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Eukaryotic Cell, July 2006, p. 1081-1090, Vol. 5, No. 7
1535-9778/06/$08.00+0     doi:10.1128/EC.00071-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

TFIIA Plays a Role in the Response to Oxidative Stress

Susan M. Kraemer,1,{dagger} David A. Goldstrohm,1 Ann Berger,3,{ddagger} Susan Hankey,3,§ Sherry A. Rovinsky,2 W. Scott Moye-Rowley,2 and Laurie A. Stargell1*

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523,1 Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242,2 Infectious Disease Genomics, Pharmacia Corporation, Kalamazoo, Michigan 490013

Received 9 March 2006/ Accepted 12 May 2006


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ABSTRACT
 
To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.


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INTRODUCTION
 
Efficient transcription by RNA polymerase II (Pol II) involves a multitude of interactions with a plethora of factors on promoter DNA, including the general transcription factors (GTFs), TFIID (comprising the TATA-binding protein [TBP] and TBP-associated factors [TAFs]), TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH (57). A fundamental step in the process of assembling the preinitiation complex is the recognition of the promoter by TFIID and the formation of a TFIID-DNA complex (9, 14, 41, 86). The general transcription factor TFIIA stabilizes the TFIID-DNA interaction (7, 34, 50) by interacting directly with the TBP and DNA (29, 74) and TAF11 (44, 65). The formation (10, 40) and stability (70) of the TFIID-TFIIA-DNA (DA) complex can be major determinants of the transcriptional output from a given gene. As such, this DA complex is an ideal regulatory target for both repressors and activators of transcription (4, 11, 17, 42, 52, 66, 67, 80, 87).

In addition to regulating the DA complex, certain transcriptional activators contribute to the assembly and stabilization of a productive preinitiation complex via protein-protein interactions with other GTFs (12, 22, 35, 54, 72), certain coactivators (22, 76), the holoenzyme or mediator complex (22, 43, 55, 60), and chromatin-remodeling or -modifying enzymes (60-62). Moreover, certain activators can influence steps subsequent to preinitiation complex formation in the transcriptional process: activators can enhance promoter clearance (49), aid in release of Pol II pausing (6), and increase the elongation rate of Pol II (89). Thus, activators can exert an influence on numerous steps in the transcriptional process, and specific interaction profiles appear to be unique for each activator.

Albeit much is known about a subset of specific activator proteins, the mechanism of action for many activators remains to be elucidated. For example, it is uncertain how the basic-leucine zipper transcriptional activator Yap1, which is the key regulator of the oxidative stress response in yeast (59), functions at the transcriptional level. Numerous studies have revealed many of the specifics of the redox-dependent nuclear targeting of Yap1 during the oxidative stress response (5, 13, 19, 30, 36, 38, 45, 48, 63, 82, 84, 85, 88). In contrast, although the regions of Yap1 involved in activating transcription have been mapped (47, 83), little is known about the molecular details of transcriptional activation by Yap1. Yet, a Yap1-dependent increase in transcription of the genes required for detoxification is essential for cell survival under oxidative stress conditions (15, 39, 68, 69). Moreover, transcriptional regulation of the oxidative stress response is conserved from yeast to humans (for review, see reference 16), and it has been implicated in numerous health-related aspects such as aging (25) and cancer (77). Therefore, understanding the mechanisms by which cells respond to oxidative stress and discovering the key players involved in this response will be fundamental to our understanding of how cells survive and flourish in an aerobic environment.

Here we identify and characterize an interaction between the general transcription factor TFIIA and Yap1. We find that genome-wide expression profiling of a structurally-designed set of TFIIA mutants indicates enrichment for genes regulated by Yap1. Consistent with this, the TFIIA mutants exhibit a variety of oxidative stress phenotypes. In addition, TFIIA interacts with Yap1 both in vivo and in vitro, supporting a direct role for the interaction with regard to transcriptional activation. Taken together, these results define a new and fundamental role for TFIIA in the mechanisms of how cells defend themselves against reactive oxygen species (ROS).


