From the Molecular Biology Program and
¶ Department of Physiology and Biophysics, University of Iowa,
Iowa City, Iowa 52242 and the § Department of Molecular
Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, Higashi-Hiroshima, 739-8526 Japan
Received for publication, November 27, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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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 gene
YOR1. 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 (3-5) or
YOR1 (6, 7). Overexpression of these ABC transporters is
required for the Pdr phenotype and most commonly results from genetic
lesions that activate function of the
Cys6-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/Pdr3p-independent 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 3× 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.
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 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 wild-type 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 5' to
3'. The plasmids pXTZ111 to 119 were constructed by replacing the
upstream activation sequence of the CYC1 gene with a DNase I Footprint Assay--
The vector pOTS-Nco12
expressing a Myc-tagged 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 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-µl
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 µg 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 µg of RNase A was added to remove
RNA. Protein was eluted from the Sepharose beads by treating with 1% SDS/0.1 M HaHCO3. Cross-links were reversed by
adding 20 µl 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 µl of TE. 1 µl 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
ATR1+40 (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 µl 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 YRR1 Activates YOR1 Gene Expression Independent of the PDRE and Its
Binding Proteins--
We have previously demonstrated that Pdr1p and
Pdr3p regulate YOR1 gene expression via directly binding to
the single PDRE (Pdr1p/Pdr3p response element) located in the
YOR1 promoter (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 wild-type 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
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 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 5'-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
A 5' deletion to 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 leads to overexpression of Pdr1p target
genes and drug hyper-resistance (8). Transformants were assayed for YRR1-dependent
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 5'-truncated versions of the
YRR1-lacZ reporter construct was prepared to localize
important regulatory sequences. Transformants were processed for
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 5'-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
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
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
Alterations at the 3'-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 ~400% increase in expression, whereas the PDR1-3
allele increased 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 mutations 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 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 requires 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.
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, 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-Cys6 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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+ 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).
S. cerevisiae strains used
269
to
230 fragment of YRR1 promoter. YRR1(
269)F (gat cCG CGG AAA TTA
GAA AAA CGT TAA AAG GTT CCA TGC A) and YRR1(
230)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 forward-oriented 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
A600 = 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 ± S.D.
-32P 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 A600 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, introduction of the
yrr1
deletion mutation into the pdr1
,
pdr3
reduced the ability of the resulting triple mutant
to tolerate oligomycin. These data indicate that, although
YRR1 does contribute to oligomycin resistance in S. cerevisiae, its contribution is masked in the presence of Pdr1p
and Pdr3p.
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Fig. 1.
Yrr1p contributes to oligomycin
resistance. Strains of the indicated genotypes were grown to an
A600 of 1 and 1000 cells/5 µl was placed on
YPGE medium containing a gradient of oligomycin (maximal
concentration = 0.3 µg/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.
-galactosidase activities were determined (Fig.
2). Expression of the wild-type
YOR1-lacZ fusion gene was increased by ~4-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.
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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 wild-type 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.
, 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.
-galactosidase activities were then
determined for each transformant (Fig.
3).
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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 5'-noncoding DNA remaining for the
5'-truncation mutants, whereas the DNA that has been deleted is shown
for the internal deletion mutants.
222 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
129 bp upstream eliminated the response to Yrr1-1p. An
internal deletion lacking the
190 to
129 region of the
YOR1 promoter was still induced in the presence of Yrr1-1p,
whereas the
299 to
50 deletion derivative lacked any response to
the YRR1-1 allele. The elevated expression of the
299 to
50 internal deletion construct has been observed before (28). These
data suggest that Yrr1p acts through the
222 to
190 bp region
upstream of the YOR1 transcription start site. Note that the
PDRE corresponds to position
218 to
209 but is not required to
mediate the response of YOR1 to Yrr1p (see above).
-galactosidase using a
chemiluminescence method owing to the low level expression of
YRR1.
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Fig. 4.
Identification of a PDRE in the
YRR1 promoter. A, the DNA sequence of
an element in the YRR1 5'-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 wild-type (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 5'
32P-end-labeled probe containing the YRR1 PDRE was prepared
as described under "Materials and Methods." This probe was
incubated with either 10 or 20 µl of crude extract from bacterial
cells expressing the DNA binding domain of Pdr1p (Pdr1p,
volume indicated by bar of increasing width), 20 µl 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).
-galactosidase activity measurements as described above (Fig.
5).
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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 5'-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.
269 to
260 and mapped to this Yrr1p-responsive region.
271 to
220 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.
269 to
230 in the
YRR1 5'-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).
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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 ~1 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."
-galactosidase activity by ~300%. Substitution and deletion mutations between positions
242 and
231 had no effect
on Pdr1p or Yrr1p regulation. However, clustered mutations located near
the PDRE (
259 and
255 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.
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Fig. 7.
C-terminal insertions activate Yrr1p or
Yor172wp function. A, strains expressing wild-type
Yrr1p or two different insertions of a 3X HA epitope tag sequence
placed between codons 695 and 696 (Yrr1p::3X HA-695) or
between codons 730 and 731 (Yrr1p::3X HA-730) 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.
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[in a new window]
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (10K):
[in a new window]
Fig. 9.
Summary of Yrr1p and Yor172wp interactions
with known PDR loci. PDR1 and
PDR3 both encode homologous zinc cluster transcription
factors that act to control the expression of both ABC
transporter-encoding genes (SNQ2, YOR1,
PDR5) and YRR1. Yrr1p and Yor172wp both control
expression of SNQ2 and YOR1 but not
PDR5. Yrr1p is autoregulated and may also be controlled by
Yor172wp. Positive effects on gene expression mediated by the
Yrr1p/Yor172wp pair are indicated by the dashed lines, whereas similar
effects of Pdr1p/Pdr3p are shown by a solid line.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Tony Weil and Steph Schroeder for providing the chromatin immunoprecipitation protocol and the HA-tagged form of Tbp1p.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed: Dept. of
Physiology and Biophysics, 5-612 Bowen Science Bldg., University of
Iowa, Iowa City, IA 52242. Tel.: 319-335-7874; Fax: 319-335-7330;
E-mail: moyerowl@blue.weeg.uiowa.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010686200
2 X. Zhang, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: ABC, ATP-binding cassette; Pdr, pleiotropic drug resistance; PDRE, Pdr1p/Pdr3p response element; HA, hemagglutinin; cre, recombinase; 4-NQO, 4-nitroquinoline-N-oxide; PCR, polymerase chain reaction; bp, base pair(s); ChIP, chromatin immunoprecipitation.
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REFERENCES |
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