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1 Institute of Microbiology, University Hospital Lausanne, Rue du Bugnon 48, CH-1011 Lausanne, Switzerland
2 Comenius University, Faculty of Natural Sciences, Department of Microbiology and Virology, 842 15 Bratislava, Slovak Republic
Correspondence
Dominique Sanglard
Dominique.Sanglard{at}chuv.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position –296 to –260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H2O2 response element (HRE), is situated at position –561 to –520. The HRE is required for H2O2-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H2O2-dependent transcription of MDR1. A minimal BRE (–290 to –273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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). The azole class of antifungals (for example fluconazole) is widely used to treat C. albicans infections. Fluconazole-resistant strains have emerged, especially in HIV-positive patients with recurrent candidiasis who have undergone prolonged fluconazole therapy (Ghannoum et al., 1996; Law et al., 1994; Marr et al., 2000; Rex et al., 1995). The molecular mechanisms proposed to explain azole resistance are diverse: (i) the cellular content of the azole target Erg11p (ergosterol biosynthesis), a 14
-lanosterol demethylase, can be elevated, (ii) the affinity of Erg11p for azole antifungal agents can be decreased, (iii) the ergosterol biosynthetic pathway can be altered by inactivation of the sterol 
5,6-desaturase and (iv) cells can fail to accumulate these agents due to the upregulation of multidrug transporters belonging to the ABC transporters and the major facilitator (MF) families. Some azole-resistant strains upregulate the ABC transporters CDR1 (Candida drug resistance) and CDR2, while others upregulate MF MDR1 (multidrug resistance 1) (Sanglard & Odds, 2002; White et al., 1998).
Overexpression and gene inactivation studies have shown that MDR1 confers specific resistance to fluconazole, benomyl, cycloheximide, benzotriazole, methotrexate, 4-nitroquinoline-N-oxide (4-NQO) and sulfomethuron methyl (Ben-Yaacov et al., 1994; Fling et al., 1991; Goldway et al., 1995; Wirsching et al., 2000b). Disruption of MDR1 causes an increased susceptibility to methotrexate, cycloheximide and 4-NQO (Goldway et al., 1995; Sanglard et al., 1996). However, its inactivation does not increase susceptibility to benomyl, suggesting that other mechanisms are involved in this resistance (Goldway et al., 1995). MDR1 is usually not expressed at detectable levels in fluconazole-susceptible C. albicans isolates but its constitutive upregulation in some fluconazole-resistant isolates reveals a correlation between azole resistance and overexpression of MDR1 (Franz et al., 1998; Sanglard et al., 1995; White, 1997; White et al., 1997).
Little is known about the transcriptional regulation of MDR1 in C. albicans. However, a dramatic transient upregulation of MDR1 can be observed in azole-susceptible strains after treatment with toxic hydrophobic agents (benomyl, methotrexate, 4-NQO, o-phenanthroline), oxidizing agents [diamide, H2O2, tert-butyl hydrogen peroxide (T-BHP) and diethylmaleate] and the alkylating agent methyl methanesulfonate (Gupta et al., 1998; Harry et al., 2005). Clinical isolates with increased MDR1 expression have not undergone amplification of the MDR1 gene (Wirsching et al., 2000a). Sequence analysis of MDR1 promoters from clinical isolates compared to wild-type promoters did not reveal cis-acting mutations (Harry et al., 2005; Wirsching et al., 2000a). Thus MDR1 upregulation in clinical strains is most likely due to transcriptional activation resulting from mutations of factors acting in trans (Wirsching et al., 2000a). Deletion analysis of the MDR1 promoter, performed with a luciferase reporter gene, suggested that regulatory functions could be assigned to two separate regions: an element required for induction by benomyl was assigned to the region between –399 and –299 upstream of the start codon and another region located between –601 and –500 was responsible for MDR1 induction by the oxidative agent T-BHP.
In Saccharomyces cerevisiae, drug resistance is also mediated by membrane transport proteins belonging to the ABC transporter and major facilitator superfamilies and the different mechanisms controlling the expression of these multidrug resistant genes are relatively well understood (Sa-Correia & Tenreiro, 2002; Taglicht & Michaelis, 1998). The homologue of MDR1, FLR1, confers resistance to multiple drugs such as fluconazole, 4-NQO, cycloheximide, methotrexate and benomyl (Alarco et al., 1997; Broco et al., 1999; Nguyen et al., 2001; Tenreiro et al., 2001). Induction of FLR1 expression by the oxidizing agents H2O2, diamide, T-BHP and diethylmaleate, as well by the alkylating agent methyl methanesulfonate (Broco et al., 1999; Jelinsky & Samson, 1999; Nguyen et al., 2001), is mediated by Yap1p (also called Pdr4p or Snq3p), a basic leucine zipper (bZip) transcription factor (Nguyen et al., 2001; Tenreiro et al., 2001). The direct targets of Yap1p, principally genes involved in oxidative stress, have a common promoter element, the palindromic YRE (Yap1p response element: TTAC/GTAA) (Fernandes et al., 1997; Kuge & Jones, 1994; Toone & Jones, 1999).
In C. albicans, Cap1p, a bZip transcription factor structurally and functionally related to S. cerevisiae Yap1p, has been implicated in multidrug resistance and oxidative stress resistance (Alarco & Raymond, 1999; Wang et al., 2006; Zhang et al., 2000). It was also shown to confer fluconazole resistance in S. cerevisiae by transcriptionally activating FLR1 (Alarco et al., 1997), suggesting it has the same sequence specificity as Yap1p. Conversely, the C. albicans MDR1 promoter harbours the putative Cap1p-binding site (TTAGTAA at –532). Together these data indicate that mechanisms regulating MDR1 and its S. cerevisiae orthologue may be grossly similar. However, while in S. cerevisiae inactivation of Yap1p abolished FLR1 transcription (Tenreiro et al., 2001), Alarco & Raymond (1999) showed that disruption of CAP1 in the azole-resistant strain FR2 did not suppress but rather increased the level of MDR1 expression. In C. albicans Cap1p may act as both a repressor and an activator of MDR1 transcription, since experiments performed by Harry et al. (2005) demonstrating the importance of the YRE in the T-BHP response suggest that Cap1p is an activator of MDR1 expression in response to oxidative stress.
