Cross-talk between Transcriptional Regulators of Multidrug Resistance in Saccharomyces cerevisiae *

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys6-Zn(II)2 transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys6-Zn(II)2 transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding geneYOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.

A common feature underlying acquisition of multiple or pleiotropic drug resistance is the overexpression of genes encoding ATP-binding cassette (ABC) 1 transporters. Multidrug resistant tumor cells often exhibit amplification of the gene encoding the Mdr1p, a prototypical drug efflux pump (1). Gene amplification is typically not seen associated with pleiotropic drug resistance (Pdr) of the yeast Saccharomyces cerevisiae (2), yet these highly drug-resistant strains commonly possess high level transcription of ABC transporter genes like PDR5 (3)(4)(5) or YOR1 (6,7). Overexpression of these ABC transporters is re-quired for the Pdr phenotype and most commonly results from genetic lesions that activate function of the Cys 6 -Zn(II) 2 transcription factors Pdr1p and Pdr3p (8,9).
PDR1 was isolated as the first locus that could be mutated to confer a Pdr phenotype in S. cerevisiae (10). Cloning and analysis of this gene indicated that Pdr1p likely acted as a transcriptional regulator (11). Hyperactive alleles of PDR1 led to marked overproduction of target genes, which in turn allowed cells to tolerate otherwise toxic levels of drugs (12). Mutant forms of a PDR1 homologue (PDR3) produce a similar effect on both drug resistance and ABC transporter expression (13,14). Biochemical experiments indicated that Pdr1p and Pdr3p bind to the same DNA element that was designated the Pdr1p/ Pdr3p response element (PDRE) (15). Interestingly, the PDR3 structural gene contains two PDREs and is regulated by Pdr1p and autoregulated at the transcriptional level (16). The presence of at least one PDRE has been found associated with every gene regulated by Pdr1p or Pdr3p.
Recently, another transcription factor has been identified that shows a partially overlapping regulatory network with Pdr1p and Pdr3p. YRR1 was cloned as a locus that could be genetically altered to give rise to high level resistance to the cell cycle inhibitor reveromycin A (17). A mutant form of this gene (YRR1-1) contained a duplicated segment of the protein in the C-terminal region of the factor and strongly activated expression of the ABC transporter-encoding genes SNQ2 and YOR1. Construction of a SNQ2-lacZ fusion gene demonstrated that Yrr1p was able to regulate the promoter of this gene (18).
In this work, we show that Yrr1p acts to regulate YOR1 transcription at the level of its promoter by a Pdr1p/Pdr3pindependent mechanism. Analysis of expression of the YRR1 gene indicates that this locus is both responsive to Pdr1p and Pdr3p and autoregulated. Two different insertions of a 3ϫ hemagglutinin (HA) epitope tag into the C terminus of Yrr1p convert this protein into a hyperactive regulator of gene expression. Chromatin immunoprecipitation experiments using these 3X-HA-tagged forms of Yrr1p demonstrate that this protein associates with both the YRR1 and YOR1 promoters in vivo.

MATERIALS AND METHODS
Yeast Strains and Media-The S. cerevisiae strains used in this study were all derived from SEY6210. The genotypes of the strains are listed in Table I. PB4 is a pdr1⌬ and pdr3⌬ mutant yeast strain as previously described (19). XZY1 and XZY2 were made by transforming a yrr1-⌬1::TRP1 gene disruption plasmid (17) digested with BamHI and PstI into SEY6210 and PB4, respectively. Trp ϩ transformants were selected and analyzed by Southern blotting analysis to confirm introduction of the disruption allele. XZY12 to XZY17 were generated by using mTn-3X HA/lacZ transposon clones from the Yale Genome Analysis Center and used as described (20). XZY12, XZY13, and XZY16 were generated with one-step transformation of plasmids of clone V41A2 (YRR1::lacZ-URA3-3X HA-695), V54F11 (YRR1::lacZ-URA3-3X HA-730), and V122G2 (YOR172w::lacZ-URA3-3X HA-774) after NotI digestion, respectively. These three strains were then processed for lox excision by inducing the expression of cre recombinase from the pB227/ GAL-cre plasmid with galactose. The resulting 3X-HA-tagged versions of XZY12, XZY13, and XZY16 were named XZY14, XZY15, and XZY17, respectively. All strains were propagated in YPD (2% yeast extract, 1% peptone, and 2% dextrose) or minimal media (21) supplemented with casamino acids at 30°C with shaking. Drug resistance tests were carried out by gradient spot test method using YPD plates containing 4-NQO or YPGE (2% yeast extract, 1% peptone, 3% glycerol, and 3% ethanol) plates containing oligomycin. Yeast transformation was carried out by a lithium acetate method (22).
