fold after 1 and 7 h in the presence of TTM, and 2.5- and 2.6-fold after 1 and 7 h in the presence of CuSO₄) (Fig 7C and 7D). In the case of Mca1 1-570-TAP, its enrichment levels were lower than enrichment levels of Mca1-TAP and Mca1 1-600-TAP (Fig 7E). Notably, Mca1 1-570-TAP constitutively associated with the mfc1⁺ promoter, exhibiting enrichment levels of 1.9- and 2.2-fold after 1 and 7 h in the presence of TTM, and 1.9- and 2.0-fold after 1 and 7 h in the presence of CuSO₄ (Fig 7C–7E). The association of Mca1-TAP, Mca1 1-600-TAP, and Mca1 1-570-TAP with the mfc1⁺ promoter was detected using primers amplifying a DNA region located between positions -183 and -32 relative to the initiator codon of mfc1⁺ (Fig 7F). To verify that Mca1-TAP, Mca1 1-600-TAP, and Mca1 1-570-TAP proteins were expressed in pat1-114/pat1-114 mca1∆/mca1∆ cells during the meiotic program, total cell extracts were analyzed by immunoblotting using anti-IgG and anti-α-tubulin antibodies. Results showed that these proteins were present after 1 and 7 h of meiotic induction in the presence of TTM or CuSO₄ (Fig 7G and 7H).

As mentioned previously untagged version of Mca1, Mca1 1–600, and Mca1 1–570 were created to avoid any interference of the TAP tag with the anti-RNA Pol II antibody (IgG-type) that was used to selectively pull down chromatin bound by Pol II. We examined the individual contribution of each version of the transcription factor (Mca1, Mca1 1–600, and Mca1 1–570) in the regulation of mfc1⁺ in the presence of TTM (150 μM) or CuSO₄ (25 μM). Steady-state transcript levels of mfc1⁺ were analyzed 1, 3, 5, and 7 h after meiotic induction. In the presence of TTM, cells expressing mca1⁺ and mca1 1–600 alleles exhibited maximal expression of mfc1⁺ transcripts that was induced 23- and 25-fold, respectively, compared to their levels of expression in copper-treated cells (7-h time point) (Fig 8A). In the case of cells expressing the mca1 1–570 allele, the steady-state levels of mfc1⁺ mRNA were dramatically decreased by 95% under copper-starved (TTM) conditions compared to transcript levels of wild-type Mca1 under the same experimental conditions (7-h time point). Furthermore, the weak levels of mfc1⁺ transcript in this mutant (Mca1 1–570) were similar to those observed in the case of cells transformed with plasmid alone (Fig 8A).

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Fig 8. The C-terminal region of Mca1 from residues 570 to 600 is required for RNA polymerase II chromatin occupancy at the mfc1⁺ gene.

A–B, Cultures of pat1-114/pat1-114 mca1∆/mca1∆ cells expressing either pBPade6 (vector alone), mca1⁺/mca1⁺, mca1 1-600/mca1 1–600 or mca1 1-570/mca1 1–570 alleles were induced to initiate and proceed through meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM). At the indicated times after meiotic induction, total RNA was extracted from culture aliquots and steady-state mRNA levels of mfc1⁺ (panel A) and mca1⁺ (panel B) were analyzed by RNase protection assays using actin (act1⁺) as an internal control. The results shown are representative of three independent experiments. C, Schematic representation of the mfc1⁺ gene in which the ChIP2 region indicates positions of the primers that were used for qPCR analysis. D–F, After 1 and 7 h of meiotic induction, aliquots of the cultures described for panels A and B were taken and chromatin was prepared and immunoprecipitated using antibodies against RNA Pol II CTD. ChIP results are presented as enrichments of mfc1⁺ (+875/+960) coding region relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments. The asterisks correspond to p<0.001 (**) and p<0.0001 (****) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g008

We assayed the levels of mRNA expression of mca1⁺, mca1 1–600, and mca1 1–570 alleles by RNase protection assays to obtain further indication that untagged Mca1, Mca1 1–600, and Mca1 1–570 proteins were expressed in mca1∆/mca1∆ cells. Results showed that wild-type and mutant mca1 constructs were clearly expressed, with transcripts constitutively detected for each indicated allele regardless of cellular copper status (Fig 8B).

