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. 2006 Jul;5(7):1081-90.
doi: 10.1128/EC.00071-06.

TFIIA plays a role in the response to oxidative stress

Susan M Kraemer  1 , David A Goldstrohm, Ann Berger, Susan Hankey, Sherry A Rovinsky, W Scott Moye-Rowley, Laurie A Stargell
Affiliations

TFIIA plays a role in the response to oxidative stress

Susan M Kraemer et al. Eukaryot Cell. 2006 Jul.

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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Figures

FIG. 1.
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.
FIG. 2.
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 10× log fold change. The black bar indicates the pool of genes used in the word search (see text).
FIG. 3.
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).
FIG. 4.
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.
FIG. 5.
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).
FIG. 6.
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.
FIG. 7.
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).
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