Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p

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Characterization of the DNA binding motif of the arsenic-responsive transcription factor Yap8p

Yulia Ilina1*, Ewa Sloma2*, Ewa Maciaszczyk-Dziubinska2, Marian Novotny3, Michael Thorsen1, Robert Wysocki2 and Markus J. Tamás1

1Department of Cell and Molecular Biology/Microbiology, University of Gothenburg, S-405 30 Gothenburg, Sweden 2Institute of Genetics and Microbiology, University of Wroclaw, 51-148 Wroclaw, Poland 3Department of Cell Biology, Charles University, 128 43 Praha 2, Czech Republic *These authors contributed equally to this work Short title: Yap8p-DNA interactions Address correspondence to: Markus J. Tamás: +46 31 786 2548 (phone); +46 31 786 2599 (fax); [email protected] (e-mail) Abbreviations used: ABC, ATP binding cassette; bZIP, basic leucine zipper; As(III), arsenite; As(V), arsenate; EMSA, electrophoretic mobility shift assay; YRE, Yap Response Element; GST, glutathione S-transferase; bp, base pair

Biochemical Journal Immediate Publication. Published on 26 Jun 2008 as manuscript BJ20080713

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2008 Biochemical Society

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DOI : 10.1042/BJ20080713

http://dx.doi.org/10.1042/BJ20080713

http://peer.ccsd.cnrs.fr/peer-00479006/fr/

http://hal.archives-ouvertes.fr

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SYNOPSIS Saccharomyces cerevisiae uses several mechanisms for arsenic detoxification including the arsenate reductase Acr2p and the arsenite efflux protein Acr3p. ACR2 and ACR3 are transcribed in opposite directions from the same promoter and expression of these genes is regulated by the AP-1-like transcription factor Yap8p. Yap8p has been shown to permanently associate with this promoter and to stimulate ACR2/ACR3 expression in response to arsenic. Here, we characterized the DNA sequence that is targeted by Yap8p. We show that Yap8p binds to a pseudo-palindromic TGATTAATAATCA sequence that is related to but distinct from the sequence recognized by other fungal AP-1 proteins. Probing the promoter by mutational analysis, we confirm the importance of the TTAATAA core element and pin-point nucleotides that flank this element as crucial for Yap8p binding and in vivo activation of ACR3 expression. A genome-wide search for this element combined with global gene expression analysis indicates that the principal function of Yap8p is to control expression of ACR2 and ACR3. We conclude that Yap8p and other yeast AP-1 proteins require distinct DNA binding motifs to induce gene expression and propose that this fact contributed towards a separation of function between AP-1 proteins during evolution. Keywords: arsenic, gene expression, DNA binding, AP-1-like proteins, yeast


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INTRODUCTION Arsenic is widespread in nature and virtually all organisms have systems in place to evade toxicity and to acquire tolerance. Arsenic resistance usually involves a multitude of proteins implicated in arsenic transport, signal transduction and transcriptional regulation, and several resistance/detoxification mechanisms have been characterized in the eukaryotic model organism Saccharomyces cerevisiae (budding yeast) [1, 2]. Pentavalent arsenate [As(V)] is likely to enter yeast cells through the phosphate transporter Pho84p [3] whereas trivalent arsenite [As(III)] enters through the aquaglyceroporin Fps1p [4, 5]. The cytosolic arsenate reductase Acr2p (systematic name YPR200c; also known as Arr2p) converts As(V) into As(III) [6, 7], which can be exported out of the cell by the plasma membrane efflux protein Acr3p (systematic name YPR201w; also known as Arr3p) [8, 9]. In addition, As(III) can be conjugated to glutathione and the As(III)-glutathione complex is sequestered into vacuoles by the ABC transporter Ycf1p [8]. Expression of YCF1, ACR2 and ACR3 is controlled by two related AP-1-like proteins, Yap1p and Yap8p (systematic name YPR199c; also known as Acr1p and Arr1p), that belong to the basic leucine zipper (bZIP) family of transcription factors [10]. Yap1p is the best-studied member of the yeast AP-1 family, and it regulates responses triggered by oxidants, by chemicals with electrophilic properties, and by several metals including arsenic [1, 11]. In response to As(III), Yap1p stimulates expression of about 70 genes, most of which encode antioxidant defence functions, functions in sulphur assimilation and glutathione biosynthesis as well as the YCF1 gene [10, 12, 13]. Yap8p contributes to arsenic tolerance by mediating arsenic-induced expression of ACR2 and ACR3. The fact that cells lacking YAP8 or YAP1 are strongly arsenic sensitive underscores their central importance for controlling tolerance systems [10, 12, 14, 15]. We previously demonstrated that Yap8p is a homodimer [16] that predominantly resides in the nucleus [10]. Moreover, using chromatin immunoprecipitation assays, we found that Yap8p was associated with the ACR3 promoter in both untreated and As(III)-exposed cells [10]. In fact, ACR2 and ACR3 share a common promoter [17] and the two genes are transcribed in opposite directions (see Fig 1). This promoter has a putative Yap response element (YRE) and deletion of this sequence (TTAATAA) reduced As(III)-dependent induction of ACR2/ACR3 expression to a similar extent as YAP8 deletion did [10]. Based on these data, we proposed that Yap8p binds to and regulates ACR3 expression through this site [10]. The preferential DNA motif recognized by Yap1p is TTACTAA but Yap1p can also bind to other sequences [18, 19]. Beside the core 7bp-long element, the nucleotides flanking the core may also influence the DNA binding capacity of these transcription factors [20, 21].

