From the Laboratoire de Génétique Moléculaire, CNRS UMR8541, Ecole Normale Supérieure, 46 rue d'Ulm 75230 Paris Cedex 05, France
Received for publication, August 21, 2002, and in revised form, January 14, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We demonstrate a genomewide approach to determine
the physiological role of a putative transcription factor, Ylr266,
identified through yeast genome sequencing program. We constructed
activated forms of the zinc finger
(Zn2Cys6) protein Ylr266, and we analyzed the corresponding transcriptomes with DNA microarrays to characterize the up-regulated genes. The direct target genes of Ylr266 were further
identified by in vivo chromatin immunoprecipitation
procedure. The functions of the genes directly controlled by
YLR266c are in agreement with the observed drug-resistance
phenotype of the cell expressing an activated form of Ylr266. These
target genes code for ATP-binding cassette or major facilitator
superfamily transporters such as PDR15, YOR1,
or AZR1 or for other proteins such as SNG1,
YJL216c, or YLL056c which are already known to
be involved in the yeast pleiotropic drug resistance (PDR) phenomenon. YLR266c could thus be named PDR8. Overlaps with
the other PDR networks argue in favor of a new specific role for
PDR8 in connection with the well known PDR regulators
PDR1/PDR3 and YRR1. This strategy to identify
the regulatory properties of an anonymous transcription factor is
likely to be generalized to all the Zn2Cys6
transcription factors from Saccharomyces cerevisiae and
related yeasts.
With the advent of postgenomic approaches that provide a nearly
complete analysis of the cell transcriptome, it has been disconcerting to discover the complexity of the cell genetic response to apparently simple physiological changes (1). This apparent complexity is
likely to reflect the action of underlying regulatory networks that
control gene-expression patterns characteristic of many different genetic changes (2). These transcriptional regulatory networks are
under the combinatorial action of transcription factors, and dissection
of the specific role of each transcription factor offers a good
opportunity to decipher the complexity of genome expression (3).
One of the main challenge further the understanding of genome functions
is to describe the set of genes that are directly regulated by the
different specific transcription factors. DNA microarrays are very
efficient tools to address such questions, but they have to be coupled
with properly designed experiments if one wishes to distinguish direct
and indirect effects of the activity of a transcription factor. Such
data are already available for several transcription factors (4)
that were previously characterized by classical biological approaches.
However, it should be kept in mind that even in Saccharomyces
cerevisisae, many direct target genes of identified or putative
transcription factors are unknown. Any experimental
approach to complete these data relies on the possibility to activate
the relevant transcription factor. We have recently designed an
approach for the artificial activation of yeast
Zn2Cys6 transcription factors. The
Zn2Cys6 family of transcription factors,
exemplified by Gal4, represents more than 25% of the yeast
transcription regulators. Our strategy to activate these factors relies
on the postulate that the DNA-binding domain of transcription factors
fused to a heterologous transcription activation domain isolated from
Gal4 is sufficient to reveal the specific transcriptional response of
the original protein. The target genes, whose expression is
constitutively and strongly activated by the chimeric protein, are then
identified by microarray analyses. We have validated this assumption
and this strategy with Pdr1 and Yrr1 (5, 6), two regulators of the
pleiotropic drug resistance
(PDR)1 phenomenon.
The purpose of the present work is to use this approach to determine
the network of genes regulated by a completely unknown transcription
factor of the Zn2Cys6 family and to deduce the
biological functions of this factor from the properties of its target
genes. The product of the unknown gene, YLR266c, was
chosen because of its structural similarities with YRR1 (5).
Yrr1 is a transcription factor (7, 8) that, when activated, confers
resistance to reveromycin A, oligomycin, and
4-nitroquinoline-N-oxide. The strategy to activate the
putative transcription factor Ylr266 was derived from our previous
experiments with the transcription factors Pdr1 and Yrr1 (5, 6).