To confirm that Mca1 and Mca1 1–600 affected transcription per se, we analyzed Pol II chromatin occupancy at the mfc1⁺ locus (positions +875 to +960) in mca1∆/mca1∆ cells expressing mca1⁺ and mca1 1–600 alleles. Consistent with the levels of mfc1⁺ mRNA that were high under low copper conditions at the 7-h time point, levels of Pol II enrichment were higher in cells that had been exposed to TTM after 7 h. Data showed 38.6- (for mca1⁺) and 40.0-fold (for mca1 1–600) enrichment at the mfc1⁺ locus relative to a 18S ribosomal DNA sequence reference (Fig 8C–8E). In the case of cells expressing the Mca1 1–570 mutant, Pol II enrichment levels were strikingly lower (by 4.5-fold) compared to those observed in mca1⁺ and mca1 1–600 cells under the same low copper conditions after 7 h (Fig 8D–8F). In fact, this decrease in the association of Pol II with the mfc1⁺ locus paralleled a weak expression of mfc1⁺ (Fig 8A). When meiotic cells were incubated in the presence of copper, there was a lack of Pol II chromatin occupancy at the mfc1⁺ locus regardless of the expressed allele (mca1⁺, mca1 1–600, and mca1 1–570) and meiotic time points (Fig 8D–8F). Taken together, these results indicated that a region of Mca1 encompassing amino acid residues 570 to 600 is required for recruitment of Pol II to the locus of mfc1⁺. In contrast, the last 97 residues of Mca1 (600–697) are dispensable for Pol II recruitment to chromatin.

Fusion of amino acid fragments 1–150 and 299–600 of Mca1 are sufficient to trigger proper and timely control of mfc1⁺ expression

Given the results of mfc1⁺ expression under the control of different Mca1 mutants, we hypothesized that residues 299 to 600 of Mca1 were required for transcriptional activation of mfc1⁺ expression. Accordingly, disruption of this region would result in poor expression of mfc1⁺ under copper starvation conditions. To test this hypothesis, we deleted the amino acid region 299 to 600 of Mca1-TAP (Fig 9A). The resulting allele was named mca1 1–298+601-697-TAP. As a complementary approach, a chimeric protein that consisted in a fusion between amino acid fragments 1 to 150 (DBD) and 299 to 600-TAP of Mca1 (denoted mca1DBD-299-600-TAP) was created to test whether it mediated proper and timely control of mfc1⁺ gene expression as a function of copper availability. The experiments were set by expressing mca1⁺-TAP, mca1 1–298+601-697-TAP, and mca1DBD-299-600-TAP alleles into pat1-114/pat1-114 mca1∆/mca1∆ cells that were synchronously induced into meiosis in the presence of TTM (150 μM) or CuSO₄ (25 μM). Aliquots of cell cultures were taken 1, 3, 5, and 7 h after meiotic induction, and steady-state levels of mfc1⁺ mRNA were analyzed by RNase protections assays. Under low copper conditions (TTM), maximal induction of mfc1⁺ was primarily observed in cells expressing mca1⁺-TAP and mca1DBD-299-600-TAP alleles after 7 h of meiotic induction (Fig 9B). In contrast, mRNA levels of mfc1⁺ remained low (96% reduction) in TTM-treated cells expressing mca1 1–298+601-697-TAP or an empty vector (Fig 9B). In the case of all the three indicated alleles expressed in mca1∆/mca1∆ cells, there was a lack of significant induction of mfc1⁺ mRNA under copper-replete conditions (Fig 9B).

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Fig 9. The central region 299–601 of Mca1 is required for copper-dependent regulation of the mfc1⁺ gene.

