Genome-wide transcriptional response to POELE and DCP treatment. Cells were treated with 0.3 mM DCP or 0.1 mM POELE for 20 min or left untreated. mRNA profiles were determined by hybridization to genome-wide yeast microarrays. (A) Specific and overlapping responses to POELE and DCP: hierarchical clustering of core POELE and DCP gene set selected as more than twofold induced in either POELE- or DCP-treated cells. Genes are indicated with binding sites and/or controlled by other transcription factors such as Pdr1/Pdr3 (Fardeau et al., 2007), Msn2/Msn4 (Chua et al., 2006), Crz1 (Yoshimoto et al., 2002), and Hsf1 (Lee et al., 2002). (B) Pdr1/Pdr3-regulated genes are induced by DCP and POELE. Genes were selected by twofold induction DCP with >1.5-fold difference to the pdr1Δ pdr3Δ deletion mutant values. Reported Pdr1/Pdr3 target genes were selected according to their induction by Pdr1 overexpression (Devaux et al., 2001) and by fluphenazine (Fardeau et al., 2007). DCP-induced genes dependent on Pdr1/Pdr3 include known PDR-target genes, as well as those induced by fluphenozine or Pdr1 overexpression. (C) Some 22 of 98 (Yoshimoto et al., 2002) Crz1-dependent genes are also induced by POELE treatment and weaker by DCP. DCP induction is not changed in wild-type versus Pdr1/Pdr3 deletion mutant. (D) HSE-containing genes in the POELE data set as identified by T-Profiler show in both POELE- and DCP-enhanced expression but no Pdr1/Pdr3 dependence is noted. (E) POELE/DCP-regulated genes also targeted by Msn2/Msn4 were selected from the Msn2/Msn4 overexpression data (Chua et al., 2006). Of 72 genes induced >1.5-fold by Msn2/Msn4 overexpression, 40 are present among the 193 core POELE/DCP-induced set (>2). The values of these genes treated with DCP are generally lower but also significantly increased according to T-Profiler. Files with the normalized values used to generate the figures are available as supplemental material and as Custer3 result files.

Deletion of PDR5 and PDR15 causes increased sensitivity to detergents. W303-1A (WT), NRY201 (pdr5Δ), NRY212 (pdr15Δ), and NRY227 (pdr5Δ pdr15Δ) were grown to exponential phase, diluted to an OD₆₀₀ of 0.4, 0.04, and 0.004 and spotted onto YPD plates containing different concentrations of DCP (A) and 1% Triton X-100, 200 μM POELE, 1% NP-40, or 10 mM benzyl alcohol (B). Growth was inspected after 3 d, and for DCP plates, after 4 d.

Induction of PDR15 and PDR5 is mainly dependent on Pdr1 and Pdr3. (A) W303-1A (WT), YA100 (pdr1Δ pdr3Δ), and msn2Δ msn4Δ growing in exponential growth phase were treated with either 100 μM POELE or 10 mM benzyl alcohol. Samples were taken before stress treatment and at the indicated times. Expression of PDR5 and PDR15 was analyzed by Northern blotting by using the appropriate probes. rRNA bands serve as loading control. (B) W303-1A (WT), YYA100 (pdr1Δ pdr3Δ), and msn2Δ msn4Δ carrying the plasmid pHW-15z were grown overnight on selective medium, shifted to YPD, and grown to the exponential phase for 4 h. Samples were either left untreated (light gray) or treated with 0.1 mM POELE (dark gray) for 90 min. β-Galactosidase activities were determined in crude cell free extracts. All experiments were carried out in triplicates.

DCP and POELE activate transcription through the PDRE. W303-1A cells carrying pDK52 (PDRE) or pDK53 (mPDRE) were shifted from selective medium to YPD and grown in exponential phase for 4 h. Samples were taken before and after 2-h treatment with 0.3 mM DCP (A) or 100 μM POELE (B), and β-galactosidase activity was determined in protein extracts. Experiments were carried out at least in triplicates.

POELE and DCP activate Pdr1/Pdr3 and Msn2/Msn4, leading to DCP resistance. (A) FY1679-28c (WT), naΔ1 (pdr1Δ), naΔ3 (pdr3Δ), and naΔ1Δ3 (pdr1Δ pdr3Δ) cells were grown on YPD to OD₆₀₀ 1.0. Then, they were diluted to an OD₆₀₀ of 0.2, 0.02, and 0.002 and spotted on plates containing increasing amounts of DCP. Cell growth was inspected after 3 d. (B) FY1679-28c (WT), naΔ1 (pdr1Δ), naΔ3 (pdr3Δ), and naΔ1Δ3 (pdr1Δ pdr3Δ) cells were grown in YPD to an OD₆₀₀ of 1.0 and diluted to an OD₆₀₀ of 0.4, 0.04, and 0.004, and then they were spotted onto plates containing increasing concentrations of POELE. Cell growth was inspected after 3 d. (C) W303-1A (WT), YYA100 (pdr1Δ pdr3Δ), and msn2Δ msn4Δ growing in the exponential growth phase were diluted to an OD₆₀₀ of 0.4, 0.04, and 0.004, and then they were spotted onto YPD plates containing the indicated concentrations of either DCP or POELE. Plates were inspected after 3-d incubation at 30°C. (D) W303-1A msn2Δ msn4Δ cells were transformed with pADH1-Msn2-GFP to express Msn2-GFP. Msn2-GFP fluorescence was monitored in living exponentially growing cells treated with 0.1 mM POELE or 0.3 mM DCP. 4,6-Diamidino-2-phenylindole staining was used to visualize nuclear DNA.

Membrane-damaging agents and detergents strongly induce Pdr5 and Pdr15. (A) W303-1A and msn2Δ msn4Δ cells were grown to an OD₆₀₀ of 1.0 in YPD and treated with 0.3 mM DCP. Samples were taken before stress and after the indicated time. PDR5 and PDR15 mRNAs were detected by Northern blotting. rRNA bands confirm equal sample loading. (B) YHW4 (WT) and YHW5 (msn2Δ msn4Δ) cells were grown to OD₆₀₀ of 1.0 in YPD and treated with 0.3 mM DCP, 100 μM POELE, 10 mM benzyl alcohol, 50 μg/ml lysoPC, 1% Triton X-100, or 0.5 M NaCl. Protein extracts were prepared and Pdr5 Pdr15-3HA levels detected by immunoblotting using anti-Pdr5 and anti-HA 16B12 antibodies, respectively. A cross-reaction of the anti-HA antibody (LC) was used to verify equal protein loading. Samples were taken before stress treatment and at the indicated times.

Differential induction of Pdr1 and Pdr3 target genes by DCP and POELE. (A) FY1679-28c (WT), naΔ1 (pdr1Δ), naΔ3 (pdr3Δ), and naΔ1Δ3 (pdr1Δ pdr3Δ) cells were grown on YPD to an OD₆₀₀ of 1.0 and treated with 0.3 mM DCP for 20 and 40 min. Samples were taken before and during stress treatment and PDR15, YOR1, PDR5, and SNQ2 expression was analyzed by Northern blotting. rRNA bands confirm equal sample loading. (B) FY1679-28c (WT), naΔ1 (pdr1Δ), naΔ3 (pdr3Δ), and naΔ1Δ3 (pdr1Δ pdr3Δ) cells were grown in YPD to an OD₆₀₀ of 1.0 and treated with 100 μM POELE for 20, 40, and 60 min. Samples were taken before and during stress treatment and PDR15 and PDR5 mRNA levels were analyzed by Northern blotting. rRNA bands confirm equal sample loading.