In this study, we used a reporter system to explore the transcriptional regulation of MDR1 in azole-resistant and azole-susceptible strains. The system has identified two different cis-acting regulatory elements and has permitted us to locate them at high resolution. The first, located between –296 and –260, was responsible for dramatic MDR1 upregulation in an azole-susceptible strain exposed to benomyl as well as for high constitutive expression in an azole-resistant strain. The second, which harbours two putative binding sites for Cap1p, is located between –561 and –520. Like the trans-acting Cap1p, this cis-acting element was not required for high constitutive expression of MDR1 in an azole-resistant strain. However, the response of MDR1 to H2O2 was dependent upon the presence of both Cap1p and its binding site within the MDR1 promoter. The benomyl-dependent response was also partially dependent upon the presence of Cap1p, raising the possibility that interactions may occur between complexes bound at the two regulatory elements.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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. Strains were grown either in complete medium YEPD [1 % Bacto peptone (Difco), 0.5 % yeast extract (Difco) and 2 % glucose (Fluka)] or in minimal medium YNB [Yeast Nitrogen Base (Difco) and 2 % glucose (Fluka)]. For solid media, 2 % agar (Difco) was added to both media. Escherichia coli DH5 (Hanahan, 1985) was used as a host for plasmid construction and propagation. DH5 was grown in Luria–Bertani (LB) broth or LB agar plates supplemented with ampicillin (0.1 mg ml–1) when required.
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Construction of promoter–lacZ fusions.
To generate MDR1–lacZ and CDR2–lacZ fusions used in drug-induction assays, the 999 bp MDR1 and 903 bp CDR2 promoters were first amplified by PCR using the Taq DNA polymerase (Roche), with primers MDR1-5'-999-KPN and MDR1-3'-PST or primers CDR2-KPN and CDR2-PST (Table 3). The generated fragments were cloned into pAU36 (Uhl & Johnson, 2001) previously digested with KpnI and PstI. The resulting plasmids (pBR1 and pDS295, respectively) were digested with either NruI for the MDR1–lacZ fusion or SnaBI for the CDR2–lacZ fusion prior to transformation into C. albicans, resulting in their integration at the MDR1 and CDR2 loci, respectively.
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). The cassette was prepared in a two-step process. C. albicans genomic DNA was used as a PCR template to amplify two distinct 500 bp fragments that overlap by the presence of the NheI restriction site. In this step, the external primers UTR3-5' and UTR5-3' were used with internal primers UTR3-3'bis and UTR5-5'bis (Table 3
) with the Expand High Fidelity system (Roche). The two purified PCR fragments were then used as a PCR template with external primers (UTR3-5' and UTR5-3'). The resulting product was next introduced into the BamHI site of pAU36 to generate pBR4. The 5' deletions of the promoter region were generated by PCR using the reverse primer MDR1-3'-PST and the different forward primers listed in Table 3. The resulting fragments were inserted at the KpnI and PstI sites of pBR4 to generate the plasmids listed in Table 3. For the construction of internal deletions and transversions within the MDR1 promoter, a similar two-step PCR strategy was used. First, external primers MDR1-5'-598-KPN and MDR1-3'-PST were used with variable primers designed for specific deletions and transversions to generate fragment pairs with a 20 bp overlap region (Table 4). After purification, the overlapping fragment pairs were used as templates with external primers to reconstitute MDR1 promoter derivatives. The fragments obtained were cloned into the KpnI and PstI sites of pBR4 to generate the plasmids listed in Table 4. All plasmids generated were linearized by NheI and their correct integration as a single copy at the MDR1 locus was verified by Southern blotting (see also Fig. 1b).
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). The resulting PCR fragments were digested with XbaI and PstI, then inserted into compatible sites of pMM43 to generate pBR101 and pBR108. The plasmid pMM43 (de Micheli et al., 2002) contains the RLUC reporter system and 198 bp of the CDR2 promoter but lacks the drug-responsive element (DRE), an element required for CDR2 upregulation. This plasmid was used as a negative control in luciferase reporter assays. As a positive control, the promoter of MDR1, 598 bp, was first amplified by PCR from plasmid pBR6 using primers MDR1-5'-598-KPN and MDR1-3'-PST. Digestion of the PCR product by XbaI and PstI generated a fragment of 592bp (–592 to +1) due to the presence of a XbaI restriction site at position –592 of the promoter. This fragment was inserted into the compatible sites of pMM43 to obtain pBR99.
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DNA amplification by PCR.
DNA fragments used in cloning steps or as probes were generated by PCR with the Taq DNA polymerase (Roche) following the instructions of the manufacturer. The reaction mixture (final volume 100 µl) was composed of 10 µl of 10x Taq DNA polymerase buffer (Roche), 10 µl of MgCl2 (Roche), 1 µl of each 100 µM forward and reverse primer, 4 µl of 10 mM deoxynucleoside triphosphates (Roche), 10 U of Taq DNA polymerase and 1 µl of 0.5–1 µg µl–1 genomic DNA. PCR was performed under the following conditions: one cycle for 4 min at 94 °C, 1 min at 54–60 °C and 2 min at 72 °C; 30 cycles for 30 at 94 °C, 1 min at 54–60 °C and 2 min at 72 °C followed by one terminal cycle for 10 min at 72 °C. After analysis of 1/10 vol. of PCR products by electrophoresis on 1 % agarose gel, DNA fragments were purified with the QIAquick PCR purification kit (QIAGEN) and resuspended in 50 µl sterile water.
Southern blotting.
