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. 2002 Apr;22(8):2642-9.
doi: 10.1128/MCB.22.8.2642-2649.2002.

New insights into the pleiotropic drug resistance network from genome-wide characterization of the YRR1 transcription factor regulation system

Stéphane Le Crom  1 , Frédéric Devaux, Philippe Marc, Xiaoting Zhang, W Scott Moye-Rowley, Claude Jacq
Affiliations

New insights into the pleiotropic drug resistance network from genome-wide characterization of the YRR1 transcription factor regulation system

Stéphane Le Crom et al. Mol Cell Biol. 2002 Apr.

Abstract

Yrr1p is a recently described Zn(2)Cys(6) transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

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Figures

FIG. 1.
FIG. 1.
Experimental strategy and physiological validation for construction of a chimeric transcription factor consisting of the DNA-binding domain of Yrr1p and the activating domain of Gal4p. (A) Schematic representation of the various YRR1 constructs used in this study. At the top, the YRR1 gene is shown with its three main regions (DNA-binding domain, inhibitory region, and activating domain). Below is the YRR1 gain-of-function mutant with a 3-HA tag insertion in the activating domain. The two chimeric constructs encoding the N-terminal amino acids of Yrr1p and the activating domain of GAL4 transcription factor are also represented. The short (Yrr1S*GAD) and long (Yrr1L*GAD) chimeras differ by 16 amino acids, as indicated in panel B. (B) Detail of an amino acid alignment including the closest homologs of YRR1 and PDR1 previously used in similar experimental approaches (9). The limit of the YRR1 region chosen for the two constructs is shown for the short (Yrr1S*GAD) and the long (Yrr1L*GAD) forms. The end of the Pdr1p chimera is shown with the beginning of the first inhibitory region for comparison. Using the PhosphoBase online analysis tool (16), we identified three sites in Yrr1p that could be phosphorylated by protein kinase C (square) and protein kinase A (circle). (C) Phenotypes observed for the Yrr1*GAD chimeras. Serial cell dilutions of various strains, from the most concentrated (on the left) to the least concentrated (on the right), were plated after induction with galactose for 20 h in the liquid phase. For the FY and W303 strains, we compared Yrr1S*GAD (S) and Yrr1L*GAD (L) with their corresponding wild-type (WT) strains. We also compared the phenotype of the gain-of-function (Gof) mutant with that of the SEY wild-type strains. Three different drugs were tested: 4-NQO at a concentration of 2 μg/ml, oligomycin at 6 μg/ml, and cycloheximide at 2 μg/ml. The plates were photographed after 72 h for cycloheximide, after 120 h for 4-NQO, and after 144 h for oligomycin. More complete drug resistance analysis is available via the Internet (http://www.biologie.ens.fr/yeast-publi.html).
FIG. 2.
FIG. 2.
Genes upregulated by YRR1. (A) Microarray results for the gain-of-function mutant and chimera experiments were analyzed by PCA (11) to determine the two key variables plotted on the two axes (PCA1 versus PCA2). This method clearly distinguished repressed genes (left, green) from activated genes (right, red) and the majority of genes not regulated by YRR1 (central, black). Genes specifically activated in the chimera experiments are marked in blue. The upregulated genes are distributed along a gradient, which may indicate the existence of different subgroups of genes. (B) Cluster analyses have been performed (12) and are shown here. The upper part of the diagram presents the results of six independent experiments in which a strain constitutively producing the gain-of-function mutant Yrr1p protein was compared with the SEY strain used as a control. The lower part of the diagram shows the results of three independent experiments in which the strain with the Yrr1*GAD construct was subjected to galactose induction for 4 h and compared with the wild-type FY strain, used as a control. Genes were identified as described in the text. The color scale is indicated in the lower part of the figure. The complete set of data is available at http://www.biologie.ens.fr/yeast-publi.html. (C) Time course of the expression of Yrr1p-regulated genes under galactose induction. Northern blot analyses were performed for each regulated gene identified by microarray experiments. Total RNA (10 μg) was deposited on the gel after induction in galactose medium for various lengths of time, from 30 min to 16 h. Three groups were defined according to the quantification profile obtained from Northern blot analysis (see the related website http://www.biologie.ens.fr/yeast-publi.html). For these three groups, mean RNA levels (arbitrary units) are plotted against the duration of galactose induction (in hours). Group 1 contains YLR046C, YLL056C, AZR1, SNG1, FLR1, YPL088W, and YLR179C, the most strongly induced genes. Group 2 comprises APD1, SNQ2, and PLB1, which display high levels of basal expression. Finally, group 3 consists of YGR035C, YMR102C, and YKL051W, which show an induction only after 2 h in galactose. Error bars are standard deviations.
FIG. 3.
FIG. 3.
Specific interactions between Yrr1p and the promoters of target genes. (A) EMSA of the main YRR1-regulated genes. Bioinformatic analysis (27) of the promoters of the 15 genes up-regulated by Yrr1p revealed a 200-bp segment with similar features in each case. These similar fragments were amplified by PCR, and EMSAs were performed with the total protein extract from an FY strain in which YRR1 was deleted (ΔY), a wild-type strain (FY), and an FY strain producing Yrr1S*GAD (FS). The precise location of the promoter region selected for each gene is indicated below the autoradiograph. Specific (narrow arrowhead) and nonspecific (thick arrowhead) DNA-binding sites are indicated. (B) Demarcation of the Yrr1p binding sites of the AZR1 and SNG1 promoters. A set of oligonucleotides was used to construct subregions of the 200-bp region of the promoter by PCR. These subregions were delimited by bioinformatic prediction for the AZR1 and SNG1 upstream sequences (Table 1). For each subpromoter region, an EMSA was done with whole-cell extract from an FY strain in which YRR1 was deleted (ΔY), the wild-type FY strain (FY), or an FY strain producing Yrr1S*GAD (FS). The intensity of the mobility shift observed is indicated, from − (no band) to ++ (intense band). The complete gels from EMSAs can be viewed at the related website http://www.biologie.ens.fr/yeast-publi.html. (C) Sequence motif depicting the DNA-binding site preferences of Yrr1p. Promoter sequence analysis of the seven genes up-regulated and involved in a specific shift revealed a core consensus sequence: (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T). Only half a dyad seems to be conserved in this consensus sequence. The numbers underneath the sequence motif indicate the sequence of the primers from 5′ to 3′. The height of the nucleotide at each position of the DNA-binding site is based on all the information available for that position, with taller nucleotides more likely to give correct binding than shorter nucleotides.
FIG. 4.
FIG. 4.
The YRR1 regulation network is linked to the PDR network. All the regulated genes found to be activated by Yrr1p fall into the three main characterized functional groups: MFS permease, ABC transporter, and metabolic genes. For all these genes, the consensus PDRE sequence is shown as an open circle. The promoters of the underlined genes gave positive results in the EMSAs presented above. Most of these genes are also regulated by Pdr1p, Pdr3p, and Yap1p. These transcription factors control the expression of other transcription factors in this series (dotted arrows) or, in some cases, their own expression (YRR1 and PDR3). Finally, the most common drugs used (4-NQO and oligomycin) are marked next to the genes associated with specific resistance.
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