The yeast ATP binding cassette (ABC) protein genes <i>PDR10</i> and <i>PDR15</i> are novel targets for the Pdr1 and Pdr3 transcriptional regulators

The widespread pleiotropic drug resistance (PDR) phenomenon is well described as the long term selection of genetic variants expressing constitutively high levels of membrane transporters involved in drug efflux. However, the transcriptional cascades leading to the PDR phenotype in wild-type cells are largely unknown, and the first steps of this phenomenon are poorly understood. We investigated the transcriptional mechanisms underlying the establishment of an efficient PDR response in budding yeast. We show that within a few minutes of drug sensing yeast elicits an effective PDR response, involving tens of PDR genes. This early PDR response (ePDR) is highly dependent on the Pdr1p transcription factor, which is also one of the major genetic determinants of long term PDR acquisition. The activity of Pdr1p in early drug response is not drug-specific, as two chemically unrelated drugs, benomyl and fluphenazine, elicit identical, Pdr1p-dependent, ePDR patterns. Our data also demonstrate that Pdr1p is an original stress response factor, the DNA binding properties of which do not depend on the presence of drugs. Thus, Pdr1p is a promoter-resident regulator involved in both basal expression and rapid drug-dependent induction of PDR genes.All living organisms have developed complex transcriptional responses for rapidly adapting genome expression to the presence of toxic compounds in the environment. These responses involve various types of cellular pathway. Genome-wide studies of drug responses in microorganisms have revealed that these responses comprise both specific effects depending on the precise chemical nature and cellular targets of the toxic compound and a general stress response (environmental stress response (ESR) 5 in the yeast Saccharomyces cerevisiae), reflecting cell adaptation to growth defects and cellular damages, regardless of the type of stress encountered by the cell (1). Inbetween these very specific and very general responses, prokaryotic and eukaryotic cells have evolved multidrug resistance (MDR) pathways, which confer resistance to a broad spectrum of unrelated chemicals, but which are restricted to the stress responses associated with organic drugs. From bacteria to humans, MDR is essentially based on the overexpression of membrane transporters able to export a large number of chemically different compounds (2-4). MDR is a major concern for human health, as it leads to antibiotic resistance in pathogens and enables cancer cells to survive chemotherapy.In the model yeast S. cerevisiae, MDR is referred to as PDR (pleiotropic drug response). The PDR network currently comprise 10 transcription factors regulating about 70 different target genes reviewed in Ref. 18). In this network, the Pdr1p transcription factor has the largest set of potential targets (about 50). Pdr1p and its functional homologue, Pdr3p, were identified in the early 1990s as regulators of the basal level of drug resistance in yeast cells (19,20). Gain-or loss-of-function alleles of PDR1 and PDR3 confer resistance or sensitivit...

Section: Pdr1p Constitutively Binds To Its Target Promoters In Vivosupporting

confidence: 64%

The Central Role of PDR1 in the Foundation of Yeast Drug Resistance

Fardeau

Lelandais

Oldfield

et al. 2007

“…In summary, our studies strongly suggest that Rdr1p acts through Pdr1p/Pdr3p binding sites. However, many genes (11,(13)(14)(15)(16)(17) (like SNQ2) that have been shown to contain PDREs and to be regulated by Pdr1p/ Pdr3p are not affected by removal of Rdr1p. Thus, the effect of Rdr1p appears to be mediated by a subset of the target genes of FIG.…”

Section: Discussionmentioning

confidence: 99%

Zinc Cluster Protein Rdr1p Is a Transcriptional Repressor of the PDR5 Gene Encoding a Multidrug Transporter