Plasmids-Plasmid pXTZ30 was constructed by transferring a BamHI fragment from YRR1-1-containing plasmid pES3 into the low copy plasmid pRS314 (23). The replacement of the PstI-SphI fragment of pXTZ30 with the corresponding one of YEp-YRR1 results in a wildtype YRR1-containing plasmid pXTZ31. A series of YRR1 promoter deletion constructs was generated by PCR. Four upstream primers, YRR1 sense (ccg gaa ttc CTT TCA GGC GTT ATT TCA GTG), YRR1up324 (cgg aat tcG CGA ATG TAG ATT TCT GCC AG), YRR1up270 (cgg aat tcA AAT CCG CGG AAA TTA G), YRR1up229 (cgg aat tcT GGG GTA GAG GCT GAT ATA CG), and one downstream primer, YRR1 antisense (ccg gga tcc ATT GTG ACG CTA TTC TTA TTG GC) were used for PCR. Note that an EcoRI restriction site was added to all upstream primers, whereas a BamHI restriction site was appended to the downstream primer as shown in lowercase letters. The PCR products were gel-purified, digested with EcoRI and BamHI, and cloned into the lacZ-containing plasmid pSEYC102 (24) that had also been digested with EcoRI and BamHI. The resulting plasmids produce a translational fusion between YRR1 and lacZ genes and were confirmed by DNA sequencing. These four plasmids were named pES5, pXTZ38, pXTZ68, and pXTZ69 in the order of deletion from 5Ј to 3Ј. The plasmids pXTZ111 to 119 were constructed by replacing the upstream activation sequence of the CYC1 gene with a Ϫ269 to Ϫ230 fragment of YRR1 promoter. YRR1(Ϫ269)F (gat cCG CGG AAA TTA GAA AAA CGT TAA AAG GTT CCA TGC A) and YRR1(Ϫ230)R (gat cTG GCA TGG AAC CTT TTA ACG TTT TTC TAA TTT CCG CG) were designed for this purpose. These two oligonucleotides were annealed, phosphorylated with T4 polynucleotide kinase and ATP, and then cloned into the BglII site of a low copy vector pRS314-ClZ after annealing. The vector pRS314-ClZ contains the minimal promoter of CYC1 fused to a lacZ reporter gene (25). The resulting plasmids were then sequenced. Three clones (pXTZ111, pXTZ115, and pXTZ117) were found to have a copy of the YRR1 oligonucleotide in the forward orientation (same orientation relative to CYC1 ATG as normally found in YRR1), whereas five plasmids had a copy of the oligonucleotide in the reverse orientation (pXTZ112, pXTZ114, pXTZ116, pXTZ118, and pXTZ119). One plasmid (pXTZ113) was found that contained a tandem repeat of the forwardoriented YRR1 oligonucleotide. Several mutations were found in these oligonucleotide sequences, and the DNA sequence of each clone is shown in Fig. 6A (see below).
␤-Galactosidase Assay-The activity levels of lacZ-encoded ␤-galactosidase were determined in two different ways. For most gene fusions, o-nitrophenyl-␤-D-galactopyranoside was used as substrate, and a standard permeabilized cell assay was employed (26). However, a luminescent ␤-galactosidase assay kit (CLONTECH) was used to determine the ␤-galactosidase activity of cells containing YRR1-lacZ due to its low expression level. Cells were grown to A 600 ϭ 1 in liquid media with shaking. Protein extracts were made by glass bead breakage. Protein concentration was determined by the Bradford method. An equal amount of protein extracts were used for the assay, and the activity of ␤-galactosidase was evaluated using a luminometer. All enzyme assays were performed at least twice on independent transformants, and the values reported are expressed Ϯ S.D.
DNase I Footprint Assay-The vector pOTS-Nco12 expressing a Myctagged Pdr1p N-terminal 248 amino acids or vector only was transformed into the Escherichia coli strain AR68. Protein extracts were then made from heat-shocked cells as previously described (19). An Asp718/SacI fragment of pXTZ118 was labeled with ␥-32 P at the Asp718-end and used as a footprint probe. The binding reaction was carried out at room temperature for 5 min followed by a 30-s DNase I digestion on ice. The reaction mix was resolved by 6% denaturing polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation.
