Cross-talk between Transcriptional Regulators of Multidrug Resistance in Saccharomyces cerevisiae

Abstract

Multiple or pleiotropic drug resistance often arises in the yeast Saccharomyces cerevisiae due to genetic alterations of the functional state of the Cys 6 -Zn(II) 2 transcription factors Pdr1p and Pdr3p. Single amino acid substitutions give rise to hyperactive forms of these regulatory proteins, which in turn cause overproduction of downstream target genes that directly mediate multidrug resistance. Previous work has identified a novel Cys 6 -Zn(II) 2 transcription factor designated Yrr1p as mutant forms of this protein confer high level resistance to the cell cycle inhibitor reveromycin A and DNA damaging agent 4-nitroquinoline-N-oxide. In the present study, we demonstrate that Yrr1p also mediates oligomycin resistance through activation of the ATP-binding cassette transporter-encoding gene YOR1. Additionally, insertion of triplicated copies of the hemagglutinin epitope in the C-terminal region of Yrr1p causes the protein to behave as a hyperactive regulator of transcription. We have found that YRR1 expression is both controlled in a Pdr1p/Pdr3p-dependent manner and autoregulated. Chromatin immunoprecipitation experiments also show that Yrr1p associates with target promoters in vivo. Together these data argue that the signal generated by activation of Pdr1p and/or Pdr3p can be amplified through the action of these transcriptional regulatory proteins on downstream target genes, like YRR1, that also encode transcription factors.A common feature underlying acquisition of multiple or pleiotropic drug resistance is the overexpression of genes encoding ATP-binding cassette (ABC) 1 transporters. Multidrug resistant tumor cells often exhibit amplification of the gene encoding the Mdr1p, a prototypical drug efflux pump (1). Gene amplification is typically not seen associated with pleiotropic drug resistance (Pdr) of the yeast Saccharomyces cerevisiae (2), yet these highly drug-resistant strains commonly possess high level transcription of ABC transporter genes like PDR5 (3-5) or YOR1 (6, 7). Overexpression of these ABC transporters is required for the Pdr phenotype and most commonly results from genetic lesions that activate function of the Cys 6 -Zn(II) 2 transcription factors Pdr1p and Pdr3p (8,9).PDR1 was isolated as the first locus that could be mutated to confer a Pdr phenotype in S. cerevisiae (10). Cloning and analysis of this gene indicated that Pdr1p likely acted as a transcriptional regulator (11). Hyperactive alleles of PDR1 led to marked overproduction of target genes, which in turn allowed cells to tolerate otherwise toxic levels of drugs (12). Mutant forms of a PDR1 homologue (PDR3) produce a similar effect on both drug resistance and ABC transporter expression (13,14). Biochemical experiments indicated that Pdr1p and Pdr3p bind to the same DNA element that was designated the Pdr1p/ Pdr3p response element (PDRE) (15). Interestingly, the PDR3 structural gene contains two PDREs and is regulated by Pdr1p and autoregulated at the transcriptional level (16). The presence of at least one PDRE has been found asso...

Yeast ABC transporters – A tale of sex, stress, drugs and aging

“…Transcription of PDR5, SNQ2, PDR10 and PDR15, as well as YOR1 (Table 2) is controlled by Pdr1p/Pdr3p and Yrr1p, the latter modulating expression of both SNQ2 and YOR1 [63,64]. PDR3 and YRR1 are also autoregulated via PDREs in their own promoters [63][64][65].…”

New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

“…The gain-of-function allele has been described elsewhere (28) and consists of an insertion of three HA tags in the C-terminal region of Yrr1p, after amino acid 730 (YRR1::3X HA-730). The yeast strains transformed with a construct encoding a fusion of the YRR1 binding domain and the GAL4 activating domain (called Yrr1*GAD) were from FY1679-28C (Mata ura3-52 trp1⌬63 leu2⌬1 his3⌬200) and W303 (MATa ura3-52 ade2-1 leu2-3,12 his3-11,15 trp1-289) backgrounds.…”

“…This response is controlled by two major transcription factors, Pdr1p and Pdr3p, which activate the production of various targets, such as the ATP-binding cassette (ABC) transporters encoded by PDR5, PDR10, PDR15, SNQ2, and YOR1; the major facilitator superfamily (MFS) permeases encoded by HXT9 and HXT11; and the lipid metabolism proteins encoded by IPT1 and PDR16 (for review, see reference 15). Other proteins, such as Pdr13p (13) and Yrr1p (28), have also been described as being involved in PDR network regulation and may act directly on the PDR genes themselves or indirectly in general drug resistance.…”

“…Promoter deletions have also demonstrated that Yrr1p interacts with the promoter region of SNQ2 (4). Furthermore, chromatin-immunoprecipitation experiments with hemagglutinin (HA)-tagged Yrr1p have shown direct binding to the promoter region of YOR1, which encodes a protein involved in oligomycin resistance (28). Thus, YRR1 is involved in complex PDR network regulation, directly activating SNQ2 and YOR1, both of which are common targets of Pdr1p, Pdr3p, and another regulator of MDR, Yap1p.…”

“…Thus, YRR1 is involved in complex PDR network regulation, directly activating SNQ2 and YOR1, both of which are common targets of Pdr1p, Pdr3p, and another regulator of MDR, Yap1p. Finally, as Pdr1p activates Yrr1p (28), it seems highly likely that YRR1 plays a major role in the PDR regulation network.…”