Abstract
Improper control of expression of ATP binding cassette transporter-encoding genes is an important contributor to acquisition of multidrug resistance in human tumor cells. In this study, we have analyzed the function of the promoter region of the Saccharomyces cerevisiae YOR1 gene, which encodes an ATP binding cassette transporter protein that is required for multidrug tolerance in S. cerevisiae. Deletion analysis of a YOR1-lacZ fusion gene defines three important transcriptional regulatory elements. Two of these elements serve to positively regulate expression of YOR1, and the third element is a negative regulatory site. One positive element corresponds to a Pdr1p/Pdr3p response element, a site required for transcriptional control by the homologous zinc finger transcription factors Pdr1p and Pdr3p in other promoters. The second positive element is located between nucleotides -535 and -299 and is referred to as UASYOR1 (where UAS is upstream activation sequence). Interestingly, function of UASYOR1 is inhibited by the downstream negative regulatory site. Promoter fusions constructed between UASYOR1 and the PDR5 promoter, another gene under Pdr1p/Pdr3p control, are active, whereas analogous promoter fusions constructed with the CYC1 promoter are not. This suggests the possibility that UASYOR1 has promoter-specific sequence requirements that are satisfied by another Pdr1p/Pdr3p-regulated gene but not by a heterologous promoter.
Highlights
Mammalian tumor cells can acquire the ability to detoxify several unrelated cytotoxic agents through alteration of a small number of genetic loci
YOR1 is transcriptionally regulated by Pdr1p and Pdr3p and inspection of the YOR1 promoter region indicated the presence of a potential Pdr1p/Pdr3p response element (PDRE) centered 215 bp upstream of the transcription start site [11]
Further reduction in the extent of YOR1 5Ј-noncoding sequence progressively reduced -galactosidase expression. These data suggest that the DNA region from Ϫ222 to Ϫ129 contains the major PDRE and is consistent with our previous identification of a candidate PDRE centered at position Ϫ215 [11]. This candidate YOR1 PDRE is identical in sequence with the site 2 PDRE present in the PDR5 promoter, which we demonstrated to function as a positive regulatory site for PDR5 transcription [15]
Summary
Mammalian tumor cells can acquire the ability to detoxify several unrelated cytotoxic agents through alteration of a small number of genetic loci. Cells containing a ⌬pdr allele are cycloheximide-hypersensitive but display no oligomycin sensitivity, indicating the presence of a second Pdr1p/3p target gene required to provide oligomycin resistance This oligomycin resistance gene, YOR1, was subsequently cloned on the basis of its ability to strongly elevate tolerance to this compound when present on a high copy plasmid [11]. YOR1 is transcriptionally regulated by Pdr1p and Pdr3p and inspection of the YOR1 promoter region indicated the presence of a potential Pdr1p/Pdr3p response element (PDRE) centered 215 bp upstream of the transcription start site [11]. These data are consistent with the YOR1 promoter receiving multiple regulatory inputs from PDR1/PDR3 and other as yet unknown factors
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