A, Schematic representation of the Mca1-TAP, Mca1 1–298+601–697, and Mca1DBD–299–600 proteins. The amino acid sequence of the indicated protein is numbered relative to its initiator codon. B, pat1-114/pat1-114 mca1∆/mca1∆ cells expressing TAP-tagged mca1⁺/mca1⁺, mca1 1–298+601-697/ mca1 1–298+601–697, and mca1DBD-299-600/mca1DBD-299-600 alleles or an empty vector (vector alone) were synchronously induced to undergo meiosis under copper-depleted (TTM, 150 μM) and copper-replete (Cu, 25 μM) conditions. Total RNA was isolated from culture aliquots and steady-state levels of mfc1⁺ and act1⁺ mRNAs were analyzed at different time points after meiotic induction. Results shown are representative of three independent RNase protection experiments. C–E, ChIP assays were performed from aliquots of cultures used in panel B after 1 and 7 h of meiotic induction. Binding of Mca1-TAP, Mca1 1–298+601–697-TAP or Mca1DBD–299–600-TAP to the mfc1⁺ promoter was calculated as the enrichment of the specific mfc1⁺ proximal regulatory region (positions -183 to -32) relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments using separate chromatin preparations. F, Schematic representation of the mfc1⁺ gene in which ChIP1 region indicates positions of the primers that were used for qPCR analysis. Black boxes depict wild-type TCGGCG sequences. G–H, Whole-cell extracts were prepared from aliquots of cultures used in panel B after 1 and 7 h of meiotic induction. Samples were analyzed by immunoblot assays using anti-IgG and anti-α-tubulin antibodies to detect TAP-tagged proteins and α-tubulin steady-state protein levels, respectively.

https://doi.org/10.1371/journal.pone.0201861.g009

Despite the fact that there was only copper starvation-dependent induction of mfc1⁺ expression in cells expressing Mca1-TAP and Mca1DBD-299-600-TAP, we tested whether Mca1-TAP, Mca1 1–298+601-697-TAP, and Mca1DBD-299-600-TAP could bind to the mfc1⁺ promoter in vivo under both low and high concentrations of copper. Results of ChIP analysis showed that in the case of cells treated with TTM after 7 h of meiotic induction, Mca1-TAP, Mca1 1–298+601-697-TAP, and Mca1DBD-299-600-TAP occupied the mfc1⁺ promoter with 2.8-, 2.3- and 3.0-fold enrichments, respectively, relative to a control 18S ribosomal DNA reference (Fig 9C–9E). Levels of TAP-tagged Mca1, Mca1 1–298+601–697, and Mca1DBD-299-600 enrichments displayed a slight increase at later time point (7 h) compared to earlier time point (1 h) (Fig 9C–9E). We subsequently assessed whether copper affected Mca1-TAP, Mca1 1–298+601-697-TAP, or Mca1DBD-299-600-TAP ability to interact with the mfc1⁺ promoter in vivo. Under copper-replete conditions, results showed that the association of Mca1-TAP, Mca1 1–298+601-697-TAP, and Mca1DBD-299-600-TAP with the mfc1⁺ promoter was respectively detected with 2.3-, 1.6-, and 2.4-fold enrichment after 7 h of meiotic induction relative to a control 18S ribosomal DNA reference (Fig 9C–9E). These results revealed that only a slight decrease of chromatin occupancy by Mca1-TAP, Mca1 1–298+601-697-TAP, and Mca1DBD-299-600-TAP at the mfc1⁺ promoter region was observed in copper-treated cells. We also noticed that the association of Mca1 1–298+601-697-TAP with the mfc1⁺ promoter was weaker than Mca1-TAP or Mca1DBD-299-600-TAP under both copper-limiting and copper-replete conditions. Immunoprecipitated chromatin by Mca1-TAP and its mutant derivatives was detected using primers amplifying a DNA region located between positions -183 and -32 relative to the initiator codon of mfc1⁺ (Fig 9F). To ensure that Mca1-TAP, Mca1 1–298+601-697-TAP, and Mca1DBD-299-600-TAP proteins were synthesized when expressed in pat1-114/pat1-114 mca1∆/mca1∆ cells during the meiotic program, whole cell extracts were analyzed by immunoblot assays using anti-IgG and anti-α-tubulin antibodies. Results showed that these proteins were present after 1 and 7 h of meiotic induction in the presence of TTM or CuSO₄ (Fig 9G and 9H).

