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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 (Zn 2 Cys 6 ) 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 Zn 2 Cys 6 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 Zn 2 Cys 6 transcription factors. The Zn 2 Cys 6 family of transcription factors, exemplified by Gal4, represents more than 25% of the yeast transcription regulators. Our ...

The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3 Delta and rom2 Delta mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3 Delta, in rom2 Delta mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast.

The PDR1 and PDR3 genes encode the main transcription activators involved in the control of multidrug resistance in Saccharomyces cerevisiae. To identify the amino acids essential for Pdr3p function, the loss-of-function pdr3 mutants were isolated and characterized. Two plasmid-borne pdr3 alleles, pdr3-E902Ter and pdr3-D853Y, which failed to complement drug hypersensitivity in the Deltapdr1Deltapdr3 mutant strain, were isolated. The E902Ter mutation resulted in a truncated protein lacking the C-terminal activation domain. The D853Y mutation allowed the expression of entire Pdr3p, but its transactivation function was lost. When overexpressed from the P(GAL1) promoter, the two mutant alleles increased the sensitivity of wild-type cells to cycloheximide and fluconazole and suppressed drug resistance in gain-of-function pdr1 and pdr3 mutant strains. The drug-sensitizing effect of overexpressed loss-of-function pdr3 mutant alleles correlated with their ability to suppress PDR5 transcription and rhodamine 6G accumulation in transformants of the wild-type and Deltapdr1 mutant strains. These results demonstrate that amino acid residue Asp853 is essential for Pdr3p function, and indicate that specific loss-of-function pdr3 mutations can convert the Pdr3p transcription activator to a multicopy suppressor of multidrug resistance.

We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a3. The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae. It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway.