Saccharomyces cerevisiae Basic Region-Leucine Zipper Protein Regulatory Networks Converge at the ATR1 Structural Gene
1997; Elsevier BV; Volume: 272; Issue: 37 Linguagem: Inglês
10.1074/jbc.272.37.23224
ISSN1083-351X
AutoresSean T. Coleman, Edith Tseng, W. Scott Moye‐Rowley,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoSaccharomyces cerevisiae cells express a family of transcription factors belonging to the basic region-leucine zipper family. Two of these proteins, yAP-1 and Gcn4p, are known to be involved in oxidative stress tolerance and general control of amino acid biosynthesis, respectively. Strains lacking theYAP1 or GCN4 structural gene have very different phenotypes, which have been taken as evidence that these transcriptional regulatory proteins control separate batteries of target genes. In this study, we provide evidence that both yAP-1 and Gcn4p control the expression of a putative integral membrane protein, Atr1p. Both yAP-1 and Gcn4p can elevate resistance to 3-amino-1,2,4-triazole and 4-nitroquinoline-N-oxide but only if the ATR1 gene is intact. Expression ofATR1 is enhanced in the presence of constitutively active alleles of YAP1 and GCN4. Regulation ofATR1 transcription by yAP-1 and Gcn4p occurs through a common DNA element related to the yAP-1 recognition element found upstream of other yAP-1-regulated genes. These data provide the first indication of overlap between the regulatory networks defined by yAP-1 and Gcn4p. Saccharomyces cerevisiae cells express a family of transcription factors belonging to the basic region-leucine zipper family. Two of these proteins, yAP-1 and Gcn4p, are known to be involved in oxidative stress tolerance and general control of amino acid biosynthesis, respectively. Strains lacking theYAP1 or GCN4 structural gene have very different phenotypes, which have been taken as evidence that these transcriptional regulatory proteins control separate batteries of target genes. In this study, we provide evidence that both yAP-1 and Gcn4p control the expression of a putative integral membrane protein, Atr1p. Both yAP-1 and Gcn4p can elevate resistance to 3-amino-1,2,4-triazole and 4-nitroquinoline-N-oxide but only if the ATR1 gene is intact. Expression ofATR1 is enhanced in the presence of constitutively active alleles of YAP1 and GCN4. Regulation ofATR1 transcription by yAP-1 and Gcn4p occurs through a common DNA element related to the yAP-1 recognition element found upstream of other yAP-1-regulated genes. These data provide the first indication of overlap between the regulatory networks defined by yAP-1 and Gcn4p. The yeast Saccharomyces cerevisiae possesses a group of sequence-specific transcription factors characterized by the presence of the basic region-leucine zipper or bZip domain as their DNA binding motif (1Landschulz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2521) Google Scholar). This family of proteins includes the proteins Sko1p/Acr1p (2Nehlin J.O. Carlberg M. Ronne H. Nucl. Acids. Res. 1992; 20: 5271-5278Crossref PubMed Scopus (90) Google Scholar, 3Vincent A.C. Struhl K. Mol. Cell. Biol. 1992; 12: 5394-5405Crossref PubMed Scopus (70) Google Scholar), Cad1p/yAP-2 (4Wu A. Wemmie J.A. Edgington N.P. Goebl M. Guevara J.L. Moye-Rowley W.S. J. Biol. Chem. 1993; 268: 18850-18858Abstract Full Text PDF PubMed Google Scholar, 5Januska A.P. Sasnauskas K.V. Janulaitis A.A. Genetika. 1988; 24: 773-780PubMed Google Scholar, 6Bossier P. Fernandes L. Rocha D. Rodrigues-Pousada C. J. Biol. Chem. 1993; 268: 23640-23645Abstract Full Text PDF PubMed Google Scholar, 7Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1994; 242: 250-256Crossref PubMed Scopus (80) Google Scholar), Gcn4p (8Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6442-6446Crossref PubMed Scopus (276) Google Scholar, 9Thireos G. Penn M.D. Greer H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5096-5100Crossref PubMed Scopus (109) Google Scholar), and yAP-1 (10Moye-Rowley W.S. Harshman K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (242) Google Scholar). In terms of the in vivo role of these factors, the most information is available for Gcn4p and yAP-1. Gcn4p is involved in regulating the transcription of genes encoding enzymes involved in biosynthesis of amino acids and nucleotides (reviewed in Ref. 11Hinnebusch A.G. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 319-414Google Scholar), while yAP-1 transcriptionally controls the expression of loci involved in oxidative stress tolerance (12Schnell N. Krems B. Entian K.-D. Curr. Genet. 1992; 21: 269-273Crossref PubMed Scopus (162) Google Scholar, 13Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar) and drug resistance (4Wu A. Wemmie J.A. Edgington N.P. Goebl M. Guevara J.L. Moye-Rowley W.S. J. Biol. Chem. 1993; 268: 18850-18858Abstract Full Text PDF PubMed Google Scholar, 6Bossier P. Fernandes L. Rocha D. Rodrigues-Pousada C. J. Biol. Chem. 1993; 268: 23640-23645Abstract Full Text PDF PubMed Google Scholar, 14Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Crossref PubMed Google Scholar). This information is consistent with the belief that Gcn4p and yAP-1 control the expression of very different sets of genes.Previous work on the function of a S. cerevisiae protein encoded by the ATR1 locus suggests a possible overlap between Gcn4p and yAP-1. ATR1 was isolated as a gene that suppressed the 3-amino-1,2,4-triazole (3-AT) 1The abbreviations used are: 3-AT, 3-amino-1,2,4-triazole; 4-NQO, 4-nitroquinoline-N-oxide; ARE, AP-1 recognition element; YRE, yAP-1 recognition element; SD medium, synthetic dextrose medium; YPD medium, yeast extract/peptone/dextrose medium; EMSA, electrophoretic mobility shift assay; UAS, upstream activating sequence. 1The abbreviations used are: 3-AT, 3-amino-1,2,4-triazole; 4-NQO, 4-nitroquinoline-N-oxide; ARE, AP-1 recognition element; YRE, yAP-1 recognition element; SD medium, synthetic dextrose medium; YPD medium, yeast extract/peptone/dextrose medium; EMSA, electrophoretic mobility shift assay; UAS, upstream activating sequence. hypersensitivity of aΔgcn4 strain when carried on a high copy plasmid (15Kanazawa S. Driscoll M. Struhl K. Mol. Cell. Biol. 1988; 8: 664-673Crossref PubMed Scopus (91) Google Scholar).ATR1 gene expression was suggested to be under Gcn4p control and evidence was presented that Atr1p might mediate 3-AT efflux (15Kanazawa S. Driscoll M. Struhl K. Mol. Cell. Biol. 1988; 8: 664-673Crossref PubMed Scopus (91) Google Scholar).ATR1 was shown to be allelic with SNQ1 (16Gompel-Klein P. Brendel M. Curr. Genet. 1990; 18: 93-96Crossref PubMed Scopus (35) Google Scholar), a member of a set of loci that elevated resistance to the DNA-damaging agent 4-nitroquinoline-N-oxide (4-NQO) when carried on a high copy plasmid. In the same high copy screen that identifiedATR1 as a 4-NQO resistance gene (17Mack M. Gompel-Klein P. Haase E. Hietkamp J. Ruhland A.R. Brendel M. Mol. Gen. Genet. 1988; 211: 260-265Crossref PubMed Scopus (19) Google Scholar), a locus designatedSNQ3 was recovered. SNQ3 was later found to be identical to YAP1 (18Hertle K. Haase E. Brendel M. Curr. Genet. 1991; 19: 429-433Crossref PubMed Scopus (59) Google Scholar). Genetic experiments argued that high copy YAP1-containing plasmids were able to suppress the 4-NQO hypersensitive phenotype of an atr1 strain (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar). This led to the prediction that these loci defined separate 4-NQO resistance pathways (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar).In this work, we have reexamined the relationship among Gcn4p, yAP-1, and ATR1 gene expression. Contrary to a previous report (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar), we find that yAP-1 is not able to influence 4-NQO tolerance unless the ATR1 gene is present on the chromosome. This observation argues that ATR1 is a target gene of yAP-1. Additionally, yAP-1 provides a major component of 3-AT resistance through its activation of ATR1 gene transcription. DNA binding experiments demonstrate that yAP-1 and Gcn4p bind to the same DNA element in the ATR1 promoter. These data indicate thatATR1 is a gene regulated by the two different bZip transcription factors, yAP-1 and Gcn4p. ATR1 is the firstS. cerevisiae gene known that is transcriptionally regulated by these two regulatory proteins. This suggests the possibility that there may be functional overlap in the roles of yAP-1 and Gcn4p in the cell.RESULTSThe resistance spectrum of yAP-1 overlaps that of Atr1p. In a systematic search for genes that conferred high level resistance to 4-NQO when present on a high copy plasmid, 2-μm plasmids carryingYAP1, SNQ2, or SNQ1 were found to be able to strongly elevate 4-NQO tolerance (17Mack M. Gompel-Klein P. Haase E. Hietkamp J. Ruhland A.R. Brendel M. Mol. Gen. Genet. 