Additionally, we have used the technique of chromatin
immunoprecipitation to confirm the in vivo interactions of
the transcription factor Ylr266 with the relevant promoters of the
direct gene targets revealed by transcriptome analyses. These
Ylr266-regulated genes encode proteins like ATP-binding cassette or
major facilitator superfamily transporters. In agreement with these
modifications, we observed that the strain expressing an active form of
Ylr266 is resistant to several unrelated drugs like ketoconazole and
oligomycin; we thus propose to name this gene PDR8.
Connections with other PDR-regulatory networks (namely Pdr1 and Yrr1)
show that Pdr8 is an element of a large regulatory program designed to
allow the cell to adapt to diverse environmental conditions.
This strategy of artificial transcriptional activation, coupled to ChIP
identification of direct targets in vivo, is the first step
to a complete comprehensive description of the transcriptional regulatory networks in yeast and could be extended to other, more complex, organisms.
Yeast Strains and Growth Conditions--
The S. cerevisiae strain BY 4742 (MATa his3D 1 leu2D 0 lys2D 0 ura3D 0)
was used in this study. Cells were grown on YPD (1% yeast extract, 2%
bactopeptone, 2% glucose) or minimal synthetic medium (0.67% yeast
nitrogen base, 2% carbon source (glucose, galactose, or glycerol + ethanol)) supplemented with appropriate amino acids at 40 mg/ml.
Osmotic or pH sensitivity and drug-resistance assays were performed by
spot tests with serial dilutions (9). Escherichia coli TG1
(K-12 Disruption of YLR266c--
The BY 4742 Plasmids and Cloning of YLR266c--
Plasmid pBFG1-PDR8 was
obtained by insertion of NotI, PstI-digested
PCR-amplified YLR266c ORF to the equivalent cloning region of pBFG1
described previously (13). For PCR amplification of YLR266c ORF, the
genomic DNA of BY 4742 strain and the following primers were used:
YLR266-NTER
(5'-GCAAGGCCTGTTAACATCGATAGCGGCCGCATGGATGGATCCCATTTTCCTATG-3') and YLR266-CTER
(5'-TGATGCGGTCCTCCTGCAGGGCCCTTATAAATCGAAATGATATTGTTTATAAAATTTC-3'). Plasmid pCB-PDR8 was prepared by insertion of NcoI,
EcoRI fragment of pBFG1-266F to pCBI, a no-HA epitope
containing version of pCB described previously (6). Plasmid
pCB-PDR8*GAD was obtained by homologous recombination in yeast of
EcoRI-digested pCBI-PDR8 with PCR-amplified
"SV40-GAL4AD" fragment of pACT2 (Clontech). The
"SV40-GAL4AD" DNA fragment contains the nuclear localization sequence of SV40 T antigen connected with GAL4p activation domain. The
primers used were 3'GAL4ADpYES
(5'-TGTAAGCGTGACATAACTAATTACATGATGCGGCCCTTATTACTCTTTTTTTGGGTTTGGTGGGGTATC-3') and 266DB/gal
(5'-TATGTGTTTGGCCCCACTTCCTGGAAAACTTTATCTTTGTTTGATAAAGCGGAATTAATTCCCGAGCCT-3') for pCBI-PDR8*GAD preparation. The expression of cloned gene was verified by Western blot analysis (12) using mouse anti-HA IgG as first
antibody (Babco) and anti-mouse IgG horseradish peroxidase-conjugated as second antibody (Promega). The signal was visualized using ECL kit
(Amersham Biosciences).
RNA Isolation and Northern Blot Analyses--
Total RNA was
prepared using a hot phenol extraction method. mRNA was purified
using Micro-FastTrack 2.0 kit (Invitrogen). Northern blot analyses were
performed with 30 mg of total RNA using standard procedures (12).
Microarray Analyses--
The microarray experiments were
performed using glass slides containing spots of open reading frames of
S. cerevisiae genome obtained from Hitachi
Software. The detailed microarray protocols are available on
request. In each experiment, 2 mg of mRNA were used for
cDNA preparation. The cDNA corresponding to cells expressing transcription factor was labeled with Cy3-dUTP and cDNA from
control cells with Cy5-dUTP. The arrays after hybridization were
scanned using Genepix 4000A scanner (Axon) and signals analyzed with
Genepix 3.1 software (Axon).
