From the Molecular Biology Program and the
§ Department of Physiology and Biophysics, University of
Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Improper control of expression of ATP binding
cassette transporter-encoding genes is an important contributor to
acquisition of multidrug resistance in human tumor cells. In this
study, we have analyzed the function of the promoter region of the
Saccharomyces cerevisiae YOR1 gene, which encodes an ATP
binding cassette transporter protein that is required for multidrug
tolerance in S. cerevisiae. Deletion analysis of a
YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to
positively regulate expression of YOR1, and the third
element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and
Pdr3p in other promoters. The second positive element is located between nucleotides 535 and
299 and is referred to as
UASYOR1 (where UAS is upstream activation sequence).
Interestingly, function of UASYOR1 is inhibited by the
downstream negative regulatory site. Promoter fusions constructed
between UASYOR1 and the PDR5 promoter, another
gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter
fusions constructed with the CYC1 promoter are not. This
suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another
Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.
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INTRODUCTION |
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Mammalian tumor cells can acquire the ability to detoxify several unrelated cytotoxic agents through alteration of a small number of genetic loci. This phenomenon, termed multidrug resistance, often involves the increased expression of an ATP binding cassette (ABC)1 transporter protein known as MDR1 (reviewed in Ref. 1). Recently, overexpression of a second ABC transporter protein has been shown to also confer a multidrug-resistant phenotype on cells. This membrane transporter protein was designated the multidrug resistance protein (Mrp) (2) and later shown to be responsible for production of a glutathione S-conjugate transporter activity in hepatocytes (3, 4). Control of the levels of expression of these mammalian ABC transporters is a critical determinant in the relative drug tolerance of an animal cell.
The yeast Saccharomyces cerevisiae also contains loci that
can be altered to give rise to multiple or pleiotropic drug resistance (reviewed in Ref. 5). Dominant mutations in the Cys6 zinc
finger transcription factors PDR1 and PDR3 result
in elevated resistance to a broad array of toxic agents including
cycloheximide and oligomycin (6-8). The ABC transporter
protein-encoding gene PDR5 is transcriptionally activated by
Pdr1p and Pdr3p and is required for Pdr1p/3p-mediated cycloheximide
resistance (9, 10). Cells containing a pdr5 allele are
cycloheximide-hypersensitive but display no oligomycin sensitivity,
indicating the presence of a second Pdr1p/3p target gene required to
provide oligomycin resistance. This oligomycin resistance gene,
YOR1, was subsequently cloned on the basis of its ability to
strongly elevate tolerance to this compound when present on a high copy
plasmid (11).
DNA sequence analysis of the YOR1 gene (11) demonstrated
that this locus encodes a protein with striking sequence similarity to
the human cystic fibrosis transmembrane conductance regulator (12), Mrp
(2), and S. cerevisiae Ycf1p (13). YOR1 is
transcriptionally regulated by Pdr1p and Pdr3p and inspection of the
YOR1 promoter region indicated the presence of a potential
Pdr1p/Pdr3p response element (PDRE) centered 215 bp upstream of the
transcription start site (11). These PDREs have been defined previously
by analysis of similar elements in the promoters of PDR3
(14), PDR5 (15), and SNQ2 (16, 17). Northern blot
and lacZ gene fusion analysis showed that YOR1
retained significant expression in a pdr1,3 strain (11).
This is a marked difference from the behavior of the
Pdr1p/Pdr3p-regulated PDR5 gene which is essentially
inactive in a
pdr1,pdr3 mutant background, explaining the
cycloheximide-hypersensitive phenotype of this strain (10). It was
anticipated that YOR1 expression would also strongly depend
on PDR1/PDR3 since a
pdr1,3 strain is
extremely oligomycin-sensitive (10, 18).
To explore the nature of the differences between the co-regulated PDR5 and YOR1 promoters, we have carried out an analysis of the transcriptional control elements of YOR1. Deletion mutagenesis of a YOR1-lacZ gene fusion defined three regions important for normal regulation of YOR1 expression, two positively acting cis-elements and one negative regulatory site. One of the positive elements corresponds to the PDRE, confirming the role of this site in YOR1 transcriptional control. The second positive element is located upstream of the PDRE and is normally repressed by the action of the negative regulatory region. The activities of these other two elements appear to be independent of PDR1 and PDR3. These data are consistent with the YOR1 promoter receiving multiple regulatory inputs from PDR1/PDR3 and other as yet unknown factors.