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MATERIALS AND METHODS
 
DNA constructs. TOA2-YCP22 contains TOA2 driven by its native promoter and terminator, which were generated from genomic DNA by PCR. An NcoI site was engineered at the ATG start codon and utilized for insertion of six myc epitopes (GEQKLISEEDLN), creating myc-TOA2-YCP22. Site-directed TOA2 mutants were created using olignonucleotide primers containing the desired mutation and PCR. All PCR products were completely sequenced. Activation domain (AD) hybrids were cloned into the 2µm LEU2-marked vector pACT2.2 (23), which contains the ADH1 promoter, a nuclear localization sequence, the hemagglutinin epitope, and the Gal4 activation domain (residues 768 to 881). AD-YAP1 and AD-YAP1 truncations were generated by PCR from genomic DNA and cloning into the pACT2.2 vector. Generation of the DNA-binding domain (DB) hybrids into the pPC97-TRP vector (79) (CEN, TRP1), which contains the ADH1 promoter, a nuclear localization sequence, and the Gal4 DNA-binding domain (residues 1 to 147), was described previously (44).

Yeast strains. TOA2 mutant derivatives were introduced into ROY100 using the plasmid shuffle technique. ROY100 is a derivative of KY114 (relevant genotype MATa ade2-101 trp1{Delta}1 ura3-52), which was created using a two-step gene knockout of the complete open reading frame (ORF) of the TOA2 gene and contains TOA2 on a 2µm URA3-marked plasmid. {Delta}yap1 was created using a one-step knockout of the open reading frame of the YAP1 gene in the strain KY320 (relevant genotype MATa ade2-101 leu2::PET56 trp1{Delta}1 ura3-52).

All strains used in the yeast two-hybrid assay were transformants of MaV103 (79). MaV103 contains the GAL1 promoter (with four Gal4 binding sites) fused to the HIS3 promoter and structural gene; both GAL4 and GAL80 are deleted in the strain.

RNA isolation and target preparation. Cells were grown in YPD medium to an optical density at 600 nm (OD600) of 0.5 to 1.0. Cells that were heat shocked at 38°C were preshocked at 38°C for 15 min and incubated at 30°C for 1 h followed by 38°C for 1 h. Total RNA was isolated from the cultures by hot phenol extraction. Poly(A)+ RNA was enriched with a QIAGEN Inc. (Valencia, CA) Oligotex mRNA kit according to the manufacturer's directions. Double-stranded cDNA was synthesized following the procedure recommended by Affymetrix from 1 µg mRNA using the GIBCO BRL Life Technologies (Frederick, MD) Superscript Choice system. In order to be able to transcribe cRNA from the resultant double-stranded cDNA, the T7 promoter was included upstream of the oligo(dT) in the primer. The T7-(dT)24 primer (GENSET Corp., La Jolla, CA) had the sequence 5'GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)243'.

The cDNA was dissolved in 3 µl nuclease-free water. A 0.5-µl aliquot was checked on a gel. With 1.5 µl double-stranded cDNA an in vitro transcription using Ambion's (Austin, TX) T7 Megascript system was performed substituting nucleoside triphosphate (NTP) labeling mix containing biotinylated CTP and UTP (biotin-11-CTP and biotin-16-UTP; Enzo Diagnostics Inc., Farmingdale, NY). The in vitro transcription reactions were incubated in a 37°C incubator for 6 h. The biotinylated cRNAs were cleaned on a QIAGEN RNeasy spin column per the manufacturer's protocol. The ethanol-precipitated samples were resuspended in 10 µl nuclease-free water and quantified spectrophotometrically. The size distribution of each sample was estimated on an RNA gel.

Hybridization and data analysis of Affymetrix GeneChip probe arrays. Each product was fragmented by heating to 94°C for 35 min in fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc) and added to a hybridization cocktail containing hybridization control cRNAs and biotin-labeled B2 oligonucleotide for grid alignment as recommended by Affymetrix. The Affymetrix Ye6100 chips were hybridized with 5 µg of biotinylated cRNA target (0.025 µg/µl) in hybridization buffer (1 M NaCl, 10 mM Tris, pH 7.6, 0.005% Triton X-100 [Sigma, St. Louis, MO] with 100 µg/ml herring sperm DNA [Promega, Madison, WI]) overnight at 45°C with rotation at 60 rpm. The probe arrays were washed and stained with streptavidin-phycoerythrin on the Affymetrix GeneChip Fluidics Station 400 using the EukGE_WS2 protocol. The microarrays were scanned with a Hewlett-Packard GeneArray controlled by the GeneChip software.