Bioinformatic Analyses--
We filtered the data to exclude
artifacts, saturated spots, and low signal spots. Assuming that most of
the genes have unchanged expression, the Cy3/Cy5 ratios were normalized
using the median of all of the ratios for each experiment (14). Each
experiment was performed at least twice, and we kept only the genes
exhibiting a reproducible up- or down-regulation. The gene-clustering
analyses were performed using "J-express" software (15). For motif
search in the promoter (between Chromatin Immunoprecipitation Assay--
The chromatin
immunoprecipitation assay was performed as adapted from Ref. 17.
Briefly, cells expressing HA-tagged Ylr266 under the control of the
GAL1 promoter were collected at different times after
galactose induction. Protein-nucleic acid complexes were fixed by
formaldehyde treatment (15 min at 20 °C). Lysates were prepared by
glass bead grinding followed by sonication to shear chromatin to an
average length of 1-3 kb. A 20-µl aliquot of the lysate was saved as
the input fraction. After incubation with mouse anti-HA monoclonal
antibody (Babco), 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 (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 NaHCO3. 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
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 PCR. The
different primer sets used for the 9 promoters are available on
request. Linearity of PCR reactions was assayed by multiple
template dilutions of input (IN) and immunoprecipitated (IP) DNA as
indicated by Larscham and Winston (18). Gel images were captured by a
fluorescent imager (Fuji) and quantified with Image Gauge (version
3.41) software. Calculation of the enrichment factor was made as
follows: 1) the values at 10 h and 14 h were normalized to a
reference product of YER184c promoter, amplified in the same reaction
mixture. YER184c is a gene determined by microarray analyses to be
transcriptionally stable in the conditions used here (data not shown).
Moreover, we checked that in the described ChIP experiments its
promoter behaves as the actin promoter ACT1 (data not
shown). Mean values were retained as the crude promoter-occupancy
value. 2) A general basal value was obtained when the transcription
factor Ylr266 was not produced. A mean basal value was derived from the
twelve independent experiments. The enrichment factor was obtained by dividing the normalized promoter-occupancy value by the mean basal value.
Design of Ylr266*GAD, an Activated Form of Ylr266, and
Overproduction of Ylr266--
We recently developed a general
genomewide strategy for the systematic analysis of regulatory networks
under the control of Zn2Cys6 transcription
factors (6). This approach is based on the conditional expression of a
chimeric gene encoding the DNA-binding region of the transcription
factor studied fused to the transcription-activating domain of Gal4. We
used a similar strategy with YLR266c to create a chimera
called Ylr266*GAD (Fig. 1). The key point
in this approach is that the chimera must contain a specific
DNA-binding domain devoid of inhibitory activity known to be contained
in the central flanking domain of this transcription factor family (5).
Based on the assumption that the inhibitory region should be highly divergent among members of the Zn2Cys6 zinc
finger family, we could define a DNA-binding domain from amino acids
1-149 that was included in the protein Ylr266*GAD (Fig. 1). Moreover,
we also overexpressed the complete form of Ylr266 under the control of
the GAL1 promoter. This allowed us to analyze the
transcriptome modifications induced by different forms of Ylr266 under
different experimental conditions (Fig. 1).
A Genomewide Search of Ylr266 Target Genes--
We investigated
the YLR266c regulation network by microarray experiments to
compare transcriptomes generated along the time-course production of
either the Ylr266*GAD or the complete transcription factor. We
performed three microarray analyses, testing the Ylr266 overproduction
against the BY4742 wild-type strain and three microarray experiments in
which the strain producing Ylr266*GAD (pCB-PDR8*GAD) was tested against
the control strain producing only the GAD domain. All of these data
were clustered, and we then assumed that the minimum set of genes
specifically activated in either conditions should represent the genes
actually activated by Ylr266. The complete analysis criteria and data
are available on request.
We identified by cluster analysis (Fig.
2A) one distinct group of
genes that was similarly activated by the artificial chimera Ylr266*GAD
and by the overproduction of the complete protein. In this group
AZR1, CTT1, GTT2, YOR1,
YJL216c, YLL056c, and YIL121w were strongly up-regulated in all
conditions and clearly distinguish from the others by principal
component analysis (Fig. 2B). In addition, eight genes are
moderately activated (Fig. 2) and are likely to be indirectly activated
by Ylr266 because no direct interactions were revealed by ChIP (see the
case of GPH1, Fig. 3). Minor quantitative
variations of gene activation levels by either forms of Pdr8 (Fig.
2A) are likely to reflect amount variations of each Pdr8
form rather than differences in specificity. This is supported by
transcriptome analyses in which different amounts of factors were
produced (data not shown).
Specific Promoter Occupancy by Ylr266 in Vivo--
To discriminate
among the putative Ylr266 target genes those which actually interact,
in vivo, with the transcription factor, a tagged version of
the wild-type form of the Ylr266 protein was progressively expressed
in vivo and covalently linked with formaldehyde to its
target sites at different times. DNA fragments ranging from 1 to 3 kb
long that were specifically cross-linked to Ylr266 were purified by
immunoprecipitation. PCR analysis of immunoprecipitated DNA confirmed
the specific association of Ylr266 with promoters of
AZR1, CTT1, YOR1, YJL216c, and YLL056c
(Fig. 3). The time-course analysis of the occupancy level of the
different promoters is a good evidence of their direct in
vivo interaction with Ylr266/Pdr8. The situation for the rest of
the up-regulated group (Fig. 2A) is less clear-cut. Some
genes are positive in Chromatin IP (SNG1, PDR15)
whereas others are negative (GPH1, YGR052w). This further supports the necessity to associate ChIP and microarray analyses to
identify direct target genes.
It is worth noting that most of the promoters that are positive in ChIP
experiments contain sequences that are good candidates to be recognized
by a zinc finger protein. Two more or less perfect CGG or CCG triplets
repeated in dyads with a constant distance in between are a consensus
signature of the binding site recognized by the
Zn2Cys6 transcription factors studied so far. A
sequence derived from TCCG(A/T/C)GGA is found in all but one of these
promoters. The AZR1 promoter, which lacks this sequence, has
nevertheless two closely related sequences TCCGCTGT and TCCGCGCT which
are precisely localized in the promoter fragment which is positive in
electrophoretic mobility-shift assays experiments (data not shown).
A Function for Ylr266--
It is noteworthy that at least seven of
the eight genes that are primary targets of Ylr266 are involved in
stress response or multidrug resistance. This last function is carried
out by ATP-binding cassette (YOR1, PDR15) or major
facilitator superfamily (AZR1) membrane transporters. Other
membrane proteins (SNG1) can also be involved. Such
modifications of the plasma membrane induced by the activation of
Ylr266 do suggest that the corresponding strain has altered drug
sensitivity. We addressed this question using ketoconazole and
oligomycin and testing serial strain dilutions (Fig.
4). Clearly, the production of Ylr266
confers resistance of the cell to both drugs. It is also very clear
that the overproduction of Ylr266 leads to sensitivity to
Na+, Li+, H+, and to cationic drugs
such as hygromycin B, whereas high concentrations of KCl have no
significant effects (Fig. 4). All of these phenotypes are probably
connected to membrane alterations induced by Ylr266. Because YIL121w,
regulated by YLR266c, belongs to the H+ antiporter DHA12
family, it is a good candidate to be involved in these phenotypes. On
the other hand, resistance to ketoconazole or oligomycin are likely to
be related to the overexpression of AZR1 and
YOR1. These regulatory properties of Ylr266 are reminiscent of the phenotype controlled by PDR connected regulators (19), we then
propose to name the ORF YLR266c as PDR8.