Untagged mca1⁺, mca1 1–298+601–697, and mca1DBD-299-600 alleles were expressed in pat1-114/pat1-114 mca1∆/mca1∆ cells to allow analysis of Pol II chromatin occupancy at the mfc1⁺ locus. This strategy was used to avoid any interference from the TAP tag with respect to the enrichment of chromatin-bound Pol II to Protein G Sepharose beads. Before entry into meiosis, cells were exposed to TTM (150 μM) or CuSO₄ (25 μM). Aliquots of cultures were retrieved at distinct time points after meiotic induction, and steady-state levels of mfc1⁺, mca1⁺, and act1⁺ mRNAs analyzed by RNase protection assays. In the case of cells expressing untagged mca1⁺ and mca1DBD-299-600 alleles, mfc1⁺ transcripts showed an expression profile similar to that observed in the case of cells transformed with mca1⁺-TAP and mca1DBD-299-600-TAP (Figs 9B and 10A). Under copper-limiting conditions, mfc1⁺ mRNA levels were strongly induced after 5 and 7 h of meiotic induction (Fig 10A). Conversely, copper supplementation resulted in strongly reduced mfc1⁺ mRNA levels in comparison to those observed with cells grown under low copper conditions (Fig 10A). In cells expressing Mca1 1–298+601–697 and an empty vector, transcript levels of mfc1⁺ remained low, irrespective of cellular copper status (Fig 10A). We next analyzed mca1⁺ mRNA levels of cells expressing the indicated mutant alleles as a function of copper availability. Results showed that untagged mca1⁺, mca1 1–298+601–697, and mca1DBD-299-600 alleles were expressed and their steady-state mRNA levels were constitutive and unresponsive to cellular copper status (Fig 10B).

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Fig 10. The central segment of Mca1 from amino acids 299–600 is required for RNA polymerase II chromatin occupancy at the mfc1⁺ gene.

A–B, Cultures of pat1-114/pat1-114 mca1∆/mca1∆ cells expressing an empty plasmid (vector alone), mca1⁺/mca1⁺, mca1 1–298+601-697/ mca1 1–298+601–697, or mca1DBD-299-600/mca1DBD-299-600 alleles were synchronously induced to undergo meiosis in the presence of TTM (150 μM) or copper (Cu, 25 μM). At the indicated times after meiotic induction, total RNA was extracted from culture aliquots and steady-state mRNA levels of mfc1⁺ (panel A) and mca1⁺ (panel B) were analyzed by RNase protection assays using actin (act1⁺) as an internal control. The results shown are representative of three independent experiments. C, Schematic representation of the mfc1⁺ gene in which the ChIP2 region indicates positions of the primers that were used for qPCR analysis. D–F, At the 1 and 7-h meiotic time points, aliquots of the cultures described for panels A and B were taken and chromatin was prepared and immunoprecipitated using antibodies against RNA Pol II CTD. ChIP results are presented as enrichments of mfc1⁺ (+875/+960) coding region relative to a 18S ribosomal DNA coding region. Data were calculated as values of the largest amount of chromatin measured (fold enrichment). Results are shown as the averages ± S.D. of a minimum of three independent experiments using separate chromatin preparations. The asterisks correspond to p<0.001 (**) and p<0.0001 (****) (paired Student’s t-test).

https://doi.org/10.1371/journal.pone.0201861.g010

To determine whether the levels of Pol II occupancy at mfc1⁺ followed transcriptional activation of mfc1⁺ by Mca1 and Mca1DBD-299-600, we probed Pol II chromatin occupancy using primers covering a coding region of mfc1⁺ (positions +875 to -960) (Fig 10C). These experiments were performed in mca1∆/mca1∆ cells expressing untagged mca1⁺, mca1 1–298+601–697, and mca1DBD-299-600 alleles. Under copper-limiting conditions, results of ChIP analysis showed that Pol II enrichment levels were consistently higher in cells expressing mca1⁺ (38.9-fold) and mca1DBD-299-600 (34.8-fold) than those observed in cells expressing mca1 1–298+601–697 (6.1-fold) after 7 h of meiotic induction (Fig 10D–10F). In contrast, there was no significant Pol II enrichment in cells expressing mca1⁺, mca1 1–298+601–697, and mca1DBD-299-600 alleles under copper-replete conditions (Fig 10D–10F). Results of chromatin immunoprecipitation experiments of mca1∆/mca1∆ cells containing an empty vector showed only background levels of Pol II bound to a mca1⁺ transcribed region (Fig 10D–10F). Taken together, these results revealed that the central segment of Mca1 that comprised amino acid residues 299 to 600 is required for Pol II-mediated maximal induction of mfc1⁺ expression in copper-limited meiotic cells.