Affymetrix GeneChip software was used to scale data across arrays, derive present/absent calls, average difference intensity (ADI) values, difference calls, and fold change values. Minor adjustments to these data were made. The resulting marginal increase (MI) and marginal decrease (MD) calls were scored as I (increase) and D (decrease), respectively. A threshold of 1.7-fold change was used in scoring increases (I) and decreases (D). Genes were scored as not expressed (NE) if the ADI in both baseline and experimental samples were less than 100 and/or if there were logic errors in the present/absent calls. Functional categories were from the Munich Information Center for Protein Sequences (MIPS; www.mips.biochem.mpg.de/proj/yeast/catalogues/funcat/index.html) (58). Four levels of categories were available and were copied to separate Excel spreadsheets. Categories were associated with ORF data in Access, and the hypergeometric distributions (75) were calculated in SAS. Hierarchical clustering was performed within Spotfire (Spotfire, Inc.), using Ward's method of calculating the incremental sum of squares, with half-square Euclidean distance as the similarity measure.

Oligonucleotide analysis was performed as described using the pattern discovery program available at http://www.ucmb.ulb.ac.be/bioinformatics/rsa-tools/ (78). Sequences 800 bp upstream of the ORF of each gene in the indicated gene pool were used for the analysis. The data shown here reflect a pool of 24 genes that represent a 3.0-fold-change cutoff. The 3.0-fold cutoff was chosen in order to reduce the size of the gene pool to aide in the computational analysis, as well as to search for motifs enriched in genes that were most significantly affected.

RNA and protein analyses. Quantitative S1 nuclease analyses were done as described previously (37), with approximately 30 to 50 µg of RNA. For the temperature shift, cells were grown in synthetic complete medium to an OD600 of 0.5 to 1.0. Cells were pre-heat shocked at 38°C for 15 min and incubated at 30°C for 1 h followed by incubation at 38°C for 1 h. Total RNA was prepared by hot phenol extraction and was quantified by OD260. Amounts of RNA in each reaction were normalized to the levels obtained from a probe to the intron of the tryptophan tRNA (tRNAW) gene. To measure expression levels of Yap1 in wild-type and TFIIA mutant strains, protein extracts were prepared from the indicated strains and immunoblot analyses was performed with polyclonal antibodies specific for Yap1 or TBP (load control).

Phenotypic studies and yeast two-hybrid assays. For phenotypic studies, 10-fold serial dilutions of strains were applied as spots to plates containing rich medium containing either glucose (YPD) or galactose (YPG) and incubated at 15, 30, or 38°C. Stock solutions of diamide (1 M) were made in dimethyl sulfoxide and stored at –20°C. A stock solution of CdSO4 (50 mM) was made in H2O and stored at –70°C. Appropriate dilutions were used for making YPD plates (see figure legends). Plates were spotted or streaked with cells within 36 h of pouring.

Both Gal4 DB and Gal4 AD plasmids were transformed into the yeast strain MaV103 using a standard lithium acetate transformation. The resulting strains were grown in the appropriate selection media, and 10-fold serial dilutions were performed. Cells were applied as spots to the appropriate plates that either contained or lacked 60 mM 3-aminotriazole (AT) and grown at 30°C for 4 to 7 days.

Nuclear localization studies. Strains expressing the various derivatives of TFIIA were transformed with a green fluorescent protein (GFP)-YAP1 fusion gene previously described (13). Appropriate transformants were grown to mid-log phase, diluted into fresh medium lacking or containing 1 mM H2O2, and allowed to grow at 30°C for 15 min. Microscopy was then performed as described previously (13). Cells were visualized using Nomarski (differential interference contrast) optics or by fluorescence for GFP and 4',6'-diamidino-2-phenylindole (DAPI) to stain DNA.