Chromatin Immunoprecipitation-Chromatin immunoprecipitation was carried out essentially as described (27) with modifications from Steph Schroeder. Briefly, cells expressing either HA-tagged Tbp1p or Yrr1p were grown to mid-log phase in YPD medium and protein-nucleic acid complexes were fixed by formaldehyde treatment. Lysates were prepared by glass bead grinding, followed by sonication. A 20-l aliquot of the lysate was saved at this step as the input fraction. Sonicated lysates were then incubated with mouse anti-HA monoclonal antibody (Babco). After antibody binding, the samples were briefly centrifuged, followed by the addition of 20 g of salmon sperm DNA along with protein A-Sepharose beads. Immunoprecipitates were extensively washed and centrifuged to recover a pellet (bound) and supernatant fraction (unbound). 20 g of RNase A was added to remove RNA. Protein was eluted from the Sepharose beads by treating with 1% SDS/0.1 M HaHCO 3 . Cross-links were reversed by adding 20 l of 5 M NaCl to all reactions and heating at 65°C for 5 h. The DNA was ethanol-precipitated, digested with proteinase K, phenol-extracted, and then resuspended in TE (100 mM Tris, pH 7.5, 1 mM EDTA) prior to PCR analysis. Both input and bound DNA were dissolved in 20 l of TE. 1 l of DNA of each reaction was used for polymerase chain reaction. The following primer sets: YRR1 up229 and YRR1 antisense, YOR1 (CCA CGG TAA TCG ACA TAT TCG TATA) and YOR1-207 (TCG ACC GGA AAT TTT GCC GGG AAT ATG), ATR1-500 (GCG GAT CCA ACA TCC AGA CTT TTA CGG G), and ATR1ϩ40 (CCT TAC TTT CCG TAA GCA CA), were used to detect the YRR1, YOR1, and ATR1 promoter fragments, respectively. PCR analyses were performed at 94°C for 4 min, followed by 25 cycles of 56°C for 1 min, 72°C for 1 min, and 94°C for 1 min. 20 l of each reaction was run on a 1% agarose gel, and images were captured by a UVP mini darkroom system.

YRR1 Confers Oligomycin Resistance in S. cerevisiae-YRR1
was originally cloned by its ability to confer reveromycin A resistance (17). Our data previously implicated YOR1 in resistance to both reveromycin A (7) and the mitochondrial ATPase inhibitor oligomycin (6). Additionally, previous work showed that the mRNA expression level of YOR1 was increased in presence of YRR1-1 (17). To further examine the effect of the loss of YRR1 on oligomycin resistance of cells, yrr1⌬ deletion alleles were made in both wild-type or pdr1⌬, pdr3⌬ strains by homologous recombination. All four yeast strains were grown in YPD medium to an A 600 of 1, and 1000 cells of each were spotted on a YPGE plate containing a gradient of oligomycin. Loss of YRR1 from an otherwise normal background did not detectably influence oligomycin resistance (Fig. 1). However,  (28). To examine if the PDRE is also required for Yrr1p function, two different YOR1-lacZ fusion plasmids were used that either contained (YOR1-lacZ) or lacked (mPDRE-YOR1-lacZ) the PDRE in the YOR1 promoter. This mutant form of the PDRE contains a two-nucleotide substitution, which blocks Pdr1p/Pdr3p binding (28). In addition, low copy number plasmids expressing either wildtype YRR1 or the gain-of-function mutant YRR1-1 were cotransformed with either one of these two YOR1-lacZ fusion plasmids. Appropriate transformants were grown in minimal medium, and ␤-galactosidase activities were determined (Fig.  2). Expression of the wild-type YOR1-lacZ fusion gene was increased by ϳ4-fold in the presence of the YRR1-1 allele. Both the loss of the PDRE from the YOR1 promoter and the loss of the PDR1 and PDR3 genes failed to eliminate the observed elevation of ␤-galactosidase activity in the presence of the YRR1-1 mutation. These data suggest that Yrr1p activation of YOR1 gene expression depends on a DNA element other than PDRE. Additionally, the hyperactive form of Yrr1p (encoded by YRR1-1) produced high level activation of YOR1-lacZ that was independent of the presence of PDR1 and PDR3.