1988; 211: 260-265Crossref PubMed Scopus (19) Google Scholar). SNQ1 was later shown to be allelic with ATR1, a gene isolated by its ability to suppress the hypersensitivity of gcn4 strains to the competitive inhibitor of histidine biosynthesis, 3-AT (15Kanazawa S. Driscoll M. Struhl K. Mol. Cell. Biol. 1988; 8: 664-673Crossref PubMed Scopus (91) Google Scholar). We evaluated the ability of yAP-1 to confer resistance to 3-AT as a first step in testing the hypothesis that yAP-1 might confer tolerance to both 3-AT and 4-NQO through control of ATR1/SNQ1 gene expression.A strain lacking the YAP1 locus was transformed with a low copy plasmid expressing wild-type yAP-1 or a gain-of-function mutant form of the protein (CSE629AAA). The CSE629AAA form of yAP-1 behaves as a constitutively active positive regulator of transcription, while the wild-type protein cycles between states of high and low activity, depending on environmental cues (29Wemmie J.A. Steggerda S.M. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 7908-7914Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The low copy vector plasmid was also transformed as a control. Appropriate transformants were placed on media containing 3-AT or 4-NQO and assayed for the ability to grow (Fig. 1).The mutant YAP1 locus encoding the CSE629AAA form of the protein supported resistance at the highest levels of 3-AT or 4-NQO tested. The wild-type YAP1 gene was able to permit growth at the lower concentrations of drugs tested, while Δyap1cells carrying the vector plasmid alone were strongly inhibited by the presence of either 3-AT or 4-NQO. These data are consistent with the notion that yAP-1 could act through ATR1 to confer resistance to both 3-AT and 4-NQO, since overproduction of Atr1p also leads to resistance to these same two compounds (16Gompel-Klein P. Brendel M. Curr. Genet. 1990; 18: 93-96Crossref PubMed Scopus (35) Google Scholar). This model predicts that loss of the ATR1 locus should decrease or eliminate the ability of yAP-1 to affect resistance to these two compounds. This prediction was tested by transforming atr1cells with a high copy YAP1 plasmid and determining drug resistance of the resulting transformants.Overexpression of YAP1 Does Not Rescue the 3-AT and 4-NQO Hypersensitivity of Δatr1 CellsIt has previously been reported that deletion of the ATR1 gene results in cells that are hypersensitive to 3-AT (15Kanazawa S. Driscoll M. Struhl K. Mol. Cell. Biol. 1988; 8: 664-673Crossref PubMed Scopus (91) Google Scholar) and 4-NQO (16Gompel-Klein P. Brendel M. Curr. Genet. 1990; 18: 93-96Crossref PubMed Scopus (35) Google Scholar). One group also reported that overproduction of SNQ3 (YAP1) in asnq1 (atr1) background still led to a 4-NQO hyperresistant phenotype (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar). We constructed an Δatr1strain that is isogenic with our wild-type strain by one-step gene disruption (34Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1098) Google Scholar). The wild-type and Δatr1 strains were then transformed with a high copy vector or the same plasmid carrying the wild-type YAP1 structural gene. Appropriate transformants were placed on media containing varying concentrations of drugs, and the relative resistance was assessed (Fig. 2).Figure 2YAP1-mediated 3-AT and 4-NQO resistance requires the presence of the ATR1 structural gene. An isogenic pair of wild-type and Δatr1 cells was transformed with a high copy vector plasmid (YEp351) or the same plasmid carrying the wild-type YAP1 gene (YEp351-YAP1). Since both yeast strains