In vitro interaction assays. Recombinant yeast glutathione S-transferase (GST)-TFIIA was purified by expressing GST-Toa2, or GST-Toa2 mutant derivatives, and untagged Toa1, in separate cultures of Escherichia coli BL21(DE3). Isopropyl-ß-D-thiogalactopyranoside (final concentration, 0.1 mM) was added at an optical density of 0.6, cells were grown for 4 h and ruptured by sonication, and insoluble material was collected by centrifugation. The Toa1 and GST-Toa2 derivatives were mixed together using a 2:1 ratio of Toa1 to GST-Toa2, denatured in a buffer containing 8 M urea, and renatured as described previously (44). The renatured proteins were dialyzed against 50 mM KCl, 5 mM MgCl2, 20 mM Tris (pH 7.5), 10% glycerol, 0.5 mM dithiothreitol. GST-TFIIA was purified using glutathione resin to ~90% pure as determined by Coomassie staining. Approximately 20 pmol of GST-TFIIA derivative or GST alone was incubated with 10 to 20 pmol of His-Yap1 protein in 200 µl of binding buffer (20 mM HEPES, pH 7.9, 20 mM Tris, pH 7.5, 200 mM NaCl, 50 mM KCl, 10 mM MgCl2, 0.025% NP40, 10% glycerol, 0.5 mM dithiothreitol) for 3 h at 4°C. Complexes were recovered by incubation with glutathione-Sepharose for 1 h at 4°C in binding buffer with 3% bovine serum albumin. Complexes were washed two times in 400 µl of binding buffer, incubated with sodium dodecyl sulfate (SDS) loading buffer, and boiled, and 10 µl of sample was separated by SDS-polyacrylamide gel electrophoresis (PAGE). Gels were analyzed by immunoblotting with antibodies specific to His-Yap1 (Santa Cruz Biotechnology) or GST (Sigma), and visualized by chemiluminescent detection (Pierce).

Electrophoretic mobility shift assays were performed using a 32P-labeled 45-bp fragment containing the adenovirus early 1B TATA box, as described previously (44). Purified recombinant yeast TBP (5 nM), TFIIA (1 nM or 5 nM), and 100 ng of poly(dG-dC) were incubated at 25°C for 30 min in 20 µl of 20 mM Tris (pH 7.5), 40 mM HEPES (pH 7.9), 100 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10% glycerol. Complexes were separated from unbound DNA by 6% nondenaturing acrylamide gel electophoresis in 0.5x Tris-borate-EDTA and quantified by phosphorimaging.


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RESULTS
 
TFIIA mutations impart growth phenotypes. TFIIA consists of two domains, a beta strand domain and a four-helix-bundle (4HB) domain (29, 74). The beta strand domain makes all of the contacts with TBP and also binds DNA upstream of the TATA element. The 4HB domain of TFIIA projects away from the TBP-DNA complex into solution. In addition, there are two large solvent-exposed patches of hydrophobic residues on TFIIA: one patch is within the beta strand domain and the other is within the 4HB domain (Fig. 1A). Hydrophobic interactions are important for many protein-protein interactions. In fact, the hydrophobic region on the beta strand domain contacts TBP, and we have previously demonstrated a role for the hydrophobic patch on the 4HB for contacting TAF11 (44, 65). In particular, replacement of the isoleucine at position 27 with lysine (I27K) abrogates an interaction with TAF11. Strikingly, replacements of methionine at position 38 with alanine (M38A) or lysine (M38K) and leucine at position 41 with alanine (L41A) or aspartic acid (L41D), two other highly conserved residues in this patch (Fig. 1B), did not result in TAF11 defects. To further determine the physiological relevance and elucidate other functional roles of the hydrophobic patch on the 4HB of TFIIA, the Toa2 mutants (under the control of the TOA2 promoter and terminator) were shuffled into a TOA2 deletion strain and tested for various growth phenotypes. Each of the alleles supported cell viability at 30°C on rich media (Fig. 1C), except for L41D, which was inviable. Several of the mutants possessed slow growth phenotypes at 30°C and/or temperature-sensitive phenotypes at 38°C. These mutant phenotypes were not the result of reduced amounts of Toa2 protein, since each strain produced amounts of Toa2 protein comparable to that producing wild-type Toa2 (data not shown). Overall, more drastic mutant phenotypes were observed with the radical substitutions, so these derivatives (I27K and M38K) were utilized throughout the rest of the study. However, the L41D substitution resulted in a lethal phenotype; thus, the L41A substitution was characterized further.


