Pdr3 is required for DNA damage induction of MAG1 and DDI1 via a bi-directional promoter element - PubMed (original) (raw)

. 2004 Sep 27;32(17):5066-75.

doi: 10.1093/nar/gkh838. Print 2004.

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Pdr3 is required for DNA damage induction of MAG1 and DDI1 via a bi-directional promoter element

Yu Zhu et al. Nucleic Acids Res. 2004.

Abstract

In order to understand how gene regulation is achieved in eukaryotes in response to DNA damage, we used budding yeast as a model lower eukaryotic organism and investigated the molecular events leading to the expression of two closely clustered damage-inducible genes, MAG1 and DDI1. MAG1 and DDI1 are co-activated by a shared 8 bp repeat sequence, UAS(DM). In this study, we screened a yeast genomic library, identified Pdr3 as the transcriptional activator and demonstrated in vivo and in vitro that Pdr3 binds UAS(DM). Pdr3 is required for the activation of a number of genes encoding membrane efflux pumps and deletion of PDR3 results in reduced basal-level expression and loss of DNA damage induction of MAG1 and DDI1. Interestingly, Pdr1, another transcriptional activator homologous to Pdr3 that is also required for the activation of multidrug-resistance genes, is not involved in the regulation of MAG1 and DDI1 expression, although it may also bind to UAS(DM). Deletion of PDR3 does not affect the expression of other well-documented DNA damage-inducible genes; hence, yeast DNA damage-inducible genes appear to have distinct effectors although to a certain extent they share a common regulatory pathway mediated by DNA damage checkpoints.

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Figures

Figure 1

Figure 1

Pdr3 binds and activates UAS_DM_ in a one-hybrid assay. (A) A diagram of UAS_DM_–lacZ and UAS_DM_–HIS3 reporter constructs. Three copies of UAS_DM_, each containing an 8 bp tandem repeat, were inserted into the promoter region of the one-hybrid reporter genes lacZ and HIS3. Both reporter genes were integrated into the genome of host strain YM4271 to create YM4271DM. (B) Multicopy PDR3, but not RPN4, is able to transactivate UAS_DM_–lacZ and UAS_DM_–HIS3. YEp-based plasmid carrying either PDR3, RPN4 or the vector alone (mock) was transformed into YM4271DM. The transformants were replica-plated onto SD minimal selective medium or SD+ 30 mM 3-AT and incubated at 30°C for 2 days (for UAS_DM_–HIS3 expression). Cells from the SD medium were used for an X-gal filter assay and incubated at 30°C for 2 h (for UAS_DM_–lacZ expression) before taking photograph.

Figure 2

Figure 2

Deletion of PDR3 results in decreased basal-level expression and loss of DNA damage induction of both MAG1 and DDI1. (A) Northern analysis. Total RNA was isolated from FY1679-28C (wt) and naΔ3 (_pdr3_Δ) after 0, 0.01, 0.05 and 0.1% MMS treatments (indicated on the top panel for 30 min.) Each lane contains about 15 μg of total RNA. The blot was sequentially hybridized and stripped with MAG1, DDI1 and ACT1 probes. (B) MAG1–lacZ and DDI1–lacZ induction by 0.05% MMS treatment for 4 h in the wild-type (FY1679-28C) and _pdr3_Δ (naΔ3) mutant transformed with either MAG1–lacZ or DDI1–lacZ. The results are the average of 3–6 independent experiments. β-gal activity is given in Miller units.

Figure 3

Figure 3

UAS_DM_ deletion is epistatic to pdr3_Δ. JD52 (wt) and its pdr3_Δ derivative WXY1090 were transformed with either YEp_MAG1_ΔDR–_lacZ (A) or YEp_DDI1_ΔDR–_lacZ (B) and the β-gal activity was determined as described. The results are the average of three independent experiments.