contained a wild-type chromosomal copy of YAP1, the dosage of this locus varied from wild type (when YEp351 was present) to high copy (when YEp351-YAP1 was present). Selected transformants were tested for drug resistance using the spot test assay as described in the legend to Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transformants carrying the 2-μm plasmid containing YAP1exhibited a large increase in the ability to tolerate both drugs. However, this increase in drug resistance was strictly dependent on the presence of the ATR1 gene. Loss of ATR1, even in cells overproducing yAP-1, led to a 3-AT- and 4-NQO-hypersensitive phenotype. These results differ from a previous study (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar) where overproduction of yAP-1 was able to suppress the 4-NQO-hypersensitive phenotype of an atr1 mutant. The reason for the difference between our data and the data of others (19Haase E. Servos J. Brendel M. Curr. Genet. 1992; 21: 319-324Crossref PubMed Scopus (25) Google Scholar) may be due to differences in the strains, atr1 alleles, or media conditions used. These data are consistent with a model invoking yAP-1 as an upstream regulator of ATR1, which in turn more directly affects drug resistance. Taken together, these observations provide genetic evidence that yAP-1 confers 3-AT and 4-NQO resistance through activation of theATR1 promoter. To provide molecular support for this model, we examined the ATR1 promoter for elements to which yAP-1 could bind and regulate transcription.Comparison of DNA Sites Known to Bind yAP-1 and the Putative ATR1 yAP-1 Recognition ElementBy computer analysis, we searched theATR1 promoter for sequence motifs that could serve as a yAP-1 recognition element (YRE). The alignment in Fig. 3 shows the comparison of a potential YRE in the ATR1 promoter with known YREs from the GSH1,YCF1, TRX2, and GLR1 genes in S. cerevisiae. The AP-1 recognition element in the SV40 early enhancer is also shown for comparison, since this site was the first binding motif shown to function in aYAP1-dependent fashion in S. cerevisiae (31Harshman K.D. Moye-Rowley W.S. Parker C.S. Cell. 1988; 53: 321-330Abstract Full Text PDF PubMed Scopus (174) Google Scholar). An ATR1 promoter element was found that showed high sequence identity to the other known YREs. This DNA segment provides a candidate element for the site of action of yAP-1 at the ATR1 promoter. This potential yAP-1 binding site is located 228 base pairs upstream of the start site for ATR1gene transcription, in a reasonable location to serve as an upstream activation sequence for ATR1 expression. To test the function of this putative YRE, we evaluated the ability of bacterially produced yAP-1 to bind this site.Figure 3Alignment of known yAP-1 recognition elements in S. cerevisiae genes. A computer alignment of yAP-1 recognition elements found in several different S. cerevisiae promoters is shown. Experimental evidence has been obtained to indicate the role of each element in yAP-1-mediated transcriptional control of each relevant promoter (see text). Theshaded residues correspond to sequence identities between the different sites. A possible consensus sequence for yAP-1 binding is shown at the bottom along with the known binding motif for Gcn4p (46Oliphant A.R. Brandl C.J. Struhl K. Mol. Cell. Biol. 1989; 9: 2944-2949Crossref PubMed Scopus (283) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bacterially Produced yAP-1 and Gcn4p Bind the ATR1 YREWe produced yAP-1 and Gcn4p in bacterial expression systems to determine if either/both of these bZip transcription factors would bind to the putative ATR1 YRE. Previous work of Kanazawa et al. (15Kanazawa S. Driscoll M. Struhl K. Mol. Cell. Biol. 1988; 8: 664-673Crossref PubMed Scopus (91) Google Scholar) provided evidence that ATR1 might be a downstream target of Gcn4p. A radiolabeled ATR1 DNA template, containing the putative YRE, was used as the template in a DNase I protection experiment assay. Bacterially produced yAP-1 and Gcn4p were used as input proteins to evaluate their relative abilities to protect the ATR1 