Figure 1
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  		FIG. 1. (A) Crystal structure of the TFIIA-TBP-DNA complex. TBP is shown in a yellow ribbon, and DNA is black. TFIIA is shown in space-filling model, with Toa1 in dark blue and Toa2 in light blue. The two hydrophobic patches on TFIIA are shown in gray: one patch contacts TBP, and the second patch is surface exposed and resides in the four-helix bundle. The amino acids in Toa2 selected for replacement by alanine or radical amino acids are shown in magenta and indicated by the arrows and the labels. The figure was created with Insight II, using the coordinates of the TBP-TFIIA-DNA structure (74). (B) Amino acid sequence alignment of Toa2 indicates that the hydrophobic patch in the four-helix-bundle domain is a conserved structural feature in lower and higher eukaryotes. Numbers indicate the starting and ending positions shown for each homolog followed by the total number of amino acids in the homolog. Asterisks denote residues that contribute to the hydrophobic patch. Rectangular boxes denote residues that were targeted for mutational analysis. Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; Xl, Xenopus laevis; At, Arabidopsis thaliana. Alignment was produced with the aid of the BLAST search engine (2). (C) Toa2 mutant derivatives confer mutant phenotypes. Strains containing the indicated Toa2 mutations or the wild type (WT) were serially diluted (from 104 to 10 cells), applied as spots to rich medium plates containing glucose (YPD), and incubated at 15, 30, or 38°C.

Whole-genome transcription profiling of the TFIIA I27K, M38K, and L41A mutants. To investigate the function of TFIIA in vivo, we analyzed genome-wide expression patterns in the I27K, M38K, and L41A Toa2 mutants by using the Affymetrix microarray system. Strains were grown at 30°C or at 38°C. Gene expression was analyzed relative to the wild-type strain grown at the corresponding temperature, and a cutoff of 1.7-fold was used to define significant changes (Fig. 2). At 30°C, 24% of the genes (1,139/4,773), were underexpressed in at least one of the TOA2 mutant strains (Fig. 3A). Given that the three mutants have molecular defects in gene expression at the "permissive" temperature of 30°C, it is clear that these do not represent conditional alleles of TFIIA function. Moreover, while a very similar number of genes, 22% (1,055/4,733), exhibited diminished expression at 38°C, the population of genes contained within these two groups does not overlap significantly. With these properties in mind, the remainder of the analysis focused on the pool of genes with altered expression at 30°C.


Figure 2
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  		FIG. 2. Hierarchical clustering analysis of the expression profiles of the three TFIIA mutants at 30°C (A) and 38°C (B). Each vertical line within each panel represents a single gene whose transcript level was increased (red) or decreased (green) twofold in any of the three mutants. This subset of genes was clustered hierarchically on the basis of the similarity of their expression profiles and plotted as a 10x log fold change. The black bar indicates the pool of genes used in the word search (see text).


Figure 3
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  		FIG. 3. Microarray analysis of genome-wide expression levels in the I27K, M38K, and L41A mutants. (A) Venn diagrams displaying genes increasing or decreasing in the I27K, M38K, and L41A mutants in relation to the number of expressed genes at 30°C (4,773 expressed genes) and 38°C (4,733 expressed genes). Intersection of the representative circles indicates the subset of genes that changed commonly in two or more mutants, and the number of genes in each subset is shown. (B) Enrichment of functional role categories for genes that were increased in expression in all three mutants. Genes were placed into 1 or more of the 107 MIPS categories. The percentage of increased genes in each category was compared to the expected percentage of the expressed genes in the category to determine if the category contained more increased genes than would be expected by chance (75). A P value of <0.05 was considered to denote an enriched category. Only categories that displayed significant enrichments are shown. (C) Enrichment of functional role categories for genes that were decreased in expression in all three mutants (calculated as in panel B).

As expected from our previous work indicating that the 4HB hydrophobic patch is multifunctional, there are subsets of genes unique to each particular allele. And, in particular, the L41A derivative possessed the most genes with altered expression patterns. Thus, for a given allele, between 11 and 27% of genes were changed at 30°C (Fig. 3A). There is also a population of genes that are commonly affected in all three mutants, presumably due to a general requirement of the hydrophobic patch in TFIIA functional activity. There were a total of 119 genes down-regulated and 94 up-regulated in all three mutants at 30°C (Fig. 3A). To determine if the expression of genes in particular metabolic pathways was specifically affected by disrupting TFIIA function, each gene in this overlapping set was associated with 1 or more of 107 MIPS categories. Actual numbers of genes in each category with decreasing or increasing expression were compared to the total number of genes in that category to determine if any category contained more changed genes than would be expected by chance (75). A P value of <0.05 was considered to denote an enriched category. We observed significant enrichment for seven categories when genes in the decreased subset were classified (Fig. 3B), and four categories were enriched in the overexpressed set (Fig. 3C). This suggests that genes in certain MIPS categories may depend on TFIIA functions in a similar manner.