Southern blots were carried out as described previously (Sanglard et al., 1999). DNA probes used in this study were generated by PCR using the following pairs of primers: for the verification of genomic insertion of pMM43-derived plasmids at the LEU2 genomic locus, CALEU2-3 (5'-CACCACCACTAAAACCCCAACAAATCA-3') and CALEU2-5 (5'-ATGTCTGTTAAAACCCAAACCATTACT-3'); for insertion of pAU36-derived plasmids at the MDR1 locus, MDR1-5'-501-KPN (5'-GCGCAAAGGTACCCAATTTTCATTTTAGGAAATTTACCGAGTTT-3') and LacZ-rev (5'-ATGTCTGTTAAAACCCAAACCATTACT-3'). Radioactive DNA probes were generated by random priming using the Megaprime DNA labelling kit (Amersham). Radioactive signals were revealed by exposure to Kodak BioMax MR film (Amersham Biosciences).
Electrophoretic mobility shift assays (EMSAs).
Protein extracts from C. albicans strain CAF2-1 were prepared from 2 l cultures in YEPD grown to mid exponential phase until the density reached 1.5x107 cells ml–1. An equal quantity of cells was exposed to benomyl (35 µg ml–1) for 30 min or left untreated as a control. The benomyl-treated or control cells were centrifuged for 20 min at 5500 r.p.m. and each cell pellet was washed with 10 ml extraction buffer [400 mM (NH4)2SO4, 10 mM MgCl2, 1 mM EDTA, 10 % (v/v), glycerol, 7 mM mercaptoethanol, 200 mM Tris/HCl pH 8]. Washed cells were centrifuged for 10 min at 5500 r.p.m. and the pellet obtained was resuspended in 10 ml extraction buffer supplemented with a protease inhibitor mix (Roche). Glass beads (10 g) were added to the cell suspension and the resulting mixture was disrupted for 5 min at 4 °C using a Macro bead-beater (Biospec Products). The homogenized solution was kept on ice for 30 min before centrifugation at 4 °C for 30 min at 13 000 r.p.m. The supernatant was transferred to a new tube and the cell pellet was reextracted with 5 ml fresh extraction buffer. The two supernatants were combined and the resulting solution was centrifuged for 1 h at 13 000 r.p.m. at 4 °C. The supernatant was recovered and ammonium sulfate was added to a final concentration of 0.3 g ml–1. After dissolution of ammonium sulfate, the protein extract was centrifuged for 10 min at 13 000 r.p.m. and 4 °C. Precipitated proteins were resuspended in 250 µl protein buffer (20 mM HEPES, 5 mM EDTA, 7 mM mercaptoethanol, 1 mM PMSF and 20 % glycerol) supplemented with protease inhibitors (Roche). Protein concentrations of the crude extracts were determined using the Bradford method.
Double-stranded oligonucleotide probes were prepared by annealing 5' end labelled oligonucleotides with their complementary strands. The sequences of the probes are described in Figs 7 and 8. Single-strand oligonucleotides (5 pmol) were incubated at 37 °C for 45 min with 10 U T4 polynucleotide kinase (Roche) and 40 µCi (1.48 MBq) [
-32P]ATP (3000 Ci mmol–1, 10 µCi µl–1) in a final volume of 10 µl. The excess of [-32P] ATP was eliminated using the Nucleotide Removal kit (Qiagen). The last elution step was performed with 100 µl of sterile water. After addition of the unlabelled oligonucleotides, the annealing of the complementary strands was performed by progressive temperature reduction after a 3 min treatment at 95 °C.
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Measurement of -galactosidase reporter activities.
Cells were grown overnight in 3 ml YEPD at 30 °C under constant agitation. Cultures were then diluted in 5 ml fresh medium to obtain a cell density of approximately 5x106 cells ml–1 and were regrown under agitation until the density reached 1.5x107 cells ml–1. At this point, 1 ml aliquots from cultures that were not exposed to drugs (azole-resistant strains FR2 or MMY412) were centrifuged at 4 °C for 5 min at 5500 r.p.m. Exposure of other strains (azole-susceptible strains DSY449 and MMY411) to different drugs was carried out for defined time periods at 30 °C with constant agitation. After drug exposure, cell density was measured and cultures were centrifuged at 4 °C and 5500 r.p.m. for 10 min. The cell pellets were washed with 1 vol. Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4) and centrifuged at 4 °C for a further 10 min. The washed cell pellets were resuspended in 150 µl Z buffer supplemented with 38.6 mM mercaptoethanol. After addition of 50 µl chloroform and 20 µl 0.1 % SDS, cells were disrupted by vortexing. To each aliquot was added 700 µl ONPG (1 mg ml–1; Sigma) in Z buffer supplemented with mercaptoethanol and preheated at 30 °C. The mixtures were incubated at 37 °C until they developed a yellow colour (20 min to 3 h). The enzyme was inactivated by the addition of 500 µl 1 M Na2CO3. The mixtures were centrifuged at 13 000 r.p.m. for 10 min and the OD420 was read. The -galactosidase activities were calculated using the following equation: -galactosidase activity (Miller units)=(1000xOD420)/(txvxOD600), where t is the time of reaction expressed in min and v is the volume of culture used in the assay expressed in ml.
Measurement of Renilla luciferase reporter activities.
Cells were grown overnight in 3 ml YEPD at 30 °C under constant shaking, then diluted in 5 ml fresh medium to obtain a cell density of approximately 1.5x107 cells ml–1. Benomyl (35 µg ml–1) was added to the cell suspensions and cultures were incubated with constant shaking at 30 °C for a further 30 min. After drug exposure, cells were centrifuged for 10 min at 5500 r.p.m. The pellet was washed twice with 1 ml cold sterile water followed by 1 ml cold RLUC buffer (0.5 M NaCl, 0.1 M K2HPO4, 1 mM EDTA, 0.6 mM sodium azide). The final pellet was resuspended in 200 µl cold RLUC buffer supplemented with protease inhibitors (Roche) and an equal volume of chilled acid-washed glass beads was added. The cells were disrupted at 4 °C for 30 s using a Minibead beater (Biospec Products) and then centrifuged at 4 °C for 10 min at 13 000 r.p.m. The supernatants containing soluble proteins were stored at –20 °C or used directly for measurement of luciferase activities.