To assess if the ability to activate the YOR1 promoter would produce a corresponding increase in oligomycin resistance, low copy number plasmids carrying YRR1 or YRR1-1 were transformed into either wild-type or a PDR1 and PDR3 deletion strain PB4. Transformants were selected and grown in minimal media, and 1000 cells were plated on YPGE plates containing oligomycin. It was found that cells expressing YRR1-1 showed strong resistance to oligomycin in comparison to either wild-type or pdr1⌬, pdr3⌬ cells transformed with YRR1. It was also found that the resistance of cell to oligomycin by YRR1-1 requires the presence of YOR1 structure gene (data not shown). These data are consistent with the idea that YRR1 confers oligomycin resistance by regulating gene expression of YOR1 in a Pdr1p/Pdr3p-and PDRE-independent manner.
Deletion Mapping of YOR1 Promoter-The above analysis suggested that Yrr1p did not act through the intact PDRE. To localize the Yrr1p binding site in the YOR1 promoter, a series of deletion derivatives of the YOR1-lacZ reporter gene was utilized. Low copy number plasmids containing the indicated YOR1-lacZ fusion with varying amounts of 5Ј-promoter sequences were transformed into a wild-type yeast strain along with a second low copy number plasmid carrying either YRR1 or YRR1-1. The ␤-galactosidase activities were then determined for each transformant (Fig. 3).
A 5Ј deletion to Ϫ222 bp upstream of the YOR1 transcription start site was as responsive to the presence of the YRR1-1 allele as the fusion gene containing 1065 bp of upstream DNA. Further deletion to Ϫ129 bp upstream eliminated the response to Yrr1-1p. An internal deletion lacking the Ϫ190 to Ϫ129 region of the YOR1 promoter was still induced in the presence of Yrr1-1p, whereas the Ϫ299 to Ϫ50 deletion derivative lacked any response to the YRR1-1 allele. The elevated expression of the Ϫ299 to Ϫ50 internal deletion construct has been observed before (28). These data suggest that Yrr1p acts through the Ϫ222 to Ϫ190 bp region upstream of the YOR1 transcription start site. Note that the PDRE corresponds to position Ϫ218 to Ϫ209 but is not required to mediate the response of YOR1 to Yrr1p (see above).
Transcriptional Control of the YRR1 Promoter-Along with localizing the Yrr1p response element in the YOR1 promoter, we were interested in examining expression of the YRR1 gene itself. Inspection of the YRR1 promoter suggested that this gene might be transcriptionally regulated by Pdr1p and/or Pdr3p. A 9 of 10 match with the consensus PDRE was detected in the YRR1 promoter (Fig. 4). To facilitate analysis of YRR1 gene expression, a YRR1-lacZ fusion gene was prepared. This fusion gene was introduced into wild-type cells along with low copy number plasmids carrying wild-type or the PDR1-3 allele of PDR1. PDR1-3 encodes a hyperactive form of Pdr1p that FIG. 1. Yrr1p contributes to oligomycin resistance. Strains of the indicated genotypes were grown to an A 600 of 1 and 1000 cells/5 l was placed on YPGE medium containing a gradient of oligomycin (maximal concentration ϭ 0.3 g/ml). The increasing drug concentration is denoted by the bar of increasing width. The plate was allowed to incubate at 30°C and was photographed.

FIG. 2. Yrr1p activation of YOR1 is independent of Pdr1p and Pdr3p.
A, wild-type or pdr1⌬, pdr3⌬ strains were transformed with low copy number plasmid expressing a gain-of-function (YRR1-1) or a wildtype form of Yrr1p along with a YOR1-lacZ fusion gene or a mutant form lacking the PDRE (mPDRE-YOR1-lacZ). Transformants were grown in minimal media to mid-log phase and then assayed for ␤-galactosidase activity as described previously (26). B, strains of the indicated genotypes carrying low copy number plasmids expressing either wild-type or hyperactive Yrr1p were tested for oligomycin resistance using a gradient plate as described above.
leads to overexpression of Pdr1p target genes and drug hyperresistance (8). Transformants were assayed for YRR1-dependent ␤-galactosidase using a chemiluminescence method owing to the low level expression of YRR1.