The objective of this work was to define the DNA binding site of Yap8p, to understand Yap8p-DNA interactions and to identify potential novel gene-targets regulated by Yap8p. We demonstrate that Yap8p binds to the pseudo-palindromic TGATTAATAATCA sequence and provide evidence for its importance for Yap8p-dependent activation of ACR3 gene expression in response to As(III). We also show that the principal function of Yap8p is to control expression of ACR2 and ACR3. Finally, a search for YAP1, YAP8 and ACR3 orthologues in fungal genomes and for YREs within the promoters of putative ACR3 genes, indicate that Yap1p and Yap8p require distinct binding sites to induce gene expression. We propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.


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EXPERIMENTAL Strains and growth conditions The Saccharomyces cerevisiae strains used in this study are listed in Table 1. Yeast strains were grown on rich YPAD medium (1% yeast extract, 2% peptone, 2% glucose, 0.004% adenine sulphate) or on minimal YNB medium (0.67% yeast nitrogen base) supplemented with auxotrophic requirements and 2% glucose as a carbon source. Growth assays in the presence of sodium arsenite (Sigma) were carried out as previously described [5]. Yeast transformations were performed by a modified lithium acetate method using PLATE mixture [22]. Plasmids and mutagenesis Plasmids used in this work are listed in Table 2. The ACR3 promoter fragments of 110bp, 130bp, 150bp, 200bp and 250bp were generated by PCR using the pRW3 plasmid as template, the forward primers SF110, SF130, SF150, SF200 or SF250, respectively, and the reverse primer SR2 [9] (primer sequences are listed in Table 3). The resulting PCR products were cleaved with EcoRI/BamHI and cloned into pSEYC102 [23], producing plasmids pES11, pES12, pES13, pES14 and pES15. The YRE and flanking nucleotides in the ACR3 promoter were mutagenized using the Altered Sites II in vitro mutagenesis system (Promega), plasmid pALTER1-ACR3 as template [10], and the mutagenic oligonucleotides listed in Table 3. Next, the mutated versions of the ACR3 promoter were transferred into EcoRI-BamHI-cleaved pSEYC102 [23] to produce ACR3-mut-lacZ fusions. The wild type ACR3 promoter in plasmid pRW3 and pEM27 plasmids was also replaced with the KpnI-KpnI fragments containing mutated versions of the ACR3 promoter from pALTER1-ACR3 to obtain the coding sequence of ACR3 gene under the control of promoters with mutated YREs.

To create the GST-YAP8 fusion, the YAP8 coding region was amplified by PCR and the resulting fragment was inserted into BamHI/SalI-digested pGEX4T-1 plasmid (GE Healthcare) generating an in-frame fusion of GST with YAP8 at the N-terminal end of Yap8p. The BamHI/SalI fragment was also inserted into the yeast expression vector pYEX4T-1 (2μ, URA3, CUP1-promoter) for expression of GST-Yap8p in yeast. All plasmids were confirmed by sequencing. Electrophoretic mobility shift assays (EMSA) Production of GST-Yap8p was induced by incubating Escherichia coli BL21 cells with IPTG (1 mM final concentration) for 3 hours at 30ºC in 2-YTG medium (1% yeast extract, 1.6% bacto-peptone, 0.5% NaCl, 2% glucose) in the presence of ampicillin. Cells were harvested and disrupted using a French press in cold PBS buffer containing protease inhibitor cocktail (Roche), DNAse (0.1 mg/ml), 5 mM DTT, and 1% TritonX-100. GST-Yap8p was purified using glutathione beads (Amersham Biosciences) according to the protocol supplied by the manufacturer.

The procedure for EMSA was based on [24]. Briefly, the probes were 5’-end labelled with [γ-32P]ATP using polynucleotide kinase (New England Biolabs). For the binding reaction, approximately 75 fmol of labelled probe (~105 c.p.m.) was incubated with 0.2 μg of GST-Yap8p in binding buffer (10 mM HEPES-NaOH, pH 7.9, 20 mM KCl, 10% glycerol, 0.1 mM EDTA, 0.5 mM DTT) containing 1 μg non-specific competitor poly(dI-dC) (Amersham). Binding reactions were incubated at 25ºC for 30 minutes and then loaded onto a 5% non-denaturing polyacrylamid gel. Electrophoresis was performed on ice (100V, 1 hour) in high ionic strength Tris-glycine buffer (50 mM Tris, pH 8.8, 380 mM glycine, 2 mM EDTA). The gels were dried and analysed using a phosphorimager (Molecular Imager FX, BioRad). For competition assays, unlabelled (cold) probe was added in ten-fold molar excess. The sequences of the oligonucleotides used as probes are listed in Table 3.