The relative luciferase activities of the different clones were measured using the Dual-Luciferase kit (Promega). Five microlitres of the crude protein extracts was added to 25 µl LAR II buffer according to the recommendations of the manufacturer and firefly luciferase activities were measured using a Lumat LB 9507 luminometer (E. G. and G. Berthold). Twenty-five microlitres of freshly prepared STOP and GLO buffer was then added to the reactions and Renilla luciferase activities were measured. Specific luciferase activities, expressed in Renilla luciferase units [RLU; i.e. U (µg protein)–1] were calculated after determination of the protein concentration of cell extracts by the Bradford method.
Statistical analysis.
For the analysis of promoter deletions, a mathematical model was fitted using non-linear least squares. The choice of the model was based on the observation that, starting from a maximal response (H1 in the equation), the response decreases to a minimum level (H0) over a very short interval of length. The centre of this interval (A) represents the critical length for loss of response, while the width of this interval indicates the abruptness of the change (the slope B). The final equation was y=H0+(H1–H0)/{1+exp[(A–x)/B]}, where y is the response and x the promoter length. For the mutant strains (cap1/), the same equation was used with parameters written in lower case (h0, h1, a, b). The fit of these equations contained eight parameters. The differences between the parameters determined for the wild type strain and the corresponding mutant were next tested using the generalized F test.
Comparisons between two sets of values (e.g. promoter bearing transversion or deletion versus wild type promoter) were performed using the nonparametric Mann–Whitney test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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). The promoter fusion (MDR1–lacZ) was integrated in the azole-susceptible mutant strain DSY449 (cdr1/) at the MDR1 locus as described in Methods. We chose to use this strain because the absence of CDR1 may reduce drug efflux and thereby enhance the sensitivity of drug-dependent MDR1 activation.
Upregulation of MDR1 can occur in C. albicans azole-resistant strains or in azole-susceptible strains exposed to diverse drugs including benomyl and H2O2 (Gupta et al., 1998; Harry et al., 2005). Therefore we tested the response of the reporter to a panel of compounds known as inducers of MDR1. In order to test the MDR1 response specificity, we also tested the response to oestradiol and fluphenazine, compounds known to induce CDR2 (de Micheli et al., 2002). For this purpose, a CDR2–lacZ reporter system was integrated in DSY449 in the same fashion as for the MDR1–lacZ construct. The relative increase of MDR1–lacZ and CDR2–lacZ reporter activities after treatment of C. albicans with these compounds is shown in Table 6. Upon exposure to benomyl, H2O2 or diamide, MDR1–lacZ reporter activities increased more than 10-fold as compared to untreated conditions, while no increase was observed for the CDR2–lacZ promoter fusion. In contrast, oestradiol and fluphenazine treatment specifically increased CDR2–lacZ reporter activities (by approximately 100-fold). Thus, the response of the two reporter genes to five different agents phenocopies the effects on the endogenous MDR1 and CDR2 genes. Exposure of C. albicans to methotrexate, sulfomethuron methyl and phenanthroline had no influence on transcription of either reporter. Based on these results, responses to the hydrophobic compound benomyl and to the oxidizing agent H2O2 were selected as readouts for the functional dissection of the MDR1 promoter. As the chemical properties of these drugs are different, we reasoned that they might reveal different modes of activation of the MDR1 promoter.
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). Correct integration was verified by Southern blot analysis (Fig. 1b).
A single transformant containing each deletion construct was chosen for testing of reporter activity in the absence and presence of benomyl. As shown in Fig. 2(a), the reporter under control of the proximal 1459 bp of the MDR1 promoter exhibited a 26.1±4.4-fold increase of activity upon exposure to benomyl. Progressive deletions (99 to 141 bp) from –1459 to –302 (with respect to the ATG start codon) did not significantly alter the relative increase of reporter activity due to benomyl exposure. However, deletion of the region between –302 and –201 resulted in a 10-fold decrease in benomyl-dependent reporter activity, indicating that the minimal domain required for dramatic benomyl-dependent upregulation must lie within this region. This element was named BRE (for benomyl response element). To further delimit the BRE, small deletions of the region between nucleotides –302 and –222 were generated (Fig. 2b). Progressive 4 to 7 bp (–302 to –279) and 18 to 20 bp deletions (–279 to –222) revealed that benomyl-dependent MDR1 transcription was significantly lowered by removal of sequence downstream of –296. The benomyl-dependent increase in reporter activity was 22.9±3.1-fold for the construct containing the MDR1 promoter region terminating at –296. It decreased to 4.7±0.7- and 6.4±1.1-fold for constructs with promoters terminating at –290 and –286, respectively. A further decline of relative reporter activity to 2.1±0.5-fold at –279 was also observed. These data indicate that the 5' extremity of the BRE lies between –296 and –279. A residual twofold increase in benomyl-dependent transcription appears to be independent of the BRE and suggests that the basal MDR1 promoter can still respond to the drug.
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). Progressive 18 to 21 bp deletions of the promoter region between –598 and –520 (Fig. 3b) located the H2O2-dependent regulatory element between –561 and –520: the relative increase of reporter activity observed under H2O2 exposure decreased from approximately 13.3±1.1-fold at –561 to 4.8±0.4- and 2.4±0.2-fold at –540 and –520, respectively. We have called the cis-acting element thus identified HRE (for H2O2 response element). The location of this element clearly distinguishes it from the BRE described above. Interestingly, two sequences matching the consensus of Cap1p-binding sites (TTAG/CTAA) were located within the HRE, raising the possibility that Cap1p, a bZip transcription factor involved in oxidative stress, might regulate H2O2-dependent expression of MDR1 by binding to this element.