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MATERIALS AND METHODS |
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Yeast Strains and Media--
Yeast transformations were
performed using the lithium acetate procedure (19). The previously
described strains used in these studies are SEY6210 (MAT;
leu2-3,-112; ura3-52; his3-
200; trp1-
901; lys2-801 suc2-
9;
Mel
), PB4 (SEY6210; pdr1-
2::hisG;
pdr3-
1::hisG) (10), and DKY7 (SEY6210;
yor1-1::hisG) (11). TCH5 (SEY6210,
yor1-
1::hisG) was used for the RNase mapping
experiments. Standard SD and YPD media (20) were used for normal yeast
growth and determination of drug resistance. Drug resistance was
assayed by spot testing on plates containing different drug
concentrations (21).
-Galactosidase assays were performed as
described previously (22).
Primer Extension Analysis--
A YOR1-specific
oligonucleotide was synthesized that corresponds to nucleotides +49 to
+75 in the YOR1 gene. This oligonucleotide was labeled at
its 5 end using [
-32P]ATP and T4 polynucleotide
kinase. The radiolabeled oligonucleotide was then annealed to 50 µg
of total RNA isolated using a rapid procedure (23) from strains of
interest. Annealing was allowed to proceed for 45 min at 65 °C, then
incubated for an additional 45 min at 45 °C, followed by reverse
transcriptase extension of the RNA-DNA complexes. Primer extension was
carried out for 1 h at 37 °C; the reaction was stopped by
phenol extraction, and the nucleic acids were precipitated. Primer
extension reactions were electrophoresed through a 6%
polyacrylamide/urea gel in parallel with dideoxy sequencing reactions
performed on a YOR1 template using the same oligonucleotide
primer.
Plasmids--
The wild-type YOR1-lacZ plasmid
pSM109-4 has been previously described (11). Briefly, pSM109-4
corresponds to a YOR1-lacZ gene fusion containing 1066 bp
upstream of the transcription start site and 213 bp of coding sequence
from YOR1. This same YOR1 DNA fragment was cloned
into pBluescript KS II+ as a BamHI/EcoRI fragment to form pTH40. 5 deletions were generated by digesting pTH40 with
EcoRI, treating this DNA with the bidirectional exonuclease Bal31, forming blunt ends with Klenow enzyme and
deoxyribonucleotides, and then ligating an EcoRI linker to
the resulting blunt-ended fragments. The ligation reaction was then
cleaved with EcoRI/BamHI and cloned into
similarly digested pSEYC102 (24). The 5
end of each deletion mutant
was sequenced to precisely determine the 5
end point. An
oligonucleotide (CCG GAA TTC GAA TAT GTC GAT TAC CGT GGG GGA T) was
used to introduce an EcoRI site upstream of the
YOR1 ATG in a PCR reaction, resulting in the plasmid pTH70. 3
promoter deletions were generated in the same fashion as the 5
deletion mutants, except pTH40 was cut initially with BamHI. These 3
deletions were cloned as EcoRI fragments into
YOR1 5
deletion mutant constructs resulting in internal
promoter deletions.
RNase Mapping Analysis of YOR1-lacZ Transcripts--
To detect
the chimeric YOR1-lacZ transcripts with no interference from
the chromosomal YOR1 gene, a strain was constructed that
lacked the region of YOR1 around the transcription start site. This was accomplished by preparing a
XhoI/HindIII subclone of YOR1 in
pBluescript. An internal BglII fragment extending from 109
to +1129 was then replaced with the BamHI/BglII
hisG-URA3-hisG fragment from pNKY51 to produce pTH140. A
SacI/KpnI fragment of pTH140 was used to
transform SEY6210 cells to URA3+. Appropriate integration
was confirmed by Southern blotting (26). The resulting transformant was
then treated with 5-fluoroorotic acid and used as host for the
YOR1-lacZ fusion plasmids. This Ura
cell was designated
TCH5. Total RNA was prepared from TCH5 transformants by hot phenol
extraction (23). A YOR1-specific probe was generated by T7
transcription of BglII-linearized pTH40, and the
PDC1 probe was produced by T3 transcription of a
BamHI/SalI PDC1 subclone in pRS426
(27). The PDC1-containing plasmid was cleaved with BglII prior to in vitro transcription. RNase
protection was carried out as described by the manufacturer
(Ambion).