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In silico analysis A BLAST search of S. cerevisiae Yap1p, Yap8p, and Acr3p orthologues was performed on 68 fungal genomes using the NCBI genomic BLAST database with fungal genomes (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi) and the tblastn program with default parameters. Putative YRE sites in the -1000 upstream regions of genes coding for Acr3p orthologues were identified using the YEASTRACT database [25]. Gene expression analyses RNA isolation, cDNA synthesis, microarray hybridization and analysis were performed as described in [13]. In brief, total RNA was isolated from exponentially growing yeast cells that were either untreated or exposed to sodium arsenite. 20 µg of total RNA was primed with 3 µg of random hexamer (Invitrogen, CA) and 3 µg of anchored oligo(dT)20 primer (ABgene, UK), and labelled in a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech, Sweden) according to standard protocols (http://cmgm.stanford.edu/pbrown). Hybridization between the microarray chip (Yeast 6.4k array from UHN Microarray Centre, Toronto, Canada) and the labelled cDNA was performed three times using cDNA from independent cultures and RNA extractions. The slides were scanned with a VersArray ChipReader (Bio-Rad), data was extracted with the ImaGene v.6 software (BioDiscovery, CA) and the microarray data was analyzed using LIMMA package (http://www.bioconductor.org). The data was normalized by subtracting a loess line from the M-value to remove intensity-dependent trends [26]. The genes were ranked by the moderated t-statistic to avoid false positives [27]. The β-galactosidase assays were performed as described in [16, 28].


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RESULTS Defining the Yap8p DNA binding site in the ACR3 gene promoter To identify the DNA motif recognized by Yap8p, we first created a number of truncations within the ACR3 promoter, fused these promoters to the lacZ gene and monitored β-galactosidase activity as a reporter of promoter activity. As(III) strongly stimulated expression from the full-length promoter (containing 353 nucleotides in front of the lacZ gene) in wild type cells, as well as from the -250, -200, -150, and -130 promoters (Fig 1A). In contrast, As(III)-dependent induction of the reporter was absent in the -110 promoter (Fig 1A). Since ACR3 promoter-lacZ expression is not stimulated in yap8Δ cells [10, 16](Fig 1B), these data indicate that the 20bp-region located between -110 and -130 is required for Yap8p-dependent induction of reporter gene expression. This region contains a pseudo-palindromic sequence TGATTAATAATCA with a putative YRE in the centre (underlined). Deletion of the TTAATAA sequence completely abolished As(III)-induced ACR3-lacZ expression (Fig 1B: mut2 construct). Similarly, altering three nucleotides in the recognition element (TGATTAATAATCA into TGATCTAGAATCA; mut4 construct) or nucleotides flanking the core (TGATTAATAATCA into TTCTTAATAAATT; mut6 construct) resulted in a lack of As(III)-stimulated induction of the reporter (Fig 1B).

To confirm the importance of this promoter element for Acr3p-mediated As(III) tolerance, we placed the mutated promoters in front of the wild type ACR3 coding region and transformed the resulting plasmids into acr3Δ cells. As shown in Fig 1C, none of the cells containing the promoter mutations grew on As(III)-containing plates whereas cells transformed with the plasmid carrying the native ACR3 promoter grew well. Hence, Yap8p-dependent ACR3 expression requires the pseudo-palindromic TGATTAATAATCA sequence.

We noted that truncations of the ACR3 promoter from -150 to -130 and -110 led to a gradual de-repression of the basal promoter activity (Fig 1A). The 40bp-region located between -150 and -130 only contains a putative binding motif (YTGAT) for the inhibitor of HO transcription Ash1p, and the Ash1p motif overlaps with that of Yap8p. The strongest de-repression was seen when truncating from -130 to -110, suggesting that Yap8p could act both as a repressor and an activator. However, the fact that we do not see de-repression of ACR3-lacZ expression in a yap8Δ mutant (Fig 1B) argues against such a role for Yap8p. Instead, the observed de-repression may be a consequence of altered promoter architecture when DNA segments are deleted.