Studies of New Transcription Factors with Activated Chimeras and
Microarrays--
In this report we present the first evidence that the
properties of a completely unknown yeast transcription factor can be inferred from its genomewide properties. Using global approaches, we
could show that eight genes are directly regulated by Ylr266/Pdr8 which
interact, in vivo, with their promoters. The drug-resistance phenotype observed when Ylr266/Pdr8 is either overproduced or activated
is in agreement with the fact that at least seven of the eight
regulated genes code for products involved in the properties of the
plasma membrane. These functional properties of YLR266c substantiate
the new name PDR8. We previously showed that known transcription factors of the zinc finger family like Pdr1 (6) or Yrr1
(5) can be engineered in a mini active form, keeping all of the
DNA-binding specificity of the original protein. This is also the case
for Pdr8, which presents further evidence that the short DNA-binding
domain (in that case 173 amino acids long) of these zinc-finger
transcription factors contain all of the specificity of the protein,
the rest of the factor being probably involved in its regulation. This
point is strongly supported by the chromatin immunoprecipitation
experiments, which demonstrate that the wild-type form of Pdr8 can
interact, in vivo, with the promoters of the genes activated
by the engineered transcription factor, thus giving credence to our
general experimental approach. Such a specificity of the DNA-binding
domain is all the more surprising because the suggested sequence
features recognized by Pdr8 in the relevant promoters are very similar
to those of the already characterized UASPDRE for Pdr1 (20)
or UASYRRE for Yrr1 (5). On the other hand, it is worth
noting that no false-positive response arise from the numerous similar
putative signals present in the genome. This again suggests that
unknown elements of the local environment of these signals exert a
discriminating role on the UAS recognition process. It is usually
believed, as in the case of the well known zinc finger transcription
factor Gal4, that chromatin obstructs most sites and leaves only a few
relevant ones accessible (21, 22).
Plasma Membrane Modifications Induced by Pdr8--
The Pdr8 target
genes cluster in specific cellular functions mainly related to plasma
membrane properties. Thus, all of the eight target genes, except
CTT1, code for proteins that have at least one transmembrane
segment. Some of these genes like AZR1 or YOR1
code for plasma membrane-localized proteins and most of them code for
small molecule transporter like SNG1, or for active transporter involved in the multidrug resistance phenomenon like AZR1 (a member of the major facilitator superfamily) or like
PDR15 and YOR1 (members of the ATP-binding
cassette superfamily). In two cases, we have a better idea of the
function of the transporter: YOR1 and AZR1 can
confer resistance to oligomycin and azoles, respectively (23, 24).
These features are in agreement with our observed phenotypes when Pdr8
is overproduced (Fig. 5). Interestingly, the set of genes regulated by PDR8 is distinct but
overlapping with other PDR regulators.
Connections with the PDR Transcriptional Regulatory
Network--
Connections with other PDR regulatory networks show that
PDR8 is an element of a large regulatory program designed to
allow the cell to adapt to diverse environmental conditions. If the PDR8 network overlaps with the control of the regulation of
CTT1 (the stress response program), it is, however, more
involved in the drug response program. PDR1/PDR3,
YRR1, and PDR8 are four zinc-finger transcription
factors for which the sets of direct target genes have been described
(5, 6, 20, and this work). Important overlap exists between the three
regulatory networks (Fig. 5). Strikingly, all of the promoters that are
recognized by these four related transcription factors contain a signal
sequence close to the UASPDRE sequences that have been
previously defined (6, 20). This is all the more surprising because
each transcription factor activates a specific set of target genes. For
instance, the PDR8 network is mostly included into the
YRR1 network because five of eight genes are controlled by
both Pdr8 and Yrr1. This may be connected to the close similarity
between the different DNA-binding domains. The phenotypes conferred by
the activation of either Yrr1 or Pdr8, however differ in agreement with
the sets of genes that are overexpressed in these two conditions.