DNase I Footprint Analysis-- DNase I footprint analysis was performed as described (28). The vector pOTS-Nco12 (29) expressing a Myc-epitope tagged version of the N-terminal 248 amino acids of Pdr1p was used to produce protein for these experiments. This plasmid was constructed by inserting an oligonucleotide encoding the Myc epitope into the NcoI site of a pOTS-Nco12 derivative expressing the N-terminal 248 residues of Pdr1p (provided by Laurence Lambert).
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RESULTS |
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Primer Extension Mapping of the YOR1 Transcription Start Site-- Previously, we localized the YOR1 transcription start site using an RNase protection assay (11). This assay provided an approximation of the site for transcription initiation accomplished by comparing the protected RNA species with an end-labeled set of DNA fragments. To locate precisely the YOR1 transcription start site, primer extension analysis was carried out. Total RNA was isolated from a wild-type strain transformed with the vector pRS315 or with pRS315 containing a dominant, gain-of-function allele of PDR1 designated PDR1-6. This gain-of-function PDR1 allele produced both elevated cycloheximide and oligomycin tolerance in addition to increasing expression of PDR5 and YOR1 (11, 30).
A 25-nucleotide YOR1-specific primer was radiolabeled with [
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5 Deletion Mapping of the YOR1 Promoter--
Low copy plasmids
carrying the YOR1-lacZ fusion with progressively larger 5
promoter deletions were transformed into a wild-type strain or an
isogenic
pdr1,3 strain.
YOR1-lacZ-dependent
-galactosidase activities
were determined for each construct (Fig.
2).
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YOR1 Promoter Internal Deletions--
Internal deletions in the
YOR1 promoter were constructed to identify cis-acting
elements that might have been missed in the 5 deletion analysis. Each
internal deletion mutant was assayed in both the wild-type and
pdr1,pdr3 strains (Fig.
3).
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Pdr1p Binds to an Element in the YOR1 Promoter--
The results of
the 5 and internal deletion analysis of the YOR1 promoter strongly
suggested that the PDRE located at
215 was likely to serve as a site
for positive control of gene expression by Pdr1p (and likely Pdr3p). To
test this idea directly, we produced Pdr1p in bacteria and assayed the
ability of this recombinant protein to bind to the YOR1
PDRE. Protein extracts were prepared from E. coli cells
carrying the empty expression vector or the same vector expressing an
N-terminal fragment of Pdr1p that contains the zinc finger DNA binding
domain. This Pdr1p fragment has previously been found to recognize the
PDREs in the PDR5 promoter (15). Protein extracts were
incubated with radiolabeled DNA templates prepared from the wild-type
YOR1 promoter or from a mutant YOR1 promoter that
lacks the PDRE. This mutant PDRE was constructed by changing 2 base
pairs that have previously been found to be essential for function of a
PDRE in the PDR5 promoter (15). The results of this DNase I
protection experiment are shown in Fig.
5.
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The PDRE Is Required for Control of YOR1 Expression by
Pdr1p--
To assess the contribution of the PDRE to YOR1
promoter function, the mutant PDRE was introduced into the context of
the YOR1-lacZ fusion gene present in pSM109-4. Both the
wild-type and mutant PDRE-containing YOR1-lacZ fusion genes
were transformed into pdr1,pdr3 cells along with a low
copy plasmid vector or this same vector plasmid carrying wild-type
PDR1 or the dominant PDR1-6 allele. YOR1-dependent
-galactosidase activities were
then determined for each transformant (Fig.
6).