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). In general, the specific -galactosidase activities measured in this strain (Fig. 4) with MDR1 promoter lengths between –999 and –302 [mean 58.2±5.5 Miller units (U)] were significantly higher than those in strain DSY449 upon its exposure to benomyl (mean 4.7±1.7 U). Moreover, reporter activities were not significantly altered when FR2 cells were exposed to benomyl (data not shown). As shown in Fig. 4(a), reporter activities were not significantly altered by promoter deletions ranging from –999 to –302, thus indicating that the cis-acting HRE located between –561 and –520 was not required for constitutive high expression of MDR1 in FR2. In contrast, removal of the promoter sequences between –302 and –201 resulted in a more than 10-fold decrease in reporter activity. Interestingly, upon progressive shortening of the MDR1 promoter from –296 to –279 (Fig. 4b), the data indicate a stepwise decrease in reporter activities: a first significant decrease between –296 (56.1±3.9 U) and –290 (40.8±4.8 U) and a second significant decrease between –286 (36.7±2.1 U) and –279 (2.6±0.2 U). This stepwise decline in reporter activities is reminiscent of that observed for the same promoter deletions in strain DSY449 exposed to benomyl (Fig. 2b), suggesting that both benomyl-induced transcription and constitutive high expression of MDR1 may be under control of the same functional subelements within the BRE.
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). For transversions T1 to T3, the residual increase in reporter activity in benomyl-treated cells as compared to the full-length promoter (22.1±2.6 fold) was between 1.4±0.02- and 2.0±0.3-fold, similar to that of constructs under control of the basal promoter (Fig. 2, promoter deletions –201 and –98). The effect of transversion T4 was also significant but less pronounced. Benomyl treatment still caused a 6.4±2.2-fold increase in the activity of this reporter, suggesting that the BRE retained some functional capacity. Consistent with these results, the deletion of the region containing the BRE (D1: –291 to –269, ) also abolished the increase of reporter activity in response to benomyl (Fig. 5b). The relative increase of reporter activity was 1.3±0.2-fold with internal deletion D1 compared with 29.0±2.7-fold with the full-length MDR1 promoter. Smaller internal deletions within this region (D2: –295 to –287; D3: –286 to –278; and D4: –277 to –269) also decreased benomyl-induced reporter activities as compared to the full-length promoter, but did not completely abolish the response. The promoter carrying deletion D2 (–295 to –287) still showed a 17.1±5.5-fold increase in relative reporter activity in response to benomyl treatment, while the promoters carrying deletions D3 and D4 retained a less pronounced response to benomyl treatment (10.0±2.5-fold and 10.7±1.5-fold increase, respectively). The results confirmed that the BRE delimited by successive 5' promoter deletion analysis (–296 to –276) is essential for the benomyl-dependent response of MDR1. It is however interesting to note that transversions T2 and T3 had significantly more severe effects on benomyl-dependent transcriptional activity than did the deletions D2 and D3.
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). The constitutive activities of MDR1 promoters bearing internal deletions D1, D2, D3 and D4 were respectively 3.5-, 1.7-, 1.7- and 2.5-fold lower than for the MDR1 promoter that had no internal deletion (Fig. 5b). As previously noted for benomyl-dependent reporter activity, partial transversions of the BRE tended to have more severe effects than the corresponding deletions. However in this case, it was transversions T3 and T4 that had significantly stronger effects than deletions D3 and D4. These results demonstrate that the region containing the BRE also plays a role in the high constitutive expression of MDR1.
The partial retention of reporter activity in constructs lacking parts of the BRE may suggest that this regulatory region is composed of several discrete functional domains but we cannot rule out the involvement of other compensatory mechanisms. Moreover, while 5' deletions only delimit the 5' extremity of regulatory elements (i.e. –296, see Fig. 2b), the results presented above aid the localization of central elements of the BRE. Deletions D2 (–295 to –287), D3 (–286 to –278) and D4 (–277 to –269) all led to a decrease of benomyl-dependent MDR1 response and of reporter activity in strain FR2, thus indicating that they all contain nucleotides important for the functionality of the BRE. Since these three deleted regions are sequential in the MDR1 promoter, it can be concluded that at least all the nucleotides of the central deleted region D3 (–286 to –278) are part of the BRE.
Cap1p, a bZip transcription factor, acts as a trans-acting factor of MDR1 regulation
The presence of two Cap1p-like binding sites within the cis-acting HRE (TTAG/CTAA; at –549 and –532) suggested that Cap1p, a bZip transcription factor involved in oxidative stress, could play a role in regulating H2O2-dependent MDR1 expression by binding to these sequences. To further explore this hypothesis, promoter deletion analysis was undertaken in MMY411 (cap1/), a strain derived from DSY449 that lacks CAP1. For all promoter fragments tested, the reporter activities obtained in the absence or presence of H2O2 treatment were not significantly different in this strain. The relative levels of reporter activity after H2O2 exposure were between 0.8±0.1- and 1.7±0.6-fold higher than that recorded when the strain was not treated with H2O2 (Fig. 3a, black columns). Thus, H2O2-dependent expression is essentially completely lost in the cap1/ strain irrespective of whether the HRE is present. Therefore Cap1p is likely to be the main positive regulator involved in the response of the MDR1 promoter to H2O2. We also examined the benomyl-dependent response of reporters under control of MDR1 promoter fragments in the cap1/ strain. Inactivation of CAP1 did not prevent the different reporter constructs from responding to benomyl. Reporter constructs carrying at least 302 nucleotides of the proximal MDR1 promoter showed a benomyl-dependent increase in activity (13.0±5.1-fold, black columns Fig. 2a). However, since the level of the responses was generally twofold lower than that recorded in the corresponding wild-type strain (25.9±5.3-fold, white columns Fig. 2a), we cannot exclude the possibility that Cap1p plays a minor role in mediating the transient response to benomyl. Finally, we observed no significant differences between the specific reporter activities measured in the azole-resistant strain FR2 (58.2±5.5 U) and the corresponding cap1/ mutant MMY412 (51.3±7.5 U, Fig. 4a), indicating that Cap1p is not necessary for constitutive high expression of MDR1 in FR2.