The presence of the PDR1-3 allele increased expression of the YRR1-lacZ fusion gene by a factor of 4 compared with introduction of a second copy of wild-type PDR1. This analysis provided strong support for the view that YRR1 represents a new target gene for Pdr1p (and likely Pdr3p).
To directly demonstrate that Pdr1p was capable of binding to the YRR1 PDRE, a DNase I protection assay was performed using a probe containing this element from YRR1 promoter (Fig. 4). Incubation of this probe with protein extracts from bacteria expressing the DNA binding domain of Pdr1p but not with extracts from cells carrying the empty expression vector led to a region of DNase I protection corresponding to the YRR1 PDRE. Together, these data indicate that expression of YRR1 is regulated by Pdr1p.
During this analysis of YRR1 expression, we tested if Yrr1p might influence transcription of its own structural gene. To evaluate this possibility, low copy number plasmids expressing either wild-type or the hyperactive YRR1-1 allele of YRR1 were introduced into wild-type cells along with the YRR1-lacZ fusion gene. A series of 5Ј-truncated versions of the YRR1-lacZ reporter construct was prepared to localize important regulatory sequences. Transformants were processed for ␤-galactosidase activity measurements as described above (Fig. 5).
The presence of the YRR1-1 allele increased expression of the YRR1-lacZ fusion by nearly 600%. This same strong increase in expression was seen with reporter constructs containing 326 or 271 of 5Ј-noncoding DNA from YRR1. However, a fusion gene extending 220 bp upstream of the YRR1 ATG was no longer significantly responsive to YRR1-1. As we found for the YOR1 promoter, the YRR1 PDRE was localized from Ϫ269 to Ϫ260 and mapped to this Yrr1p-responsive region.
These data provide two important additional findings about control of YRR1 expression. First, YRR1 is autoregulated. Second, the element responsive to Yrr1p can be mapped to positions Ϫ271 to Ϫ220 upstream of the YRR1 ATG. This region of the YRR1 promoter was selected for further analysis to gain insight into the sequence elements required for response to Yrr1p.
Separation of Yrr1p and Pdr1p Response Elements in the YRR1 Promoter-To determine whether the response elements for Yrr1p and Pdr1p were closely linked in the YRR1 promoter, an oligonucleotide corresponding to positions Ϫ269 to Ϫ230 in the YRR1 5Ј-noncoding sequence was synthesized. This oligonucleotide was used to replace the normal CYC1 upstream activation sequences in a CYC1-lacZ fusion plasmid. Several clones of this oligonucleotide were sequenced, and a number were found to contain mutant forms of the YRR1 DNA segment. Plasmids containing wild-type or mutant oligonucleotides were introduced into cells along with low copy number plasmids expressing wild-type or hyperactive alleles of PDR1 and YRR1. CYC1-dependent ␤-galactosidase activities were then determined for each transformant (Fig. 6).
Alterations at the 3Ј-end of this oligonucleotide had no effect on the ability of hyperactive forms of Yrr1p or Pdr1p to activate expression of the fusion gene. Introduction of the hyperactive form of Yrr1p led to ϳ400% increase in expression, whereas the PDR1-3 allele increased ␤-galactosidase activity by ϳ300%. Substitution and deletion mutations between positions Ϫ242 and Ϫ231 had no effect on Pdr1p or Yrr1p regulation. However, FIG. 3. Localization of the Yrr1p-responsive region in the YOR1 promoter. A series of YOR1-lacZ fusion genes lacking different segments of the YOR1 promoter was introduced into wild-type cells along with plasmids expressing either wild-type or hyperactive forms of Yrr1p. All plasmids were maintained at low copy number. Transformants were grown to mid-log phase and assayed for ␤-galactosidase activity. The solid box indicates the position of the PDRE, whereas the arrow denotes the start site for YOR1 gene transcription. The numbers on the left refer to the extent of YOR1 5Ј-noncoding DNA remaining for the 5Ј-truncation mutants, whereas the DNA that has been deleted is shown for the internal deletion mutants. FIG. 4. Identification of a PDRE in the YRR1 promoter. A, the DNA sequence of an element in the YRR1 5Ј-regulatory region matching the consensus PDRE is shown. The numbers refer to the position of this PDRE relative to the YRR1 ATG. B, a YRR1-lacZ fusion gene was transformed into wild-type cells along with plasmids carrying the wildtype (PDR1) or hyperactive form of Pdr1p (PDR1-3). Transformants were assayed for ␤-galactosidase activity in crude protein extracts using a chemiluminescent reagent (CLONTECH) as recommended by the manufacturer. C, a 5Ј 32 P-end-labeled probe containing the YRR1 PDRE was prepared as described under "Materials and Methods." This probe was incubated with either 10 or 20 l of crude extract from bacterial cells expressing the DNA binding domain of Pdr1p (Pdr1p, volume indicated by bar of increasing width), 20 l of crude extract from cells carrying the empty expression vector (Vector), or with buffer alone (No Protein). Samples were then digested with DNase I for 30 s, deproteinized, and electrophoresed through a denaturing polyacrylamide gel. Location of the PDRE was established by comparison with Maxam-Gilbert sequencing reactions on the same fragment (data not shown). clustered mutations located near the PDRE (Ϫ259 and Ϫ255 in pXTZ112, 114, 115, 117) eliminated the ability of the corresponding oligonucleotide to be regulated by Yrr1p in every case. Importantly, these mutant oligonucleotides did maintain their ability to respond to Pdr1p with the exception of pXTZ117, which contains a 1-bp deletion in the PDRE. These data are consistent with the view that the Yrr1p response element and the PDRE are linked but separable in the YRR1 promoter.