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Normal Oligomycin Tolerance Requires the Presence of a Functional YOR1 PDRE-- The 2-base pair substitution mutation in the PDRE of a YOR1-lacZ fusion gene blocked the ability of this promoter to respond to PDR1. To evaluate the consequence of loss of this sequence element on YOR1-mediated oligomycin tolerance, the mutant PDRE was introduced into the context of the wild-type YOR1 structural gene. This altered YOR1 structural gene was introduced into yor1 cells along with a low copy plasmid carrying the PDR1-6 allele or the vector plasmid alone. An equivalent wild-type YOR1 gene was transformed in a parallel fashion to provide a control for normal YOR1 function. Appropriate transformants were placed on rich medium lacking or containing oligomycin (Fig. 7).
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Evidence for Promoter-specific Function of the Pdr1p-independent
YOR1 UAS--
Along with the PDRE, the analysis described above
pointed to the presence of a second positive transcriptional control
element located between 299 and
535 (UASYOR1). To
explore the function of this positive regulatory region, we constructed
promoter fusions between various upstream fragments of YOR1
and two different UAS-deficient promoter-lacZ fusion
constructs. The two promoter-lacZ constructs were the well
characterized CYC1-lacZ fusion gene (22) or a similarly
deleted PDR5-lacZ fusion that we have previously shown to
lack its UAS (10). The YOR1 promoter fragments were placed upstream of these test promoters and introduced into
pdr1,pdr3 cells along with a low copy plasmid carrying
the wild-type or PDR1-6 alleles of PDR1 or the
vector alone. YOR1 promoter fragments were inserted upstream
of the heterologous promoters in the same relative orientation as in
the native YOR1 promoter.
-Galactosidase activities of
appropriate transformants were then determined (Table I).
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DISCUSSION |
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Although both PDR5 and YOR1 lie under the umbrella of PDR1/PDR3 gene regulation, these two loci do not exhibit common promoter structures. Both PDR5 and YOR1 contain PDREs, with PDR5 having three elements and YOR1 only one. The sequence of the YOR1 PDRE is identical to PDR5 PDRE site 2 (15). PDR5 expression is strictly dependent on the presence of either PDR1 or PDR3, whereas YOR1 expression exhibits a large component that is PDR1/PDR3-independent. This finding suggested the presence of other positive regulatory elements for transcription in the YOR1 promoter, a suggestion we have confirmed in the current study.
The second positive regulatory element for YOR1 expression
(UASYOR1) lies between positions 535 and
299. The
function of this element was not detected by 5
deletion analysis as
UASYOR1 activity is under negative control by an upstream
repression sequence (URSYOR1) lying between
115 and
50.
YOR1 appears to represent a new example of a PDR
gene in terms of having this fairly complex arrangement of interacting
positive and negative transcriptional control elements superimposed on
the PDRE-mediated control of expression. Studies of the PDR5
promoter have not indicated the presence of any regulatory sequences
other than the PDREs (10, 15). Removal of the PDREs from the
PDR3 promoter eliminates the ability of this gene to elevate
the cycloheximide resistance of a
pdr1,pdr3 cell (14).
This comparison suggests that YOR1 is likely to integrate
transcriptional control signals from factors other than Pdr1p/Pdr3p.
Identification of these other factors will provide new insight into the
physiological role of Yor1p.
Another unique feature presented by the YOR1 promoter is the
presence of a single PDRE in its 5-noncoding region. All other known
PDRE-containing promoters contain at least 2 (PDR3) to 3 (PDR5) PDREs. Interestingly, these multiple PDRE-containing
promoters also exhibit markedly more responsiveness to Pdr1p/Pdr3p than does YOR1. This observation is consistent with our previous
finding that each PDRE in the PDR5 promoter contributes
approximately equally to the overall response of PDR5 to
Pdr1p levels (15). These data suggest that the degree of Pdr1p/Pdr3p
responsiveness of a given promoter can be largely predicted by the
number of PDREs found in the promoter of interest.