Figure 4

Figure 4

EMSA using the UAS_DM_ probe. Each reaction contained 1 μg of poly(dI–dC), 2 μg of BSA, 5 ng of labeled UAS_DM_ probe and various amount of yeast cell extract in buffer A. The source of yeast crude extract used in each reaction is indicated. Yeast strains used in this study: FY1679-28C (wt), yYA14 (_pdr1_Δ), naΔ3 (_pdr3_Δ) and naΔ1Δ3 (_pdr1_Δ pdr3_Δ). All strains are isogenic to FY1679-28C. Cell extracts with increasing amounts are 5, 10 and 20 μg. Lane 0, no protein control. Lanes 1 and 2 contain 20 μg wild-type cell extract plus 10 μg proteinase K (lane 1) or 1 μg UAS_DM competitor (lane 2).

Figure 5

Figure 5

DNA damage induction of MAG1–lacZ and DDI1–lacZ in the pdr1 and pdr3 mutants. (A) MMS-induced MAG1–lacZ expression. (B) MMS-induced DDI1–lacZ expression. Yeast strains used in this study are the same as in Figure 4. The results are the average of at least three independent experiments.

Figure 6

Figure 6

In vivo and in vitro binding of Pdr3 to UAS_DM_. (A) Interaction of Pdr3 and UAS_DM_ by a ChIP assay. The ChIP experiment was performed as described using TN7-87A9 cells expressing Pdr3-HA. After HA immunoprecipitation and extensive washing, the chromatin-containing samples were used as templates for 30-cycle PCR using UAS_DM_-specific (MAG1-1/MAG1-8, 0.3 kb) and non-specific (SSU1-2/SSU1-3, 1.4 kb) primer pairs. Templates used: lane 1, DNA after immunoprecipitation; lane 2, DNA before immunoprecipitation (input DNA); lanes 3 and 4, total yeast genomic DNA. Primers used in the PCR reaction: lanes 1–3, MAG1-1/MAG1-8 plus SSU1-2/SSU1-3; lane 4, SSU1-2/SSU1-3. Lane 5 contains molecular size marker (in kb). Lane 1 contains the ChIP result and lanes 2–4 serve as various controls. (B) Physical interaction of bacterially produced GST–Pdr3 with UAS_DM_ in EMSA. All reactions contained 5 ng of labeled UAS_DM_ probe. Lane 1, no protein control; lanes 2–4, 1, 2 and 4 μg cell extracts, respectively, from the pGEX–PDR3 (GST–Pdr3) transformant; lane 5, 2 μg cell extract from the pGEX (GST) vector transformant; lane 6, same as in lane 4 but the sample was incubated with 10 μg proteinase K for 10 min prior to loading.

Figure 7

Figure 7

Deletion of PDR3 and RPN4 does not affect PHR1 and RNR2 expression. (A) PHR1–lacZ expression. (B) RNR2–lacZ expression. Yeast strains used in this study are: JD52 (wt), EJY140 (_rpn4_Δ), WXY1090 (_pdr3_Δ) and WXY1091 (_rpn4_Δ _pdr3_Δ).

Figure 8

Figure 8

DNA damage induction of MAG1–lacZ and DDI1–lacZ in the pdr3 and rpn4 mutants. (A) MMS-induced MAG1–lacZ expression. (B) MMS-induced DDI1–lacZ expression. The results are the average of at least three independent experiments. Yeast strains used in this study are the same as in Figure 7. The results are the average of at least three independent experiments.

Figure 9

Figure 9

Signal transduction cascade of DNA damage induction of selected genes in S.cerevisiae. Data are assembled from various literatures. Only three sets of target genes, whose promoters have been dissected and binding proteins identified, are presented. ‘P’, promoter region with defined target sequences. ‘X’, an unknown protein is expected to act as the target for Pds1 phosphorylation and mediate the regulation of MAG1–DDI1 expression. ‘?’, while Pdr3 is defined as an effecter, how Rpn4 relates to Pdr3 in the regulation of MAG1 and DDI1 is currently unclear.

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