Importance of the cis-acting HRE for MDR1 regulation
As the response of MDR1 to benomyl was partially reduced in the strain lacking CAP1, the deletion of a region (–552 to –521) within the HRE (delimited between –561 and –520) was performed in the full-length MDR1 promoter and reporter activities were measured under benomyl exposure in DSY449. As shown in Fig. 6(a), deletion of the region between –552 and –521 within the full-length MDR1 promoter did not alter the response to benomyl: the increase in reporter activity after benomyl exposure (25.7±5.0-fold) was not significantly different from that in the control (30.1±3.1-fold). The simultaneous deletion of the BRE and HRE resulted in a similar effect to the deletion of the BRE alone (2.1±0.5-fold versus 1.3±0.2-fold increase in reporter activity). Consistent with the results shown in Fig. 2(a), these results indicate that the upregulation of MDR1 under benomyl exposure is not dependent on the HRE. The HRE was also not involved in high constitutive expression of MDR1 in strain FR2. In this strain, reporter activities were identical in the presence (66.4±5.2 U) or absence (73.6±3.7 U) of the HRE (Fig. 6b). Moreover, reporter activities after the simultaneous deletion of BRE and HRE (23.6±2.6 U) were also similar to those observed in the absence of the BRE (16.3±0.7 U) (Fig. 6b).
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), the increase in reporter activity measured with the promoter lacking the BRE (5.6±1.0-fold) was approximately twofold lower than that with the full-length promoter (9.4±2.0-fold). In the absence of HRE, as well as with the simultaneous deletion of HRE and BRE, the relative increase of reporter activity in response to H2O2 exposure was 2.9±0.1-fold and 2.0±0.7-fold, respectively, which represents a threefold and 4.6-fold lower response than that recorded for reporters under control of the full-length promoter. This suggests that not only the HRE but also the BRE could mediate H2O2-dependent responses of MDR1. In summary, our results show that the HRE is not essential for MDR1 upregulation by benomyl or in an azole-resistant isolate but is required for H2O2-dependent regulation of this gene. In the presence of H2O2, Cap1p is likely to act in trans on the MDR1 promoter through the HRE. While we cannot exclude an indirect role for Cap1p in the benomyl response, it does not act through either of the cis-acting elements that we have identified in MDR1.
The BRE and HRE are functional in a heterologous promoter
To further assess the cis-acting role of the BRE in response to benomyl, the element was placed in a minimal 198 bp CDR2 promoter lacking its own regulatory element (DRE). The CDR2 promoter is not responsive to benomyl and is therefore convenient for addressing the involvement of the BRE in this process. A BRE–CDR2 chimeric promoter was constructed and used with a Renilla luciferase reporter system. Because the sequences immediately flanking the BRE may modulate its function, a 50 bp MDR1 promoter sequence was used which spanned nucleotides –309 to –260. The reporter system was inserted at the LEU2 genomic locus of strains DSY449 and FR2. As shown in Fig. 7(a), the presence of the BRE conferred benomyl responsiveness to the minimal CDR2 promoter. Up to 12.0±2.1-fold increase in reporter activity could be measured in benomyl-treated cells as compared to the control, untreated cells. Activity was reduced however as compared to the native 592 bp MDR1 promoter (72.8±5.6-fold). When two copies of the BRE were fused to the CDR2 promoter, the increase in reporter activity (96.8±46.4-fold) was comparable to that of the MDR1 promoter. To assess the role of the HRE in the benomyl-dependent response, a 41 bp MDR1 promoter sequence spanning nucleotides –556 to –516, and containing the HRE with Cap1p-binding sites, was placed upstream of the BRE–CDR2 promoter. The increase in reporter activity induced by benomyl (17.6±1.6-fold) was not significantly different from that observed with the BRE–CDR2 promoter (12±2.1-fold). When the HRE alone was fused to the CDR2 promoter, almost no increase in reporter activity could be detected (1.6±0.5-fold), indicating that the HRE is not involved in the benomyl response of MDR1. In FR2, the BRE–CDR2 chimeric promoter mediated high reporter activity. As shown in Fig. 7(b), the specific luciferase activity obtained with the BRE–CDR2 fusion (1.9±0.45x106 RLU) was only 2.5-fold lower than that obtained with the MDR1 promoter (4.9±1.0x106 RLU). Insertion of two copies of the BRE upstream of the minimal CDR2 promoter gave reporter activities (5.8±1.6x106 RLU) similar to those measured with the MDR1 promoter. Since the specific reporter activities measured with the HRE–BRE–CDR2 and the BRE–CDR2 fusions (1.7±0.3x106 and 1.9±0.5x106 RLU, respectively) were similar and only background reporter activity was detected with the HRE alone (<104 RLU), the HRE was not considered to be involved in the high levels of expression of MDR1 in FR2. The data presented in Fig. 7 also contribute to a more precise localization of the functional BRE. Placing the 5'extremity at position –296 as previously determined, the 3' extremity can be proposed to be at position –260, since this corresponds to the position identified in the testing of BRE functionality.
We investigated whether the HRE could confer an H2O2-dependent response to the minimal CDR2 promoter in strain DSY449 (Fig. 7c). However, when the HRE alone was fused to the minimal CDR2 promoter, the relative increase in reporter activity (1.9±0.7-fold) was not different from the background activity increase measured with the basal CDR2 promoter (2.0±1.0-fold). The relative increase in reporter activities after H2O2 exposure obtained with the BRE–CDR2 promoter was similar (2.9±1.8-fold) to that with the HRE–CDR2 promoter. However, when the HRE was placed upstream of the BRE–CDR2 promoter, a 43.0±3.1-fold increase in reporter activity generated by H2O2 exposure was measured, which was 1.6-fold higher than with the MDR1 promoter (27.3±3.7-fold). Thus, consistent with our observation that deletion of either element reduced the H2O2-dependent response of the MDR1 promoter, both elements are required to convey significant H2O2-dependent upregulation to a heterologous promoter.
Trans-acting factors bind to the BRE
To assess the possible interaction between the BRE and putative transcription factors present in C. albicans protein extracts, EMSAs were performed with double-stranded probes corresponding to several regions of the BRE. In this experiment, it was possible to observe the formation of a DNA–protein complex with labelled probe 1 (–296 to –259; Fig. 8a), which contained the BRE delimited by 5' promoter deletion analysis and insertion into another promoter context (between –296 and –260). This complex was present at higher levels, as revealed by the intensity of the band, when a protein extract from benomyl-exposed cells was used in comparison to that observed with proteins from untreated cells. The protein–DNA complex was specific for the BRE, since competition with increasing amounts of unlabelled BRE resulted in the disappearance of the complex (Fig. 8a). Furthermore, in the presence of a molar excess of an unlabelled probe, corresponding to a promoter region of the RTA4 gene, the formation of the protein–DNA complex could be still observed. Finally, using this labelled RTA4 as a probe (probe 2), formation of a protein–DNA complex with a similar migration pattern was not observed.