The presence of the negatively acting URSYOR1 element was
detected by an increase in YOR1-lacZ expression that
occurred upon deletion of the 115 to
50 region of the
YOR1 promoter. We believe that this increase in expression
is due to an unmasking of the UASYOR1 rather than a change
in spacing of promoter elements for two reasons. First, several other
YOR1 internal deletions also exhibit this enhancement of
expression (Fig. 3) while each varies significantly in terms of its
position relative to the transcription start site. Second, when these
same internal deletion end points are used to construct chimeric
promoters with PDR5, all are capable of conferring
significant Pdr1p/Pdr3p-independent levels of lacZ expression (Table I). The simplest explanation of these results is that
the
535/
299 UASYOR1 is activated upon removal of the URSYOR1 site. Additionally, the
115 to
50 promoter
segment appears to contribute to 5
end production in YOR1
transcription.
Other S. cerevisiae promoters have been found to have this arrangement of positive elements controlled by linked negative regulatory sites. The CAR1 gene encodes arginase and is highly repressed unless arginine is present as the nitrogen source (31). CAR1 also has a UAS element that is under control of a downstream URS. The negative regulation of this downstream URS is overcome in CAR1 when the inducer of CAR1 expression is present. Interestingly, this URS does not respond to the presence of inducer when it is placed between the UAS and TATA region of the heterologous CYC1 gene (32). The inability of this CAR1 promoter segment to function in a heterologous environment is reminiscent of the activity we have seen for the YOR1 promoter fragments placed upstream of CYC1 or PDR5. We do not yet know if the YOR1-PDR5 chimeras are active due to the presence of an additional positive site or the absence of a negatively acting element that is found in CYC1.
The behavior of the YOR1 UAS region placed upstream of
CYC1-lacZ was unexpectedly complex. This UAS region was not
able to stimulate CYC1-dependent
-galactosidase activity to an appreciable extent, irrespective of
the genetic background examined. Even in the presence of the
PDR1-6 allele, the YOR1-CYC1-lacZ fusion genes
were essentially inactive. This was especially surprising considering
that we previously have shown that an oligonucleotide corresponding to
a PDRE present in the PDR5 promoter is capable of acting as
a UAS when placed upstream of the same CYC1-lacZ test
promoter (15). There are two important differences between the current
study and these previous experiments. First, a different PDRE was used
in each experiment. Second, the YOR1 PDRE was provided in
the context of its wild-type upstream sequence while the
PDR5 PDRE was inserted as an oligonucleotide. These
differences are likely to explain the distinctions in the behavior of
these PDREs as UAS elements for CYC1.
One of the major differences between the PDR5 and CYC1 fusion vectors used to test YOR1 UAS function is likely to be the TATA element provided by each fusion plasmid. The inability of the YOR1 UAS to stimulate CYC1-lacZ expression might be explained by an inability of this UAS region to productively function with the TATA elements contributed by CYC1. Differential TATA utilization has been seen for the HIS3 (33) and HIS4 (34) promoters, consistent with the idea that certain TATA elements are only capable of responding to particular types of transcriptional regulatory proteins. Alternatively, the presence of some feature of the CYC1 promoter may act to inhibit the productive interaction between the YOR1 fragments and the CYC1 TATA region. Further experimentation is required to discriminate between these possible explanations for the unusual promoter specificity of the YOR1 UAS.
Recent experiments have demonstrated that YOR1 is required for resistance to the drug reveromycin A and that YOR1 transcription was inducible by this drug (35). It will be of great interest to determine which DNA sequences are involved in this observed transcriptional induction of YOR1. The work presented here provides important information about candidate regulatory elements that may mediate reveromycin A induction of YOR1 expression. Future work will determine if this drug inducibility of YOR1 acts through the PDRE or the other Pdr1p/Pdr3p-independent transcriptional control elements we have identified.
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ACKNOWLEDGEMENTS |
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We thank Laurence Lambert for constructing and providing the Myc-Pdr1p expression plasmid and David Katzmann for a critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM49825 (to W. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 319-335-7874; Fax: 319-335-7330; E-mail: moyerowl{at}blue.weeg.uiowa.edu.
1 The abbreviations used are: ABC, ATP binding cassette; UAS, upstream activation sequence; bp, base pair; PDRE, Pdr1p/Pdr3p response element; PCR, polymerase chain reaction.
2 T. Hallstrom, unpublished data.
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REFERENCES |
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