promoter DNA from DNase I cleavage.As can be seen in Fig. 4, inclusion of either bacterially expressed yAP-1 or Gcn4p led to protection of a region in the ATR1 promoter that corresponded to the YRE. To evaluate the role of the nucleotides corresponding to the putative YRE in yAP-1 and Gcn4p DNA binding, a clustered base substitution mutation in the ATR1 YRE was prepared and used in the DNase I protection assay. Loss of these nucleotides in the YRE prevented binding of both yAP-1 and Gcn4p to the YRE. These data suggest that both yAP-1 and Gcn4p bind to the same DNA element in theATR1 promoter.Figure 4Recognition of a common element in theATR1 promoter by yAP-1 and Gcn4p. The ability of bacterially produced yAP-1 or Gcn4p to bind to the ATR1promoter was assessed by DNase I protection analysis. DNA templates corresponding to the wild-type (wtYRE) or mutant (mYRE) ATR1 promoters were labeled with32P and incubated with 1 or 5 μl (denoted by theincreasing width of the bar) of recombinant yAP-1 or Gcn4p. Protein was omitted from the reaction as a control (no protein). The radiolabeled wild-type ATR1 template was subjected to the purine- and pyrimidine-specific Maxam-Gilbert chemical sequencing reactions (AG and CT, respectively) to allow the location of the binding site to be determined. The bound promoter DNA segments are shown on the left side with the size of protected areas indicated by the bars. The boldface sequence motif corresponds to the location of the YRE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATR1 Expression Responds to Gene Dosage of YAP1 and GCN4To determine if the ability of yAP-1 and Gcn4p to bind to theATR1 promoter translated into an effect on in vivo regulation of ATR1, the expression of anATR1-lacZ gene fusion was evaluated in strains with different gene dosages of YAP1 and/or GCN4. TheATR1-lacZ fusion gene, containing 713 base pairs ofATR1 5′-flanking DNA upstream of the transcription start site, was introduced into appropriate S. cerevisiae strains, and the β-galactosidase activity was determined (Table II).Table IIAnalysis of ATR1-lacZ expression in strains with varying gene dosage of selected bZip transcription factorsStrainRelevant genotype2-aOnly the alleles of the bZip-encoding genes of interest are shown.ATR1-lacZexpression2-bValues represent β-galactosidase activities that were determined and reported as described previously (21). Cells were grown in SD medium containing appropriate supplements.units/A600SEY6210Wild type5 ± 0.4SM13Δyap11.7 ± 0.1EE7Δgcn42.4 ± 0.3EE8Δyap1, Δgcn41.1 ± 0.1SEY6210 (YEp351-YAP1)2-μm YAP116 ± 0.7SEY6210 (pSC6)GCN4C11 ± 1SEY6210 (pJAW101)2-μm CAD16 ± 0.22-a Only the alleles of the bZip-encoding genes of interest are shown.2-b Values represent β-galactosidase activities that were determined and reported as described previously (21Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (871) Google Scholar). Cells were grown in SD medium containing appropriate supplements. Open table in a new tab The ATR1-lacZ reporter fusion present in wild-type cells produced 5 units/A 600 of β-galactosidase activity. Removal of either the YAP1 or GCN4 gene reduced ATR1-dependent expression to 1.7 and 2.4 units/A 600, respectively. A strain lacking bothYAP1 and GCN4 produced only 1.1 units/A 600 of activity. The introduction of a multicopy plasmid overproducing yAP-1 elevated production of β-galactosidase activity to 16 units/A 600. A low copy plasmid carrying a constitutively active form of theGCN4 locus increased ATR1-dependent enzyme activity to 11 units/A 600. No effect onATR1 expression was seen in response to changing the gene dosage of the CAD1 locus, a gene encoding a bZip protein related to yAP-1 and Gcn4p (4Wu A. Wemmie J.A. Edgington N.P. Goebl M. Guevara J.L. Moye-Rowley W.S. J. Biol. Chem. 1993; 268: 18850-18858Abstract Full Text PDF PubMed Google Scholar, 5Januska A.P. Sasnauskas K.V. Janulaitis A.A. Genetika. 