Genes that are underexpressed in the TFIIA mutants are regulated by Yap1. In order to determine if there is a common promoter element that is dependent on wild-type TFIIA functions for full activity, we performed motif analysis (78) on the set of genes that are underexpressed in all three mutant strains. Analyses of 800 bp of upstream sequences from this class of genes showed several overrepresented motifs, including AAGAGC, TTACTAA, CAAGA, CGGAG, GACGAA, and AAGCG. One of these motifs, TTACTAA, is the binding site for the transcriptional activator Yap1 (1, 24, 69).

Previous studies have demonstrated that overexpression of Yap1 results in increased expression of a pool of over a dozen genes, most of which are involved in tolerance to oxidative stress (21). Interestingly, a majority of these genes are down-regulated in the three TFIIA mutants (Table 1). When compared to genome-wide expression analysis of other factors involved in transcription, this pool of Yap1-regulated genes are not uniformly down-regulated in an srb10, swi2, or taf145 mutant (Table 2) (33). Thus, this phenomenon is specific to the TFIIA mutants.


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  		TABLE 1. Expression levels of a majority of genes up-regulated by overexpression of Yap1 are down-regulated in the TFIIA mutants


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  		TABLE 2. Genome dependence on functions of TFIIA compared to those of other transcription factors

TFIIA mutants display defective growth phenotypes related to the oxidative stress response. To further delineate a connection between Yap1 function and TFIIA, we tested the TFIIA mutants for phenotypes associated with Yap1 function and the oxidative stress response. When grown on H2O2, all three mutants are severely defective for growth, comparable to a strain with YAP1 deleted, whereas wild-type strains grow robustly (Fig. 4). In addition, when grown on other oxidative stress-inducing agents (diamide or CdSO4) a strain containing the I27K mutation fails to grow, and the other two mutants grow poorly. In contrast, all three mutant strains grow well on sorbitol, an inducer of osmotic stress. Taken together, these results indicate that these TFIIA mutants are compromised for growth on a variety of agents that specifically induce the oxidative stress response.


Figure 4
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  		FIG. 4. Cells containing Toa2 derivatives display mutant growth phenotypes under conditions that induce the oxidative stress response. Strains containing the indicated Toa2 mutations or the wild-type Toa2 (WT) were serially diluted (from 104 to 10 cells); applied as spots to control plates (YPD) or plates containing H2O2 (2 mM), diamide (1 mM), CdSO4 (75 µM), or sorbitol (1 M); and incubated at 30°C.

Nuclear localization of Yap1 is normal in the TFIIA mutant strains. The above observations suggest an important link between TFIIA and Yap1. However, there are several trivial phenomena that would also produce a decrease in expression of Yap1-dependent genes in the TFIIA mutant strains. One is diminished expression of Yap1. To test this hypothesis, Yap1 mRNA levels were measured (Fig. 5A). We found no changes from the wild type in mRNA expression levels for Yap1 in the three mutants, whereas the Yap1-regulated genes FLR1, GTT2, and YKL071W were diminished (and in accord with the array data). We also examined the protein level of Yap1 by immunoblotting, and as was the case for the mRNA, protein levels were similar to those of the wild-type strain (Fig. 5B).


Figure 5
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  		FIG. 5. Yap1 levels are unchanged in the three TFIIA mutant strains. (A) YAP1 transcript levels remain unchanged, while expression levels of the Yap1-dependent FLR1, GTT2, and YKL071W genes are greatly reduced. Thirty to 50 µg of total RNA was hybridized with a 100-fold excess of the indicated probe and treated with S1 nuclease. The Pol III-transcribed tRNAW gene served as a loading control. (B) Protein levels of Yap1 in the wild type (WT) or strains containing I27K, M38K, or L41A are indistinguishable. Strains were harvested after incubation at 30°C. Protein extracts were subjected to SDS-PAGE and immunoblotting with anti-Yap1 antibodies or anti-TBP antibodies (load control).