We also found that truncation of the ACR3 promoter from -200 to -150 resulted in a 2-fold reduction of As(III)-induced ACR3-lacZ expression (Fig. 1A), suggesting that additional regulatory elements might contribute to ACR3 control. The -200 to -150 region contains two 13bp-long pseudo-palindromic sequences ACTTTTCAAAAGT (from -187 to -175) and TGATTTAAAATCA (from -172 to -159) that might serve as potential regulatory elements. However, mutagenesis of the first sequence had no effect on ACR3 promoter activity (A. Marchwicka and R. Wysocki, unpublished data). The significance of the latter sequence has not been tested yet. Analysis of the -200 to -150 region using the YEASTRACT database [25] revealed two putative Ash1p binding motifs and one putative Fkh1p/Fkh2p motif. Since the focus of this study was Yap8p-DNA interactions, we did not further study whether these transcription factors contribute to As(III)-dependent ACR3 expression. Differential regulation of ACR3 promoter by Yap1p and Yap8p Previous studies implicated both Yap1p and Yap8p in the control of ACR3 expression; YAP1 or YAP8 deletion reduced ACR3 expression levels in response to As(III) [10, 12, 13, 15] (Fig 1B) whereas YAP1 overexpression stimulated ACR3 expression [29, 30]. Hence, Yap1p might recognize the Yap8p site in the ACR3 promoter. In contrast, there are no reports on Yap8p affecting expression of Yap1p-dependent genes. To get more insight into how these two AP-1


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factors control ACR3 expression, we made another set of promoter mutations. First, we changed the Yap8p core site (TTAATAA) into a Yap1p site (TTACTAA: mut3 construct) and monitored ACR3 promoter-lacZ expression. ACR3-mut3-lacZ expression was strongly stimulated by As(III) in wild type cells; in fact β-galactosidase activity appeared somewhat higher in ACR3-mut3-lacZ than in ACR3-lacZ (wild type promoter) transformed cells (Fig 2A). Interestingly, whereas there was basically no induction of ACR3-lacZ in the yap8Δ mutant (Yap1p is present), ACR3-mut3-lacZ expression was clearly induced in yap8Δ cells (Fig 2A). Hence, the mut3 promoter (TTACTAA) is likely to be recognized by Yap1p. In addition, the mut3 promoter is also recognized by Yap8p as ACR3-mut3-lacZ expression is induced in yap1Δ cells to the same extent as ACR3-lacZ. To test the role of the flanking sequences for Yap1p and Yap8p recognition of mut3, we created two additional mutations (mut8 and mut9; see Fig 2A). We found that mutation of nucleotides flanking the core TTACTAA element abolished Yap8p-dependent activation of ACR3-lacZ expression since no ACR3-mut8-lacZ or ACR3-mut9-lacZ expression was detected in yap1Δ cells. In contrast, Yap1p was still capable of driving As(III)-induced ACR3-lacZ expression from the mut8 promoter but not from the mut9 promoter since ACR3-mut8-lacZ expression but not ACR3-mut9-lacZ expression was detected in yap8Δ cells (Fig 2A). However, the activity of the mut8 promoter was five times lower than that of the mut3 promoter. Similarly, the mut8 promoter exhibited ~20% activation relative to the wild type promoter. Taken together, these results confirm the importance of the nucleotides flanking the core TTAA/CTAA consensus for recognition by Yap1p and Yap8p. We note that Yap8p recognition of the element is more sensitive to changes in the flanking nucleotides than Yap1p.

We hypothesized that Yap1p and Yap8p might control ACR3 expression under different phases of the cell’s adaptation to As(III) and that we might miss the dynamics of this regulation by monitoring gene expression/β-galactosidase activity at a single time-point after As(III) exposure. Therefore, we monitored β-galactosidase activity driven by the ACR3 wild type and mut3 promoters in a time-course experiment (Fig 2B). Our data show almost identical induction profiles of ACR3-lacZ in wild type and yap1Δ cells whereas there is only a very weak Yap1p-dependent stimulation of reporter activity in yap8Δ. Hence, Yap8p appears to be (almost) fully responsible for ACR3 induction in wild type cells whereas Yap1p might get access to the ACR3 promoter in the absence of Yap8p. The situation for the mut3 promoter was different (Fig 2B); Yap8p is not as potently activating ACR3 expression from the mut3 promoter as from the wild type promoter (see induction profile in yap1Δ). In contrast, the mut3 promoter appears to be more accessible for Yap1p than the wild type promoter (see induction profile in yap8Δ). We also note that As(III)-dependent induction of ACR3-mut3-lacZ was faster and stronger than that of ACR3-lacZ in wild type cells. Collectively, these data suggest that in vivo, Yap8p controls ACR3 expression and that Yap1p has a negligible role. Electrophoretic mobility shift assays confirm the Yap8p DNA binding site The data above strongly suggested that Yap8p binds to the TTAATAA core sequence within the ACR3 promoter and that this binding is influenced by flanking nucleotides. To confirm this, we first fused Yap8p to GST, expressed, and purified the heterologous protein from E. coli. Next, we evaluated the DNA binding capacity of GST-Yap8p by electrophoretic mobility shift assays (EMSA). To make sure that the GST-tag does not influence the function of Yap8p and/or affect its DNA binding, we expressed GST-Yap8p in yap8Δ cells and scored growth of the transformants. The fusion protein appeared to be functional since it restored As(III) tolerance to the yap8Δ mutant (Fig 3A). The EMSA revealed formation of a protein-DNA complex when GST-Yap8p was incubated with a 386bp 32P-labelled DNA fragment derived from the ACR3 promoter as a probe, whereas no mobility shift was detected when


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GST alone was mixed with this probe (Fig. 3B). When unlabelled (cold) probe was added in excess, no complex formation was detected (Fig. 3B) confirming that the observed binding is specific for Yap8p. Hence, GST-Yap8p is capable of binding to the ACR3 promoter.