The C Termini of YRR1 and Its Homologue YOR172W Regulate Factor Function-An invaluable resource in the analysis of S. cerevisiae genes is the availability of a collection of transposon insertions into a large fraction of the genome (20). This series of insertion mutations was found to contain two different in-frame insertions of E. coli lacZ into YRR1, one after codon 695 and one after codon 730. These insertions are in a region of the protein in which a gain-of-function mutation was previously isolated (17). The transposon used to make these muta-tions also contains loxP sequences that can be cleaved by the cre protein expressed in yeast to evict the lacZ cassette and associated URA3 marker, leaving behind a 3X hemagglutinin (3X HA) epitope tag inserted into the coding sequence. These two different transposon insertions were integrated in place of the wild-type YRR1 locus in an otherwise wild-type strain, the lacZ/URA3 sequences removed by expressing cre in appropriate integrants and the resulting mutant YRR1 loci designated YRR1::3X HA-695 (3X HA insertion after codon 695) and YRR1::3X HA-730 (3X HA insertion after codon 730).
Strains expressing wild-type or the two different 3X HA insertion variants of Yrr1p were assayed for their ability to confer resistance to oligomycin and 4-NQO using a gradient plate of each drug (Fig. 7). Both the Yrr1p::3X HA-695 and Yrr1p::3X HA-730 gave rise to an increased ability to grow in the presence of both oligomycin and 4-NQO. To determine whether this increased oligomycin resistance correlated with an elevation in YOR1 expression, a YOR1-lacZ fusion plasmid FIG. 5. Deletion mapping the Yrr1p-responsive region in the YRR1 promoter. A series of YRR1-lacZ fusion genes, varying in the extent of YRR1 5Ј-noncoding DNA, were introduced into wild-type cells along with low copy number plasmids expressing either wild-type or the hyperactive YRR1-1 form of Yrr1p. YRR1-dependent ␤-galactosidase activities were determined using the chemiluminescence assay as above. The numbers denote the position relative to the YRR1 ATG (indicated on the figure), and the solid box represents the position of the YRR1 PDRE.
FIG. 6. Localization of Yrr1p and Pdr1p response elements in the YRR1 promoter. A, the various oligonucleotides analyzed for their ability to activate expression of a CYC1-lacZ fusion gene are shown. Expression of the CYC1-lacZ fusion in the absence of an inserted oligonucleotide was ϳ1 unit/optical density. Wild-type refers to the DNA sequence from the normal YRR1 promoter. The actual sequences of each oligonucleotide evaluated here are shown below next to the plasmid that contains each. Note that pXTZ113 contains two oligonucleotides. Orientation refers to the placement of each oligonucleotide relative to the CYC1 promoter. Forward means the oligonucleotide is placed in the same orientation as at YRR1, whereas reverse indicates that the oligonucleotide is cloned in the opposite orientation as it would be at YRR1. The numbers below the sequence show the positions in the native YRR1 control region. Mutant residues are boxed and deletions indicated by a minus sign. The location of the PDRE is shaded. B, the ␤-galactosidase activities of the reporter plasmids from above are listed. The isogenic strains SEY6210 (YRR1) or the mutant XZY15 (containing the hyperactive YRR1::3X HA-730) were used to vary the activity of the Yrr1p factor. Low copy number plasmids were used to introduce a single extra copy of PDR1 or PDR1-3. Transformants were grown to mid-log phase and assayed as described under "Materials and Methods." was introduced into these backgrounds. The Yrr1p::3X HA-695 and Yrr1p::3X HA-730 led to an increase in YOR1-lacZ expression to 25 and 31 units/optical density, respectively, whereas the presence of wild-type Yrr1p produced 11 units/optical density. These data indicate that insertion of foreign sequences into the C-terminal region of Yrr1p activate the function of this transcriptional regulatory protein.