Typically, genes like FLR1 or SNQ2 which are
activated by Yrr1 and not regulated by Pdr8, are involved in the
resistance to benomyl (25, 26) or to
4-nitroquinoline-N-oxide (27). This is in agreement with the
observed phenotypes of the strains activated either for Yrr1 or for
Pdr8 and which are, respectively, resistant or sensitive to these two
drugs. On the other hand, a common resistance to azole derivatives like
ketoconazole could be observed in the two type of strains in
connection, probably, with the fact that they both overexpress
AZR1 (24). These are examples of the strong overlaps that
characterize the regulatory networks controlled by Pdr1/Pdr3, Yrr1, and
Pdr8. This however should not overlook the phenotype specificity
controlled by each transcription factor. This family of binuclear
cluster transcription factors appears to contain a battery of
structurally related proteins that specifically control the responses
of the yeast cell to its variable toxic environment. Recent systematic
analyses of strains carrying deletions of zinc cluster genes have
suggested that, directly or indirectly, one gene might be a repressor
and seven new genes might be activators of the PDR phenomenon (28). It
remains to determine how the regulatory networks of these twelve
PDR-related transcription factors actually overlap and to decipher
their own intricate regulation (8,
29).2 All of this information
strongly suggests that the yeast S. cerevisiae has
elaborated a highly sophisticated system to cope with the wide variety
of biotic toxicants present in the environment. More generally, this
study, which demonstrates the possibility to decipher the set of target
genes directly connected to virtually any transcription factor, opens
the way to a systematic analysis of the elementary networks that
compose the yeast transcriptional network.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
REFERENCES
(lac-pro) supE thi hsdD5/F' traD36 proA+B+ lacIq lacZ
M15) was used
for plasmid constructions.
ylr266c:LEU2 strain
was prepared by one-step gene replacement. The LEU2
disruption marker was PCR-amplified from plasmid pRS425 described
previously (10) using oligonucleotides YLR266-up
(5'-CATCAATCATAAATACATTTATAAATCGAAATGATATTGTAGATTGTACTGAGAGTGCAC-3') and YLR266-down
(5'-GAATAAGAAAGAGGCAAACGGCACTTAGTTTGTTGGGATGCTGTGCGGTATTTCACACCG-3'). The primers used contain a 20-bp flanking region around the gene LEU2 in pRS425 and 40 bp upstream and downstream from
YLR266c initiation and stop codons, respectively. The prepared PCR
fragment was used for yeast transformation using LiCl procedure (11). Yeast colony PCR (11) and Southern blot analysis (12) allowed us to
determine the correctly disrupted clones. The primers used for yeast
colony PCR were YLR266-pro (5'-CACGTGGTTGGTACGGGAC-3') a 19-bp upstream
region of YLR266c ORF and LEU2-pro (5'-TCTAAAAGAGAGTCGGATGC-3') a 20-bp
internal region of LEU2 ORF.
800 and +1) of the up-regulated
genes, we used Regulatory Sequence Analysis tools (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Experimental strategies to express activated
forms of the putative transcription factor Ylr266. The upper
line schematizes the general structure of the protein Ylr266 by
analogy with the known transcription factors of the
Zn2Cys6 family. On the right,
pCB-PDR8 represents two N-terminally tagged version of the gene which
can be expressed under the control of the GAL1-10 or
PGK1 promoters, respectively. On the left, a
short engineered form of the same gene is represented. PCB-PDR8*GAD
contains the sequence coding for the DNA-binding domain (149 aa) of
YLR266c. It contains the sequence coding for three HA epitopes, a
nuclear targeting signal (from SV40), and the Gal4 activation domain.
Most of these sequences and the host vector pCB has been previously
described (6).
View larger version (32K):
[in a new window]
Fig. 2.
Genes up-regulated by
YLR266c. A, genes up-regulated by the
two activated forms of Ylr266. Cluster analyses (B) have
been conducted and represented as previously described (30).
Columns 1 and 2 represent, respectively, the
global results of the analyses conducted with pCB-PDR8 and pCB-PDR8*GAD
(Fig. 1). B, microarray results for the expression of the
two different forms of YLR266c described in Fig. 1 were analyzed by PCA
analysis (31). This method clearly distinguishes five genes, which are
also distinguished in the cluster analysis of the up-regulated genes as
being strongly up-regulated in the two conditions (A,
left).
View larger version (49K):
[in a new window]
Fig. 3.