Next, we analyzed the binding of GST-Yap8p to the various mutated promoters by using 32P-labelled oligonucleotides (51 nucleotides long; see Table 3) as probes. First, we checked whether ten-fold excess of the cold probes mut2, mut3, mut4 or mut6 could disrupt the protein-DNA complex formed in extracts containing GST-Yap8p and the oligonucleotide corresponding to the wild type ACR3 promoter sequence. As shown in Fig 3C (upper panel), the protein-DNA complex was not visible after addition of the unlabelled wild type and mut3 probes whereas addition of mut2, mut4 and mut6 did not affect protein-DNA complex formation, indicating that GST-Yap8p binds to the wild type and mut3 sequences but not to mut2, mut4 and mut6. Indeed, GST-Yap8p and the mut3 oligonucleotide formed a specific protein-DNA complex (Fig 3C, lower panel, lanes 1-3). In contrast, this complex was not formed when GST-Yap8p was mixed with mut4 or mut6 oligonucleotides (Fig 3C, lower panel, lanes 4-9). We conclude that Yap8p can bind to the wild type and mut3 sequences (TGATTAATAATCA and TGATTACTAATCA) and that the EMSAs fully support the gene expression and phenotypic data above. Genome-wide search for putative Yap8p-controlled genes To identify Yap8p gene-targets in addition to ACR2/ACR3, we performed global gene expression analysis and compared the expression profiles of the yap8Δ mutant to that of wild type cells after As(III) exposure (0.2 mM As(III) for 1 hour). This concentration was used since we previously showed that 0.2 mM As(III) triggers a robust transcriptional response in yeast and furthermore allows a direct comparison between this study and published genome-wide expression profiles of wild type cells and mutants lacking other transcription factors [13]. We found 15 genes that displayed at least a two-fold lower expression in yap8Δ compared to wild type (Supplementary Table 1). Since one cannot firmly conclude that these genes are direct Yap8p-targets solely based on expression analysis, we searched for TTAATAA and/or TTACTAA in the promoters of the genes showing altered expression in yap8Δ. This search identified six genes; ACR2, ACR3, YOL155c, LEU1, PMA1 and CLG1 (two YREs). To test whether Yap8p binds these promoters in vitro, we performed EMSA with oligonucleotides corresponding to those gene-promoters and GST-Yap8p. We found that GST-Yap8p did not bind to the promoters of CLG1, YOL155c, LEU1 and PMA1 in the EMSAs (data not shown). We note that even though the promoters of these genes contain TTAATAA or TTACTAA, the flanks are different from the bona fide Yap8p motif and the extended elements are not pseudo-palindromic. Moreover, growth of mutants lacking CLG1, YOL155c or LEU1 (deletion of PMA1 is lethal) was unaffected by As(III) (data not shown). Hence, these genes are not likely to be Yap8p-targets or to play a major role in As(III) detoxification in vivo. An in silico search for TGATTAATAATCA or TGATTACTAATCA in the S. cerevisiae genome identified only two additional genes, FRM2 and DRE2, with promoters containing the respective Yap8p motif. Although expression of FRM2 is strongly induced by As(III), this induction is Yap8p-independent [13] (supplementary Table 1). Nevertheless, GST-Yap8p could bind to the FRM2 promoter in the EMSA (Fig 3D). However, deletion of FRM2 did not produce any As(III) sensitivity (data not shown). In contrast, DRE2 expression is not responsive to As(III) [13] and the DRE2 null mutation is lethal. Taken together, our data suggest that the physiological function of Yap8p may be restricted to control the expression of ACR2 and ACR3 in response to As(III) exposure. Distribution of YAP1, YAP8 and ACR3 homologues in fungal genomes and a search for putative YREs in the promoters of ACR3 genes


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The data above seems to indicate first, that Yap8p has evolved for a highly specific function in S. cerevisiae since it controls expression of only two genes that share a common promoter, and second, that with a one base pair change in the ACR3 promoter Yap1p is capable of taking over the role of Yap8p, at least to some extent. To get insight into how these AP-1 proteins have evolved in various organisms with respect to arsenic resistance, we made a database search for the presence and distribution of YAP1, YAP8 and ACR3 genes in fungal genomes. Whereas Yap1p orthologues were found to be ubiquitously present in fungi, genes encoding putative Yap8p-like proteins were only found in the genomes of three closely related species; Saccharomyces paradoxus, Saccharomyces kudriavzevii and Kluyveromyces lactis (Table 4). However, homologues of the ACR3 gene were identified in 20 fungal genomes. We next asked whether the promoters of these ACR3 genes contain a YRE and whether the nucleotide sequence of these elements depend on the presence or absence of Yap8p orthologues. Thus, we searched the ACR3 promoters for putative Yap8p and/or Yap1p binding sites. Importantly, the full TGATTAATAATCA element was always identified in the promoters of ACR3 genes in organisms possessing Yap8p orthologues. Further, we found the presence of several variants of YREs in the other ACR3 promoters, but with no apparent conservation of the flanking nucleotides (Table 4). We conclude that in evolution the appearance of Yap8p coincided with base pair substitutions in the centre and flanks of the TTACTAA sequence in the ACR3 promoter resulting in the formation of the TGATTAATAATCA element which specifically responds to Yap8p, but no longer to Yap1p, during arsenic stress.


