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

Abstract

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...

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