Labelled probes harbouring transversions identical to those used in promoter analysis (T1 to T4: probes 3–6) were also incubated with C. albicans protein extracts. However, as illustrated in Fig. 8(b), no specific band could be detected, thus corroborating the negative effects of these transversions upon reporter activities. Moreover, EMSAs carried out with labelled probes harbouring internal deletions (deletion D5: probe 7, –282 to –277 and deletion D6: probe 8, –285 to –274) showed no specific binding (Fig. 8b). This result is consistent with the finding that deletions within this region strongly reduced reporter activities (Fig. 5b). These results show that the promoter regulatory region previously delimited (–296 to –260) corresponds to that which can bind sequence specific protein complexes in vitro.
To further delimit the BRE sequence necessary for the binding in trans of specific protein complexes, band-shift assays were performed with probe lengths reduced at both the 5' and 3' extremities of probe 1 (–296 to –259) containing the BRE. Removal of 5 and 6 bp at the 5' extremity of probe 1 (respectively probes 9 and 10) and of 5 to 13 bp at its 3' extremity (probes 15–17) did not abolish the formation of a DNA–protein complex (Fig. 9a). The intensity of bands for protein complexes with probes lacking 7 bp at the 5' extremity (probe 11) or 14 bp at the 3' extremity (probe 18) was slightly reduced as compared to the full-length probe 1. Finally, removal of at least 8 bp at the 5' extremity (probes 12–14) or at least 15 bp at the 3' extremity (probes 19–22) resulted in the absence of protein complex formation (Fig. 9a). The results show that the minimal protein-binding region of the BRE is likely to be from –290 to –273. As expected, a specific DNA protein complex could be revealed (Fig. 9b) by using a labelled probe covering this region (probe 23).
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to lie within the region (–601 to –500), might also be attributable to the HRE. The HRE contains two YRE-like sequences, one that perfectly matches the consensus sequence (–532 to –526: TTAGTAA) and a second that differs from the consensus by two nucleotides (–549 to –543: TAACTAT). Deletion of the HRE almost abolished the H2O2-dependent response of the MDR1 promoter (Fig. 3), suggesting strongly that the YRE-like sequences play a central role in this induction. The HRE within the MDR1 promoter may be a downstream target of Cap1p, the functional homologue of the S. cerevisiae Yap1p, a bZip transcription factor binding to the YRE consensus sequence (Zhang et al., 2000). In accordance with this hypothesis, the deletion of CAP1 abolished the H2O2 response of MDR1. However, the absence of the HRE did not alter the response of the MDR1 promoter when C. albicans was exposed to benomyl. In contrast, the promoter of FLR1 from S. cerevisiae (a functional homologue of MDR1) was shown to be activated by benomyl via a YRE (Nguyen et al., 2001). Such species-specific differences serve to illustrate that the signal transduction pathways regulated by CAP1 and YAP1 are likely to have evolved significant differences despite the evidence for functional conservation of some of their downstream targets.
Surprisingly, the HRE had no impact on high constitutive expression of MDR1, since absence of this element did not alter MDR1 expression (Fig. 6). Moreover, when fused to the promoter CDR2, the HRE was unable to confer a high reporter activity in FR2. In the background of an FR2-derived mutant lacking CAP1, MDR1–lacZ reporter activities were also unchanged as compared to the parent strain. These results highlight the existence of distinct transcriptional programmes responsible for MDR1 upregulation. While the influence of the HRE appears restricted to the H2O2 response, the deletion of CAP1 also decreased, but did not eliminate, the benomyl-dependent response of MDR1 (Fig. 2). Thus, our work reveals that there is some degree of crosstalk between the elements that mediate distinct responses. As the benomyl response of MDR1 is dependent on the BRE, we speculate that Cap1p may cooperate with unknown factor(s) acting on the BRE.
The second cis-acting element of the MDR1 promoter, the BRE, was responsible both for the benomyl-dependent response of MDR1 and for high constitutive expression of this gene in the azole-resistant strain FR2. The delimitation of the minimal regulatory element that constitutes the BRE requires still further promoter analysis. The promoter truncations we examined have only delimited the 5' extremity of the regulatory elements (i.e., –296, see Fig. 2); internal deletions and transversions (Fig. 5) demonstrated that nucleotides between –286 and –278 are part of the BRE, but do not give a precise position of the 3' extremity of the regulatory element. Nevertheless, the 3' functional extremity of the BRE can be deduced from results obtained with the BRE–CDR2 promoter fusion. They suggest that the position of the 3' extremity must lie upstream of position –260, therefore defining the functional BRE between –296 and –260. In the light of results obtained by EMSA with oligonucleotides corresponding to the sequence of the BRE, the minimal oligonucleotide length still enabling sequence-protein binding was determined to be located between –290 and –273 (Figs. 8 and 9) and thus lies fully within the functionally defined BRE. The convergence of both methods to this specific region of the promoter strengthens its implication as a regulatory element. In vivo and in vitro footprinting analysis of the BRE can now be applied to aid precise delimitation of this regulatory element and the further characterization of the proteins that bind to it.