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DISCUSSION S. cerevisiae has eight AP-1-like proteins - Yap1p to Yap8p – that represent a distinct and fungal-specific family of bZIP transcription factors. These proteins have a high degree of conservation within the basic region that makes contact with DNA [18]. The preferred DNA binding motif for Yap1p, Yap2p, Yap3p and Yap4p is centered around the consensus TTACTAA, however, the strongest activation through this site is observed for Yap1p and much weaker for Yap2p and Yap3p [18]. The reason for this observation and how the AP-1 proteins show specificity towards different subsets of target genes is not fully understood.

Here, we show that Yap8p recognizes and binds to an extended YRE of 13bp with the pseudo-palindromic sequence TGATTAATAATCA. We demonstrate that the core TTAA/CTAA is required but not sufficient for Yap8p binding and that the nucleotides flanking the core element strongly influence the binding capacity of Yap8p. Importantly, mutations in the flanks were not tolerated since such mutations abolished Yap8p-DNA interactions and consequently, no induction of ACR3 expression in response to As(III) was observed. In contrast, Yap8p could still interact with the TTACTAA sequence provided that the flanks were unchanged (i.e. TGATTACTAATCA). The importance of the full 13bp sequence for Yap8p-dependent ACR3 expression is further underscored by the fact that organisms having a Yap8p orthologue also contained the TGATTAATAATCA sequence in the promoters of the respective ACR3 gene. Conversely, in organisms that do not have a Yap8p orthologue, the Yap8p-binding site was not conserved but showed a number of alterations in the nucleotide sequence. Finally, flanking sequences clearly influence Yap8p DNA binding in vivo: chromatin immunoprecipitation assays demonstrated that Yap8p is present on the ACR3 promoter (contains TGATTAATAATCA) but not on its own promoter (contains TTCTTAATAAATT) [10].

Yap5p was recently shown to activate transcription of CCC1, encoding a protein involved in iron import into the vacuole [31]. Interestingly, the CCC1 promoter contains two YREs with the consensus TTACTAA sequence, however, Yap5p activates CCC1 transcription only from one of these YREs [31]. The two motifs differ from each other in the flanking nucleotides: CAATTACTAATGT for the nonfunctional site and ATATTACTAACAT for the functional site. Hence, in analogy to Yap8p, the flanking nucleotides have an influence on the DNA-binding capacity of Yap5p.

The importance of the flanking sequences was also inferred from global gene expression analysis: Yap1p and Yap2p was found to control activation of nonoverlapping sets of genes in response to H2O2 and it was shown that mutations of nucleotides flanking the core YRE decreased expression of their respective target genes [20]. Indeed, Yap1p appears to have a strong preference for a C immediately preceding the core motif (i.e. CTTACTAA) whereas Yap2p has a strong preference for a T immediately following the core motif (i.e. TTACTAAT) [21]. Taken together, evidence is accumulating that suggests that the yeast AP-1 proteins control distinct sets of gene targets and that extended YREs may provide specificity towards different subsets of genes. By employing ChIP-chip technology (i.e. chromatin immunoprecipitation followed by intergenic microarray assays), phylogenetic footprinting and computational identification of cis regulatory sites of individual Yap-proteins, it should be possible to reveal the mechanism of specificity towards different subsets of target genes.

The three-dimensional structure of Yap8p has not been determined yet, so the precise details of the protein-DNA complex are not known. Nevertheless, the amino acids within Yap8p that are likely to be involved in binding to the TGATTAATAATCA sequence can be deduced from local alignments of the DNA-binding motifs of AP-1 proteins (Fig 4). The alignments show that three amino acids that are very well conserved in Yap1p to Yap7p, are substituted in Yap8p proteins; a conserved asparagine is substituted by a leucine, a conserved alanine is substituted by a serine, and on a position where either arginine or lysine


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appears in Yap1p to Yap7p, asparagine is found in all but one of the Yap8p sequences (Fig 4). The role of these residues (Asn86, Ala89 and Arg91) has been revealed in the crystal structure of the bZIP domain of the Schizosaccharomyces pombe (fission yeast) AP-1 protein Pap1p bound to DNA [32]. The majority of interactions between DNA and Pap1p are either non-specific (i.e. to the DNA backbone) or mediated through conserved water molecules. This is true also for Asn86 that forms numerous contacts with DNA bases. Similarly, the side chain of Arg91 of chain B and the main chain of Ala89 are in a contact with a phosphate group of DNA (also through water molecules). All three substitutions observed in Yap8p proteins will therefore influence the capacity to bind DNA and could be the key to their altered DNA-binding preference. Other differences in the bZIP domains among the AP-1 proteins exist and those differences may for example affect dimerization properties and further contribute to distinct DNA binding characteristics of these proteins [32]. Ultimately, structural analysis will be required to fully answer this question. Interestingly, the structure of Pap1p shows that (like for Yap8p) it also recognizes a 13 nucleotide long DNA sequence. This observation further supports the idea that the flanking regions outside the canonical core element contribute to binding specificity.