Hellauer¹,

Akache²,

MacPherson³

et al. 2002

The yeast PDR5 gene encodes an efflux pump that confers multidrug resistance. Expression of PDR5 is positively regulated by the transcription factors Pdr1p and Pdr3p that recognize the same pleiotropic drug resistance elements (PDREs) in the PDR5 promoter. Pdr1p and Pdr3p belong to the Gal4p family of zinc cluster proteins. The function of RDR1 (YOR380W), which also encodes a member of this family, is unknown. To identify target genes for Rdr1p, we have performed wholegenome analysis of gene expression with DNA microarrays. Our results show that Rdr1p is a transcriptional repressor of five genes, including PDR5. A ⌬rdr1 strain has increased resistance to cycloheximide, as expected from the overexpression of PDR5. In addition, the activity of a PDR5-lacZ reporter is increased in a ⌬rdr1 strain. All (but one) genes affected by removal of Rdr1p contain PDREs in their promoters. We tested if the effect of Rdr1p is mediated through PDREs by inserting this DNA element in front of a minimal promoter. Activity of this reporter was increased in a ⌬rdr1 strain. Moreover, mutations known to reduce binding of Pdr1/ Pdr3p abolished the induction observed in the ⌬rdr1 strain. Thus, we have identified a transcriptional repressor involved in the control of multidrug resistance.Multidrug resistance is a widespread phenomenon that allows organisms ranging from bacteria to humans to defend themselves against a variety of toxic compounds. Drug resistance is mediated through membrane-bound transporters that act as drug efflux pumps. This process has been extensively studied in the yeast Saccharomyces cerevisiae. Two major classes of multidrug transporters have been identified: the major facilitator superfamily (MFS) 1 (1) and the ABC (ATP binding cassette) family of transporters (2-4). ABC transporters act in an ATP-dependent manner, and their overexpression leads to increased drug resistance. Members of the family of ABC transporters include Pdr5p, Snq2p, and Yor1p (2).The transcriptional activators Pdr1p and Pdr3p control the expression of many ABC transporters (3, 4). These activators belong to a family of transcriptional regulators called zinc cluster or binuclear cluster proteins (5-7). Members of this family contain six highly conserved cysteines that coordinate binding to two zinc atoms to allow proper folding of the DNA binding domain. The cysteine-rich region (or zinc finger) is usually followed by a short linker sequence that bridges the zinc finger to a dimerization domain (6). This domain is involved in recognition of specific DNA sequences through interaction of the zinc finger with DNA (for references see Ref. 8). Pdr1p and Pdr3p both recognize CGG triplets oriented in opposite directions (CCGCGG) to form an everted repeat (9). This motif is found in the target genes of Pdr1p and Pdr3p (10 -17). For example, three binding sites called PDREs (pleiotropic drug response elements) for Pdr1p and Pdr3p have been mapped in the PDR5 promoter (10, 12). Thus, Pdr1p and Pdr3p recognize the same DNA sequences in target promoters f...

“…The genotypes of yeast strains used in this study are listed in Table I ( [32][33][34][35]. To monitor drug-induced PDR5 transcription, cycloheximide treatment at a final concentration of 0.2 g/ml (ϳ0.71 M) for 45 min was used, which was far below the drug concentration required for translation inhibition (28.13 g/ml or 100 M).…”

Section: Methodsmentioning

confidence: 99%

On the Mechanism of Constitutive Pdr1 Activator-mediated PDR5 Transcription in Saccharomyces cerevisiae

Gao

Wang

Milgröm

et al. 2004

Drug resistance as a result of overexpression of drug transporter genes presents a major obstacle in the treatment of cancers and infections. The molecular mechanisms underlying transcriptional up-regulation of drug transporter genes remains elusive. Employing Saccharomyces cerevisiae as a model, we analyzed here transcriptional regulation of the drug transporter gene PDR5 in a drug-resistant pdr1-3 strain. This mutant bears a gain-of-function mutation in PDR1, which encodes a transcriptional activator for PDR5. Similar to the well studied model gene GAL1, we provide evidence showing that PDR5 belongs to a group of genes whose transcription requires the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex. We also show that the drugindependent PDR5 transcription is associated with enhanced promoter occupancy of coactivator complexes, including SAGA, Mediator, chromatin remodeling SWI/ SNF complex, and TATA-binding protein. Analyzed by chromatin immunoprecipitations, loss of contacts between histones and DNA occurs at both promoter and coding sequences of PDR5. Consistently, micrococcal nuclease susceptibility analysis revealed altered chromatin structure at the promoter and coding sequences of PDR5. Our data provide molecular description of the changes associated with constitutive PDR5 transcription, and reveal the molecular mechanism underlying drug-independent transcriptional up-regulation of PDR5.Transcriptional regulation of the ATP binding cassette (ABC) transporter genes plays a pivotal role in the development of a drug resistance phenotype in mammalian (1, 2) and yeast cells (3-5). Constitutive transcriptional up-regulation of drug transporter genes occurs in certain mutations involving various transcriptional activators. However, the molecular mechanism underlying this enhanced transcription is not fully understood.In Saccharomyces cerevisiae, the multiple/pleiotropic drug resistance (MDR/PDR) phenotype is primarily regulated via two transcriptional activators encoded by homologous PDR1 and PDR3 genes. These factors are responsible for activating the majority of drug transporter genes, including PDR5 (5-7). Both activators belong to the Gal4 superfamily with the Zn2Cys6 DNA binding domain (8). The amino-terminal of their DNA binding domains recognize promoters with the Pdr1/Pdr3 response element (PDRE), 1 5Ј-TCCGCGGA-3Ј (9), such as that with PDR5.The Pdr5 transporter belongs to the ABC transporter superfamily. In the drug-resistant pdr1-3 and pdr3-7 strains, PDR5 transcription is the highest among target genes activated by Pdr1 and Pdr3 (10). The pdr1-3 and pdr3-7 strains are analogous to other mutants bearing gain-of-function mutations in PDR1 (11) and PDR3 (12) that up-regulate PDR5 transcription. The pdr1-3 allele bears a F815S mutation located near the transcription activation domain at the COOH terminus of Pdr1, suggesting that the mutation may alter the function of the activation domain.Activation of transcription in eukaryotes is mainly controlled by activator-mediated recruitments of transcription fact...