1988; 24: 773-780PubMed Google Scholar, 6Bossier P. Fernandes L. Rocha D. Rodrigues-Pousada C. J. Biol. Chem. 1993; 268: 23640-23645Abstract Full Text PDF PubMed Google Scholar, 7Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1994; 242: 250-256Crossref PubMed Scopus (80) Google Scholar).To determine if the YRE was required for the observed effect of yAP-1 and Gcn4p on ATR1 gene expression, a mutant form of theATR1-lacZ fusion gene was prepared using the clustered base substitution in the YRE. Neither yAP-1 or Gcn4p was able to bind to this mutant YRE in vitro (Fig. 4). This mutant YRE-containing ATR1-lacZ fusion gene (mYRE-ATR1-lacZ) was transformed into cells along with a low copy vector plasmid or low copy plasmids carrying constitutively active alleles of YAP1(CSE629AAA) or GCN4 (GCN4c). The wild-typeATR1-lacZ fusion was assayed in parallel as a control for the behavior of the normal locus (Table III). Loss of the YRE blocked the ability of the constitutive alleles of either YAP1 orGCN4 to normally stimulate expression ofATR1.Table IIIRegulation of ATR1 by yAP-1 and Gcn4p requires the presence of the YREFusion gene3-aThe indicated fusion genes contained either wild-type (ATR1-lacZ) or mutant (mYRE-ATR1-lacZ) versions of the ATR1 YRE.β-Galactosidase activity3-bβ-Galactosidase activities produced by the indicated fusion genes present in wild-type cells along with a low copy vector or constitutively active alleles of YAP1 (CSE629AAA) orGCN4 (GCN4c) were determined and reported as described above.VectorCSE629AAAGCN4cunits/A600ATR1-lacZ5 ± 0.416 ± 0.511 ± 1mYRE-ATR1-lacZ2.5 ± 0.25 ± 0.45.5 ± 0.43-a The indicated fusion genes contained either wild-type (ATR1-lacZ) or mutant (mYRE-ATR1-lacZ) versions of the ATR1 YRE.3-b β-Galactosidase activities produced by the indicated fusion genes present in wild-type cells along with a low copy vector or constitutively active alleles of YAP1 (CSE629AAA) orGCN4 (GCN4c) were determined and reported as described above. Open table in a new tab These data indicate that both yAP-1 and Gcn4p act to stimulate expression of ATR1. Additionally, the transactivation ofATR1 by both yAP-1 and Gcn4p requires the YRE. The phenotypic effects of loss of the YRE from the ATR1 promoter were next evaluated.3-AT and 4-NQO Resistance Phenotypes of the ATR1 Promoter MutantsTo examine the phenotypic consequences of the mutations in the ATR1 YRE, the clustered base substitution mutation was transferred into the context of the wild-type ATR1 gene. A Δatr1 (SCY2) yeast strain was transformed with a low copy number plasmid bearing the wild-type or the mutant YRE-containingATR1 gene. Along with the plasmids containing the two different ATR1 alleles, a low copy vector plasmid or this same plasmid expressing constitutively active forms ofYAP1 or GCN4 was also introduced. The resulting transformants were assayed for their ability to complement the 3-AT- and 4-NQO-hypersensitive phenotype of the Δatr1strain (Fig. 5).Figure 5The yAP-1 recognition element is required for normal ATR1 gene function. A Δatr1 strain (SCY2) was transformed with a low copy plasmid carrying either the wild-type ATR1 gene or a mutant variant lacking the YRE. The presence of the normal YRE in the plasmid-borne copy of ATR1is indicated at the top of each panel as YRE +, while the presence of the mutant YRE is denoted as YRE −. Along with each ATR1 derivative, a second plasmid was introduced corresponding to a low copy vector plasmid or the same vector plasmid carrying a constitutively active form of either YAP1(CSE629AAA) or GCN4 (GCN4c). The vector plasmids used were either pRS315 for YAP1 or pRS314 forGCN4. Appropriate transformants were assayed for drug resistance by spot test assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transformants containing the wild-type ATR1 gene (pSC8) were able to grow normally on both 3-AT and 4-NQO. The constitutively active alleles of both YAP1 and GCN4 produced elevated resistance to both of these chemicals in the presence of the wild-typeATR1 locus. However, transformants carrying anATR1 gene with a mutant YRE could not grow as well on either cytotoxic agent as those containing an ATR1 allele with a wild-type YRE. Moreover, the mutations in the YRE blocked the hyperresistance conferred by the constitutively active YAP1and GCN4 alleles. These