Global gene expression analysis combined with an in silico search for putative Yap8p gene-targets identified a number of candidates. However, most of these gene-promoters lack the Yap8p DNA binding motif and those with a YRE in their promoters were not bound by Yap8p in EMSA. The only promoter that Yap8p could bind to (beside the ACR2/ACR3 promoter) was that of FRM2. FRM2 encodes a nitroreductase-like protein of unknown function and its expression is strongly stimulated by As(III) both at gene- and protein levels [13]. However, YAP8 deletion did not affect FRM2 expression (supplementary Table 1) and the frm2Δ mutant was not As(III) sensitive (data not shown). Frm2p levels are elevated in response to cadmium in a Yap2p-dependent way, and frm2Δ is cadmium sensitive [33]. Hence, despite the fact that Yap8p binds to the FRM2 promoter in vitro, Frm2p does not appear to mediate Yap8p-dependent arsenite resistance. Instead, our data indicate that the principal function of Yap8p is to control expression of ACR2 and ACR3.

What would the evolutionary advantage be to have only ACR2 and ACR3 regulated by Yap8p when a large number of genes are already regulated by Yap1p in response to As(III) [10, 13]? It could be important for the cell to separate between 'general' oxidative stress and metal-specific responses. For instance, it is probably not economical to turn expression of every gene on when the cell experiences metal-derived oxidative stress, and high levels of Acr3p in the plasma membrane might not be favourable under oxidative stress conditions. Hence, these two proteins have acquired different roles in As(III)-exposed cells: the Yap1p-mediated response may be aimed at relieving the cells from the consequences of the stress (i.e. arsenite-induced oxidative stress generation) whereas the Yap8p-mediated response is aimed at removing the cause of that stress (i.e. arsenite export through Acr3p).

To conclude, we have defined the DNA binding motif that is recognized by Yap8p and through which this protein activates transcription in response to arsenite. Gene duplication followed by functional divergence is a major driving force for the evolution of phenotypic complexity [34]. Our analysis indicates that Yap8p and other yeast AP-1 proteins require distinct DNA binding motifs to induce gene expression and we propose that this fact contributed towards a separation of function between AP-1 proteins during evolution.


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Acknowledgements We thank Erik Kristiansson and Olle Nerman (Chalmers University of Technology, Gothenburg) for expert help with microarray data analysis, Yujun Di (Gothenburg) for the pGEX4T-1-GST-YAP8 plasmid, Dominik Piotrowiak (Wroclaw) for pDP1 plasmid, Joanna Dodzian (Wroclaw) for pJD3 and pJD4 plasmids, and Karin Lindkvist (Gothenburg) for helpful suggestions during the course of this work. We gratefully acknowledge financial support from the Swedish Research Council (VR), Carl Trygger Foundation and Swedish National Research School in Genomics and Bioinformatics to MJT, from the Ministry of Science and Higher Education research funds in the years 2006-2009 (PBZ-Min-015/P05/2004 and N30105931/1785) to RW, and from the Czech Ministry of Education, Youth and Sports (grant LC07032) to MN.


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16 Di, Y. and Tamás, M. J. (2007) Regulation of the arsenic-responsive transcription factor Yap8p involves the ubiquitin-proteasome pathway. J Cell Sci 120, 256-264


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20 Cohen, B. A., Pilpel, Y., Mitra, R. D. and Church, G. M. (2002) Discrimination between paralogs using microarray analysis: application to the Yap1p and Yap2p transcriptional networks. Mol Biol Cell 13, 1608-1614

21 Tan, K., Feizi, H., Luo, C., Fan, S. H., Ravasi, T. and Ideker, T. G. (2008) A systems approach to delineate functions of paralogous transcription factors: role of the Yap family in the DNA damage response. Proc Natl Acad Sci USA 105, 2934-2939

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26 Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. and Speed, T. P. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30, e15

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30 Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D. and Brown, P. O. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell 11, 4241-4257

31 Li, L., Bagley, D., Ward, D. M. and Kaplan, J. (2008) Yap5 is an iron-responsive transcriptional activator that regulates vacuolar iron storage in yeast. Mol Cell Biol 28, 1326-1337

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33 Azevedo, D., Nascimento, L., Labarre, J., Toledano, M. B. and Rodrigues-Pousada, C. (2007) The S. cerevisiae Yap1 and Yap2 transcription factors share a common cadmium-sensing domain. FEBS Lett 581, 187-195