Promoter-occupancy of Ylr266 in
vivo. Occupancy of a variety of promoters was measured in
vivo by performing chromatin immunoprecipitation experiments with
a -HA antibody. Cells containing HA-tagged Ylr266 under
control of GAL1-10 promoter were analyzed at different
times after galactose induction and prior to immunoprecipitation (lane
Start). Top lane, control corresponds to the same
analysis of a strain carrying a vector devoid of the Ylr266c
gene. The presence of promoter fragments listed at left was analyzed by
the use of PCR and the appropriate primers (see "Experimental
Procedures"). Each specific promoter analysis was conducted in
parallel with the analysis of the promoter of YER184c, a promoter known
to be independent of Ylr266. The results corresponding to this negative
control are indicated by an asterisk. A quantitative value
of the enrichment factor was obtained by taking into account the two
experiments after 10 and 14 h, respectively, of protein production
and the different negative controls (see "Experimental
Procedures").
View larger version (67K):
[in a new window]
Fig. 4.
The phenotypes of the strains overexpressing
YLR266c are in agreement with the corresponding transcriptome
analyses. Wild-type strain BY4742 and the isogenic strains either
deleted or overexpressing YLR266c were grown in YPD and cells,
resuspended in water, serially diluted, and spotted on galactose
plates, complemented as indicated.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
CONCLUSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 5.
Overlaps of Ylr266 controlled gene network
with the regulatory networks controlled by PDR1/PDR3
and YRR1. The set of genes directly activated by the
four transcription factors have been compared. All of the
Ylr266-up-regulated genes but CTT1 are shared with
PDR1/PDR3 and/or YRR1 networks. The
common genes (see boxes) are in agreement with the common
phenotypes (drug names in italic) when the corresponding
transcription factors are activated.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Goffeau for many stimulating discussions and advice. We thank V. Tanty and C. Blugeon for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from CNRS (Puces a ADN 2A2112) and Association pour la Recherche contre le Cancer (5691).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.
Both authors contributed equally to this work.
§ Present address: Comenius University, Dept. of Microbiology and Virology, Faculty of Natural Sciences, Mlynska Dolina B2, 842 15 Bratislava, Slovak Republic.
¶ Supported by a European Commission long-term fellowship (Combating of MDR, QLK2-CT.2001.02377).
To whom correspondence should be addressed. E-mail:
jacq@biologie.ens.fr.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M208549200
2 A. L.-D., unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDR, pleiotropic drug resistance; ChIP, chromatin immunoprecipitation; ORF, open reading frame; HA, hemagglutinin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Marc, P., Devaux, F., and Jacq, C. (2001) Nucleic Acids Res. 29, 63-64 |
2. | Hughes, T. R., Marton, M. J., Jones, A. R., Roberts, C. J., Stoughton, R., Armour, C. D., Bennett, H. A., Coffey, E., Dai, H., He, Y. D., Kidd, M. J., King, A. M., Meyer, M. R., Slade, D., Lum, P. Y., Stepaniants, S. B., Shoemaker, D. D., Gachotte, D., Chakraburtty, K., Simon, J., Bard, M., and Friend, S. H. (2000) Cell 102, 109-126[Medline] [Order article via Infotrieve] |
3. | Simon, I., Barnett, J., Hannett, N., Harbison, C. T., Rinaldi, N. J., Volkert, T. L., Wyrick, J. J., Zeitlinger, J., Gifford, D. K., Jaakkola, T. S., and Young, R. A. (2001) Cell 106, 697-708[CrossRef][Medline] [Order article via Infotrieve] |
4. | Devaux, F., Marc, P., and Jacq, C. (2001) FEBS Lett. 498, 140-144[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Le Crom, S.,
Devaux, F.,
Marc, P.,
Zhang, X.,
Moye-Rowley, W. S.,
and Jacq, C.
(2002)
Mol. Cell. Biol.
22,
2642-2649 |
6. |
Devaux, F.,
Marc, P.,
Bouchoux, C.,
Delaveau, T.,
Hikkel, I.,
Potier, M. C.,
and Jacq, C.
(2001)
EMBO Rep.