data indicate that the YRE is necessary for the YAP1- andGCN4-dependent resistance to 3-AT and 4-NQO conferred by ATR1.The ATR1 YRE Functions as an Upstream Activation Sequence When Placed Upstream of a Heterologous PromoterTo determine if theATR1 YRE was sufficient to serve as an upstream activation sequence in S. cerevisiae, the UAS region of aCYC1-lacZ fusion gene was replaced with an oligonucleotide corresponding to the ATR1 YRE. The ATR1 YRE oligonucleotide was fused to CYC1-lacZ in each of the two possible orientations, and the resulting fusion plasmids were transformed into isogenic wild-type, Δyap1,Δgcn4, and Δyap1,gcn4 strains. β-Galactosidase activities were determined for each transformant (Table IV).Table IVThe ATR1 YRE can function as a UASFusion gene4-aThe lacZ gene fusion responsible for the observed β-galactosidase activity is listed.YRE orientation4-bA single copy of the ATR1 YRE was inserted in the same (forward) or opposite (reverse) orientation relative to theCYC1 promoter as in the ATR1 promoter.β-Galactosidase activity4-cβ-Galactosidase activities produced by the indicated fusion genes present in isogenic wild-type or Δyap1transformants were determined and reported as described above.Wild typeΔyap1Δgcn4Δyap1Δgcn4units/A600CYC1-lacZNone2.6 ± 0.22.5 ± 0.32.5 ± 0.22.6 ± 0.3YRE-CYC1-lacZForward53 ± 716 ± 449 ± 63.5 ± 0.8YRE-CYC1-lacZReverse65 ± 513 ± 3.562 ± 14 ± 0.64-a The lacZ gene fusion responsible for the observed β-galactosidase activity is listed.4-b A single copy of the ATR1 YRE was inserted in the same (forward) or opposite (reverse) orientation relative to theCYC1 promoter as in the ATR1 promoter.4-c β-Galactosidase activities produced by the indicated fusion genes present in isogenic wild-type or Δyap1transformants were determined and reported as described above. Open table in a new tab The YRE-CYC1-lacZ gene fusions were able to produce 4.8-fold (pSC2) and 3.3-fold (pSC11) more activity then that of the vector only (pLGΔBS) in the wild-type background. Loss of the YAP1gene reduced the level of YRE-stimulated β-galactosidase expression produced by either reporter plasmid. Removal of the GCN4gene had no significant effect on the transactivation supported by theATR1 YRE, as long as the YAP1 gene was still present. The contribution of Gcn4p to ATR1 YRE function could clearly be seen in a strain lacking both YAP1 andGCN4. This double mutant strain produced β-galactosidase levels that were essentially the same as those of theCYC1-lacZ fusion gene lacking any UAS. This analysis suggests that, under these conditions, yAP-1 is the major contributor to YRE-mediated transactivation of ATR1. These data also indicated that the ATR1 YRE was able to act as an orientation-independent UAS that is responsive to yAP-1 levels in the cell.The Relative Affinity of yAP-1 Is Higher Than Gcn4p for the ATR1 YREThe data presented here indicate that yAP-1 has a quantitatively greater role in ATR1 expression than Gcn4p. One possible explanation for this greater effect of yAP-1 onATR1 expression is that yAP-1 has a higher relative affinity for the YRE than does Gcn4p. To investigate this possibility, we carried out electrophoretic mobility shift assays (EMSAs) to compare the affinities of yAP-1 and Gcn4p for the ATR1 YRE. Oligonucleotides corresponding to the ATR1 YRE and the SV40 ARE were radiolabeled and used in EMSA to investigate the relative affinities yAP-1 and Gcn4p (Fig. 6).Figure 6Comparison of the relative affinity of yAP-1 and Gcn4p for the ATR1 YRE. Relative affinities of yAP-1 and Gcn4p were measured for the ATR1 YRE by EMSA. The yeast strains SEY6210 (YAP1) and SM13 (Δyap1) were grown in YPD medium prior to preparation of whole cell extracts. 30 μg of whole cell extracts or 1 μl of a crude extract containing bacterially produced Gcn4p was used in a final reaction volume of 20 μl. Oligonucleotides corresponding to the ATR1 YRE and the SV40 ARE were end-labeled and used as probes. The first laneof each panel corresponds to EMSA done in the absence of protein. Varying concentrations of the ATR1 YR
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