Analysis of the S. cerevisiae genome detected the presence of a protein encoded by the YOR172w locus that shared strong sequence identity with Yrr1p (41%) identity (17). Search of the transposon library indicated that a single insertion mutation had been isolated in which the transposon was placed after codon 774 of the 786 codon open reading frame. This transposon was integrated into a wild-type strain in place of the YOR172w locus, and the lacZ/URA3 sequences were eliminated by expression of cre recombinase in appropriate transformants. The resulting Yor172wp::3X HA-774-expressing strain was then assayed for drug resistance in comparison to an isogenic wild-type cell (Fig. 7).
Expression of Yor172wp::3X HA-774 led to a modest but reproducible increase in both oligomycin and 4-NQO tolerance. These findings suggest that, like Yrr1p, Yor172wp also re-quires an intact C terminus for normal regulation.
YRR1 Interacts with Both the YRR1 and YOR1 Promoters-A simple model that could explain the effect of Yrr1p on expression of YOR1 and YRR1 would be provided by the ability of this factor to directly bind to and activate these promoters. We tried a variety of bacterial expression systems but were not able to reproducibly detect Yrr1p DNA binding activity. We turned to the technique of chromatin immunoprecipitation (ChIP) (27) to determine if Yrr1p was capable of associating with these putative target promoters in vivo.
Three different strains were used to assess in vivo association of Yrr1p with the YOR1 and YRR1 promoters: a wild-type strain expressing no HA-tagged proteins (SEY6210), a strain expressing an HA-tagged Yrr1p (XZY15), and a strain expressing an HA-tagged form of TATA-binding protein (Tbp1p) as its sole source of this essential protein (DPY11). Three different primer pairs were used to evaluate the specificity of the ChIP assay. Primers corresponding to the promoter regions of YRR1 and YOR1 as well as a primer set that would amplify the promoter of the ATR1 gene (29), a locus not under Yrr1p or Pdr1p/Pdr3p control (data not shown). ChIP was performed using anti-HA antibody essentially as described (27) and analyzed by PCR amplification of total DNA prior to immunoprecipitation (input) or specifically immunoprecipitated DNA (Anti-HA IP; Fig. 8).
Both the YRR1 and YOR1 promoters are detected in DNA recovered from ChIP reactions performed on chromatin lysates from the cells expressing the HA-tagged proteins but not on control lysates from wild-type cells. Importantly, the ATR1 promoter was identified in the immunoprecipitates from the HA-Tbp1p expressing strain but not from the HA-Yrr1p-expressing cells. These data provide support for the view that Yrr1p associates in vivo with target promoters and through this association leads to an increase in gene expression. DISCUSSION These data illustrate important new connections in the pleiotropic drug resistance pathway in S. cerevisiae. The finding that Pdr1p (and likely Pdr3p) regulate expression of YRR1 suggest a potential new complexity in the analysis of Pdr1p/ Pdr3p target genes. Genome microarray experiments have suggested the presence of a large number of genes that increase in expression in response to activated forms of Pdr1p or Pdr3p (30). Although many of these genes contain at least one PDRE, were grown to mid-log phase and tested for resistance to oligomycin and 4-NQO using a gradient plate assay. The increasing drug concentrations are indicated by the bar of increasing width. B, strains expressing the wild-type or 3X HA-tagged forms of Yrr1p were transformed with a wild-type YOR1-lacZ fusion plasmid. Transformants were assayed for ␤-galactosidase levels using a permeabilized cell assay as before (26). C, cells expressing wild-type (YOR12w) or the 3X HA-tagged form (YOR172w::3X HA-774) form of Yor1172wp were assayed for resistance to oligomycin and 4-NQO using a gradient as above.