2,
493-498 |
7. |
Cui, Z.,
Hirata, D.,
Tsuchiya, E.,
Osada, H.,
and Miyakawa, T.
(1996)
J. Biol. Chem.
271,
14712-14716 |
8. |
Zhang, X.,
Cui, Z.,
Miyakawa, T.,
and Moye-Rowley, W. S.
(2001)
J. Biol. Chem.
276,
8812-8819 |
9. |
Goossens, A.,
de La Fuente, N.,
Forment, J.,
Serrano, R.,
and Portillo, F.
(2000)
Mol. Cell. Biol.
20,
7654-7661 |
10. | Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, 115-132[CrossRef][Medline] [Order article via Infotrieve] |
11. | Adams, A., Gottschling, D. E., Kaiser, C. A., and Stearns, T. (1997) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
12. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
13. | Chardin, P., Camonis, J. H., Gale, N. W., van Aelst, L., Schlessinger, J., Wigler, M. H., and Bar-Sagi, D. (1993) Science 260, 1338-1343[Medline] [Order article via Infotrieve] |
14. |
Marc, P.,
and Jacq, C.
(2002)
Bioinformatics
18,
888-889 |
15. | Dysvik, B., and Jonassen, I. (2001) Bioinformatics 17, 369-370[Abstract] |
16. | van Helden, J., Andre, B., and Collado-Vides, J. (2000) Yeast 16, 177-187[CrossRef][Medline] [Order article via Infotrieve] |
17. | Strahl-Bolsinger, S., Hecht, A., Luo, K., and Grunstein, M. (1997) Genes Dev. 11, 83-93[Abstract] |
18. |
Larschan, E.,
and Winston, F.
(2001)
Genes Dev.
15,
1946-1956 |
19. | Balzi, E., and Goffeau, A. (1995) J. Bioenerg. Biomembr. 27, 71-76[Medline] [Order article via Infotrieve] |
20. | DeRisi, J., van den Hazel, B., Marc, P., Balzi, E., Brown, P., Jacq, C., and Goffeau, A. (2000) FEBS Lett. 470, 156-160[CrossRef][Medline] [Order article via Infotrieve] |
21. | Marmorstein, R., Carey, M., Ptashne, M., and Harrison, S. C. (1992) Nature 356, 408-414[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Ren, B.,
Robert, F.,
Wyrick, J. J.,
Aparicio, O.,
Jennings, E. G.,
Simon, I.,
Zeitlinger, J.,
Schreiber, J.,
Hannett, N.,
Kanin, E.,
Volkert, T. L.,
Wilson, C. J.,
Bell, S. P.,
and Young, R. A.
(2000)
Science
290,
2306-2309 |
23. |
Decottignies, A.,
Grant, A. M.,
Nichols, J. W.,
de Wet, H.,
McIntosh, D. B.,
and Goffeau, A.
(1998)
J. Biol. Chem.
273,
12612-12622 |
24. | Tenreiro, S., Rosa, P. C., Viegas, C. A., and Sa-Correia, I. (2000) Yeast 16, 1469-1481[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Nguyen, D. T.,
Alarco, A. M.,
and Raymond, M.
(2001)
J. Biol. Chem.
276,
1138-1145 |
26. | Tenreiro, S., Fernandes, A. R., and Sa-Correia, I. (2001) Biochem. Biophys. Res. Commun. 280, 216-222[CrossRef][Medline] [Order article via Infotrieve] |
27. | Servos, J., Haase, E., and Brendel, M. (1993) Mol. Gen. Genet. 236, 214-218[Medline] [Order article via Infotrieve] |
28. |
Akache, B.,
and Turcotte, B.
(2002)
J. Biol. Chem.
277,
21254-21260 |
29. | Delahodde, A., Delaveau, T., and Jacq, C. (1995) Mol. Cell. Biol. 15, 4043-4051[Abstract] |
30. |
Eisen, M. B.,
Spellman, P. T.,
Brown, P. O.,
and Botstein, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14863-14868 |
31. | Raychaudhuri, S., Stuart, J. M., and Altman, R. B. (2000) Pac. Symp. Biocomput. 1, 455-466 |