Thus, the level of the activator is not responsible for the diminished gene expression in the mutant TFIIA strains. Since Yap1 is regulated via nuclear localization during the response to oxidative stress (82), we next tested whether Yap1 was appropriately targeted in the TFIIA mutant strains. Intracellular localization studies using GFP-tagged Yap1 indicate that the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress agents (Fig. 6). Taken together, these results suggest that the expression of this pool of genes is dependent on a functional requirement between Yap1 and TFIIA.


Figure 6
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  		FIG. 6. Yap1 is localized appropriately during the response to oxidative stress in the TFIIA mutant backgrounds. The wild-type (WT) strain or TFIIA mutant (I27K, M38K, and L41A) strains were transformed with a GFP-Yap1-expressing plasmid and cultured under normal conditions (no stress) or under conditions for induction of the stress response (1 mM H2O2 for 15 min). GFP-Yap1 is present in the cytoplasm under no stress and localizes to the nucleus after stress in all of the strains tested. Photographs were taken using green (GFP) filters with an Olympus BX60 fluorescence microscope with a Hammamatsu ORCA charge-coupled device camera.

TFIIA interacts with Yap1 in vitro and in vivo. To test the hypothesis that TFIIA and Yap1 interact in vivo, we tested for an interaction using the yeast two-hybrid assay. The DNA-binding domain of Gal4 was fused in frame with Toa2 (DB-Toa2), creating the bait for the two-hybrid assay (44). The Gal4 activation domain was fused in frame to Yap1 (AD-Yap1). Fusion proteins were expressed in a yeast strain with the HIS3 gene under the control of the GAL1 promoter (which contains four Gal4 binding sites). Interactions between Yap1 and TFIIA were determined by examining activation of the HIS3 gene, which was assayed by growth in the presence of aminotriazole, a competitive inhibitor of the HIS3 gene product (32). Strains in which the HIS3 gene is highly expressed, due to interactions between the DB fusion protein and the AD fusion protein, will grow on high concentrations of AT. A strong interaction was observed between Toa2 and Yap1 when compared to the negative controls of strains containing the DB vector and AD-Yap1, DB and AD vectors, or DB-Toa2 and the AD vector (Fig. 7A). In contrast, this interaction was diminished in all three mutant derivatives of Toa2 (I27K, M38K, and L41A).


Figure 7
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  		FIG. 7. TFIIA mutants are compromised for interaction with Yap1 both in vivo and in vitro. (A) The two-hybrid assay was used to show an interaction between Yap1 and TFIIA in vivo. The Gal4 activation domain fused to Yap1 and Gal4 AD alone were tested for the ability to interact with a DNA binding domain fusion to wild-type Toa2, mutant Toa2 derivatives (as indicated), or DB alone. Approximately 104 cells were applied as spots onto either 0 or 50 mM aminotriazole. (B) Yap1 interacts directly with TFIIA in vitro. Recombinant GST, GST-TFIIA, and GST-TFIIA mutant derivatives (as indicated) were mixed with His-tagged Yap1. Bound material was analyzed by 10% SDS-PAGE followed by immunoblotting with anti-GST and anti-His tag antibodies. (C) TFIIA mutant derivatives are fully functional for interaction with the TBP-TATA complex in vitro. These studies were done under conditions in which the TBP-DNA interaction is not stable unless TFIIA is present in the complex. For all reactions, concentrations of TATA DNA (9 nM) and TBP (5 nM) were held constant. The concentration of the indicated TFIIA derivative was varied (5 or 1 nM).