FIG. 8. Yrr1p associates with in vivo target promoters. PCR was performed using primer pairs that specifically detect the promoters of YRR1, YOR1, or ATR1. DNA templates were either subcloned versions of each promoter (Positive Control), total chromatin (Input), or immunoprecipitated chromatin (Anti-HA IP). Chromatin samples were prepared from wild-type cells (No HA-tagged protein), cells expressing a 3X HA-tagged Yrr1p or a strain expressing a 3X HA-tagged Tbp1p. Equal volumes of each PCR were electrophoresed through a nondenaturing 1% agarose gel containing ethidium bromide and were photographed. not all do. These data provide a possible explanation for this observation, because activation of Yrr1p expression by Pdr1p or Pdr3p could lead to an increase in transcription of downstream target genes via Yrr1p binding to an element other than a PDRE.
With this possibility in mind, it is interesting to note that in the two genes identified here are Yrr1p targets, both contain PDREs. Additionally, previous work (17) has provided evidence that Yrr1p activates expression of the ABC transporter-encoding locus, SNQ2, which in turn leads to the increase in 4-NQO resistance seen in cells containing activated forms of Yrr1p. SNQ2, like YRR1 and YOR1, also contains a PDRE. Examination of these three genes cannot be viewed as representative of all Yrr1p target genes, because all three loci are involved in drug resistance. Less directed approaches such as microarray analyses must be undertaken to give a more accurate picture of the necessary linkage of Yrr1p response with a PDRE.
A second striking feature of the observed linkage of the PDRE with the Yrr1p response element is the finding that these two recognition sequences appear to be tightly physically linked. We have identified two base pairs, critical for Yrr1p activation, that are located immediately adjacent to the YRR1 PDRE. A 33-bp region containing the YOR1 PDRE appears to be necessary for the Yrr1p responsiveness of this gene. A similar tight linkage has been found for SNQ2 (18). At least for these three Yrr1p/Pdr1p-coregulated genes, the possibility exists that these regulatory proteins may directly communicate during gene regulation.
Even though the actions of Pdr1p and Yrr1p occur through sites that are physically close on target promoters, we provide evidence that their actions are not through the same element. First, a mutant YOR1 promoter lacking a functional PDRE can still be activated by Yrr1p. Second, the presence of the YRR1-1 allele completely bypasses the requirement for Pdr1p or Pdr3p in terms of oligomycin resistance and YOR1-lacZ expression. Finally, mutant forms of the YRR1 promoter can be generated that fail to respond to Yrr1p but are normally activated by Pdr1p.
Although the ChIP analysis demonstrates that Yrr1p interacts with target promoters, a consensus binding site for Yrr1p has not yet been identified. Efforts to produce forms of Yrr1p in bacterial expression were not successful even though similar constructs were used to produce active forms of either Pdr1p or Pdr3p (15,19). Inspection of the three known target promoters has not revealed any striking candidates for shared recognition elements. The identification of the precise binding site for Yrr1p is a high priority.
The finding that insertions of random sequences into the C termini of either Yrr1p or Yor172wp lead to an increase in function of these factors suggests that these proteins may normally be negatively regulated. The insertion of this extraneous sequence leads to a loss of the ability to respond to this negative signal and results in a constitutively active protein. Previously, a hyperactive form of YRR1 (YRR1-1) was identified that contained a duplication of amino acids 695-706 (17). Our observation that insertion of the 3X HA sequence after either position 695 or 730 indicates that there is not likely to be a special significance to the previously reported duplication but rather that this region of Yrr1p must be structurally intact for normal regulation of function. Other Zn(II) 2 -Cys 6 transcription factors like Gal4p (31), Pdr1p (32), Leu3p (33), or Hap1p (34) also possess central regulatory domains that can be mutationally altered to change the function of the resulting factor. We have previously reported that the activity of Pdr3p is tightly linked to the status of mitochondria (35) and find that Yrr1p also appears to be involved in this regulatory circuit. 2 Together, these data illustrate the close communication between Pdr1p, Pdr3p, and Yrr1p activity (Fig. 9). Coordination of the activity of these transcription factors is a critical factor for normal drug resistance as shown by the interlocking systems of transcriptional control of the Pdr3p and Yrr1p structural genes. Identification of the precise regulatory signals controlling these factors will provide important new insight into the physiological basis underlying these multidrug resistance regulatory proteins.