The functional cis-acting BRE identified in our work (–296 to –260) lies close to a cis-acting benomyl-responsive region, as delimited by a separate study (Harry et al., 2005). In that study, removal of the region between –399 and –299 led to a 20-fold diminution of the benomyl-dependent increase in reporter gene activities, whereas a weaker diminution (two- to fourfold) was observed when the region between –299 and –200 was absent from the MDR1 promoter. Thus, while both studies indicate that the BRE-containing sequences are important, we have not seen evidence of a second, more distal benomyl-responsive element. This discrepancy could be the result of several factors, including the strain background or the type of gene reporter system used to assess promoter activity. Above all, the genomic site of reporter gene insertion might contribute to such differences. In the present study, the MDR1–lacZ reporter system was integrated at the MDR1 genomic locus, whereas the Renilla luciferase reporter system used in the study of Harry et al. (2005) was inserted at the ADE2 genomic locus. A very recent study by Hiller et al. (2006) also shows the existence of several cis-acting domains in the MDR1 promoter. This promoter analysis was performed with a GFP reporter system integrated at the ACT1 locus in an azole-susceptible strain exposed to benomyl (3 h at 50 µg ml–1) and in an azole-resistant isolate expressing MDR1 at high levels. This experimental context is similar to the one used here, except that the drug exposure time was longer and the gene reporter system was different and not integrated at the MDR1 locus. The authors identified three different regulatory regions (region 1: –397 to –301; region 2: –588 to –500; region 3: –287 to –209). In their study, region 2 was implicated in the benomyl-dependent MDR1 response and regions 1 and 3 were required for constitutive high expression of MDR1. Thus while there is some consensus of agreement on the delimitation of regulatory regions, there are also significant discrepancies between the findings of Hiller et al. (2006) and the other two studies, this work and Harry et al. (2005). Most notably, our results clearly implicate the HRE in the H2O2 response of MDR1, while Hiller et al. (2006) found that the region encompassing this element (region 2) was responsible for benomyl-dependent MDR1 upregulation, a feature not confirmed in our study. The discrepancies between these different studies could have several origins, as mentioned above. The details of which regulatory elements respond to particular signals may also depend on chromatin-mediated factors and the gene's global context within the genome. Thus we believe it is of importance to have integrated the reporter constructs at the MDR1 locus. Moreover, the demonstration that we can recapitulate the functions of the HRE and BRE in the context of another promoter further validates the transcriptional cis-acting roles that we assign to them. Furthermore, we demonstrated protein binding to the BRE, thus confirming that this cis-acting element may interact with as yet unknown factor(s).
A BRE-like sequence is present in the promoter of genes co-regulated with MDR1 and contains a Mcm1p-binding site
In a previous study, we determined, by transcript profiling experiments, that MDR1 was co-regulated with other genes in an azole-resistant strain and in a susceptible strain exposed to benomyl (Karababa et al., 2004). We therefore expected to identify a common regulatory cis-acting motif resembling the BRE in the promoters of these genes. Alignment of promoter sequences revealed that nine of the 16 commonly upregulated genes indeed contain one or two sequences that match the signature of the BRE (Fig. 10a). Conserved sequence 1 (–287 to –267) in the MDR1 promoter matches the previously delimited BRE (–296 to –260) and differs by 4 nucleotides from the consensus established after promoter sequences alignment (5'-TCCMMWTTWGKAAAKTYHCCG-3'). While the presence of the BRE signature in these promoters is therefore consistent with their co-regulation, additional work will be required to confirm that the BRE is important for the regulation of these genes. A second BRE-like sequence (–496 to –476) was identified in the MDR1 promoter (see Fig. 10a); however, promoter deletion analysis revealed that this sequence is not involved in MDR1 regulation either in DSY449 exposed to benomyl or in FR2. Furthermore, EMSA with a probe corresponding to this second sequence failed to reveal protein-binding activity (data not shown).
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; Passmore et al., 1989; Spellman et al., 1998; Wynne & Treisman, 1992) was localized within this sequence. A similar promoter context exists in the MDR1 promoter of Candida dubliniensis (Fig. 10b), a species related to C. albicans in which MDR1 upregulation has been demonstrated (Moran et al., 1998). The presence of the putative Mcm1p-binding site in the BRE suggests that Mcm1p could act as a trans-acting factor on the BRE and thus be involved in the regulation of MDR1 via the BRE. Interestingly, this sequence overlaps the region deleted in Fig. 5 (D3: –286 to –278), which was considered to be a central part of the BRE. In S. cerevisiae, MCM1 is an essential gene of the MADS box transcription factor family involved in a variety of cellular processes, including chromatin remodelling, arginine response and mating as well as cell cycle regulation (Keleher et al., 1988; Passmore et al., 1988). In C. albicans, MCM1 is crucial for the determination of cell morphology and is autoregulated by a feedback mechanism. Depletion of Mcm1p leads to constitutive induction of hyphae. However, overexpression under specific conditions also results in enhanced hyphae formation. It has been hypothesized that Mcm1p might act as a mediator recruiting regulatory factors required for morphogenesis (Rottmann et al., 2003). MCM1 is essential in C. albicans (Rottmann et al., 2003), and thus experimental approaches to validate its involvement in MDR1 regulation are difficult. Chromatin immunoprecipitation (ChIP) experiments and EMSA with a labelled BRE probe using tagged Mcm1p are currently being undertaken in our laboratory and should address whether Mcm1p is effectively a trans-acting factor involved in the regulation of MDR1 through the BRE.
In summary, the work presented here has enabled the precise delimitation of two cis-acting regulatory elements (HRE and BRE) in the MDR1 promoter. We have shown that Cap1p is likely to be one trans-acting factor for the HRE; however, confirmation of its role and the full identification of the trans-acting components with which it interacts will necessitate further investigation. Future studies will also clarify whether mutations in MCM1 can be responsible for high constitutive expression of MDR1 in a manner analogous to the recent finding that mutations in TAC1, a key trans-acting regulator of the ABC-transporter genes CDR1 and CDR2, are responsible for the high CDR1/CDR2 expression of clinical strains (Coste et al., 2006).
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Received 3 July 2006;
revised 17 August 2006;
accepted 5 September 2006.
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0.05) from those obtained with the minimal CDR2 promoter in Mann–Whitney tests.
0.05). (b) Precise delimitation of the domain responsible for the high reporter activity in strain FR2. Promoters with 5' deletions (4 to 7 bp between –302 and –279 and 18 to 20 bp between –279 and –222) between –302 and –222 were analysed. The domain containing the BRE is represented by a black box. Asterisks indicate values significantly different (P