We next tested the hypothesis that Yap1 and TFIIA interact directly in vitro by using recombinant proteins. His-tagged Yap1, GST-tagged TFIIA, and GST-tagged mutant TFIIA derivatives were expressed in E. coli and used in a GST-pull-down assay. We found that wild-type TFIIA interacts with Yap1 in the pull-down assay, whereas all three of the mutant derivatives are compromised for interaction with Yap1 (Fig. 7B). To ensure that the bacterially expressed mutant TFIIA derivatives are folded properly, electrophoretic mobility shift assays with TBP and a DNA fragment containing a canonical TATA element were performed. As expected from the location of the substitutions on the four-helix-bundle domain and not the TBP-TFIIA interface, all three TFIIA mutants were fully functional in the electrophoretic mobility shift assays (Fig. 7C). Thus, the loss of interaction with Yap1 in the pull-down assay is not due to a general defect in the TFIIA mutants. The results presented here represent the first in vitro characterization of Yap1 with respect to the general transcription machinery and provide strong evidence that the loss of response to stress-inducing agents in the TFIIA mutants is a direct result of a compromised interaction between Yap1 and TFIIA.


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DISCUSSION
 
Aerobically growing organisms must employ numerous defense mechanisms to combat harmful exposure to reactive oxygen species. ROS are produced during normal metabolism, including respiration, beta-oxidation of fatty acids, and exposure to radiation, light, metals, and redox cycling drugs (71). ROS can damage DNA, proteins, and lipids (27, 31). When the redox balance within the cell is upset by increasing levels of ROS, oxidative stress occurs. In higher eukaryotes, oxidative stress has been linked to numerous conditions, including cancer, as a number of free radical-generating compounds are carcinogenic in animals and certain tumor-promoting agents stimulate the production of ROS (3, 25, 26). In fact, elevated levels of ROS circulating in the bloodstream are detected in cancer patients (18, 56). In addition, an underlying feature of ataxia telangiectasia is oxidative stress (81), and individuals with this disease are at a high risk for developing lymphoreticular cancers and breast cancers (73). As such, understanding the regulation of the oxidative stress response will certainly provide new insights into the characterization and treatment of numerous health-related conditions.

As in humans, the oxidative stress response in the yeast Saccharomyces cerevisiae is regulated at the level of transcription (15, 39, 68, 69). This response is modulated through the function of Yap1, a basic-leucine zipper transcriptional activator (59). Here we describe a link between Yap1-dependent gene activation and fully functional TFIIA activity. Using whole-genome expression profiling of TFIIA mutant strains, we observed enrichment for the Yap1 binding site in the upstream promoter region of the pool of genes with decreased expression in all three mutants. In addition, this pool of genes is up-regulated by overexpression of Yap1 (21). Consistent with the analysis of strains with yap1 deleted or mutant strains, which are hypersensitive to numerous oxidants (8, 28, 46, 51), the TFIIA mutant strains also exhibit growth defects under conditions that require a functional response to oxidative stress.

Through the use of in vivo yeast two-hybrid analyses and in vitro pull-down assays, we have also established that a protein-protein interaction exists between Yap1 and TFIIA. Moreover, this interaction is diminished significantly in the three TFIIA mutants. Thus, it is likely that Yap1 is acting through TFIIA to stimulate the expression of these Yap1-dependent genes. This is not an unprecedented function of TFIIA, since TFIIA has been shown to be a critical aspect of activated transcription by the human activator Zta (20, 42, 53, 64). With the discovery of a similar functional dependence between Yap1 and TFIIA, this now allows for the use of the powerful genetic and biochemical approaches afforded by the yeast system to fully characterize this interaction. In addition, our findings now establish TFIIA as a critical component in the response to oxidative stress. Further study of the Yap1-TFIIA interaction will ultimately lead to a better understanding of how transcriptional activators depend on TFIIA for functional activity, as well as how aerobically growing cells survive and flourish in an oxidative stress-inducing environment.


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ACKNOWLEDGMENTS
 
We would like to thank Pharmacia Corporation (Infectious Disease Genomics), Kalamazoo, MI, for allowing A.B. and S.H. to pursue these studies.

This work was supported by grants from the National Institutes of Health to L.A.S. (GM056884) and W.S.M. (GM57007).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-5068. Fax: (970) 491-0494. E-mail: Laurie.Stargell{at}Colostate.edu. Back

{dagger} Present address: Seattle Biomedical Research Institute, Seattle, WA 98109. Back

{ddagger} Present address: NephRx Corporation, Kalamazoo, MI 49008. Back

§ Present address: Western Michigan University, Kalamazoo, MI 49008. Back


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Eukaryotic Cell, July 2006, p. 1081-1090, Vol. 5, No. 7
1535-9778/06/$08.00+0     doi:10.1128/EC.00071-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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