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34 Ohno, S. (1970) Evolution by Gene Duplication, Springer-Verlag, New York 35 Thomas, B. J. and Rothstein, R. (1989) Elevated recombination rates in

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Sebastia, L. and Lacroute, F. (1991) A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7, 609-615


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TABLES Table 1 Saccharomyces cerevisiae strains used. Strain Genotype Source/Reference

W303-1A MATa ura3-1 leu2-3/112 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0 [35]

RW117 W303-1A yap8Δ::loxP [10] RW124 W303-1A yap1Δ::loxP [10] RW120 W303-1A yap1Δ::loxP-kanMX-loxP yap8Δ::loxP [10] BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 EUROSCARF BY4741 clg1Δ BY4741 clg1Δ::kanMX6 EUROSCARF BY4741 leu1Δ BY4741 leu1Δ::kanMX6 EUROSCARF BY4741 frm2Δ BY4741 frm2Δ::kanMX6 EUROSCARF BY4741 yol155cΔ BY4741 yol155cΔ::kanMX6 EUROSCARF


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Table 2 Plasmids used in this study Plasmid Description Source / Reference pSEYC102 CEN vector with lacZ reporter gene, URA3 [23]

pES11 110bp-long ACR3-lacZ fusion in pSEYC102 This work





pEM19 353bp-long ACR3-lacZ fusion in pSEYC102 [10]

pEM20 ACR3-mut2-lacZ fusion in pSEYC102 [10]

pDP1 ACR3-mut3-lacZ fusion in pSEYC102 This work

pEM30 ACR3-mut4-lacZ fusion in pSEYC102 This work

pJD3 ACR3-mut6-lacZ fusion in pSEYC102 This work

pES9 ACR3-mut8-lacZ fusion in pSEYC102 This work

pES10 ACR3-mut9-lacZ fusion in pSEYC102 This work

pFL39 CEN vector, TRP1 [36]

pRW3 ACR3 cloned in pFL39 [9]

pRW33 ACR3 behind its own promoter lacking the TTAATAA sequence (mut2) in pRW3 [10]

pEM31 ACR3 behind its own promoter containing the mut4 mutation in pRW3 This work

pJD4 ACR3 behind its own promoter containing the mut6 mutation in pRW3 This work

pGEX4T-1-GST-YAP8 Plasmid for GST-YAP8 expression in E. coli This work

pYEX4T-1-GST-YAP8

Plasmid (2μ, URA3, CUP1-promoter) for GST-YAP8 expression in yeast This work


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Table 3 Primers and oligonucleotides used in this study Name Sequence 5’ to 3’ Oligonucleotides for EMSA ACR3 TCTTAATTATCTTTTTGTTTGATTAATAATCAACTTTAGCGGCAACGCTCC mut2 TCTTAATTATCTTTTTGTTTGA-------TCAACTTTAGCGGCAACGCTCC mut3 TCTTAATTATCTTTTTGTTTGATTACTAATCAACTTTAGCGGCAACGCTCC mut4 TCTTAATTATCTTTTTGTTTGATCTAGAATCAACTTTAGCGGCAACGCTCC mut6 TCTTAATTATCTTTTTGTTTTCTTAATAAATTACTTTAGCGGCAACGCTCC mut8 TCTTAATTATCTTTTTGTTTTCTTACTAATCAACTTTAGCGGCAACGCTCC mut9 TCTTAATTATCTTTTTGTTTGATTACTAAATTACTTTAGCGGCAACGCTCC mut10 TCTTAATTATCTTTTTGTTTTCTTACTAAATTACTTTAGCGGCAACGCTCC YOL155c TCATCGAAAAGTGAGGTGTAGCTTACTAAAGCATTCCTCTTTATCCAAGTC LEU1 ACTTACATGAACGTATACAAATTTACTAACACTACTTGAAAATATGAACCA CLG1 (1) TAAGTCTTCGAGTCGTTTTTTTTTATTAACTTTAATCCTTTTTCTGTGTGT CLG1 (2) GGAATTTTTTTTTTCTTTAACATTATTAAGACAGTTATTGAGTTAATTCGT PMA1 TAAATTTTTTCTTTAACAATCGTTAATAATTAATTAATTGGAAAATAACCA FRM2 AATGTCTGCGAGTGTTACTTGATTATTAATCAATATTTGTCAATGCTCTTC PCR primers SF110 GGAATTCCAGCGGCAACGCTCCTTAC SF130 GGAATTCCTTTGATTAATAATCAACTTTAG SF150 GGAATTCCTGCTCTTAATTATCTTTTTG SF200 GGAATTCCAGTTTTTATACCGACTTTTC SF250 GGAATTCCTACGCTTGCTGGATTGTCAAG SR2 CGGGATCCCCATTCACCATATTAACCTTAG FP300 CGGGATCCTCAATTAGGCCCTTGAGTTGC RP300 GCCATGACAATGACTTGATCG ACR3FPr CCTTCCTCTGATTTTCAATTAGGCCC ACR3RPr GCGATTCACCATATTAACCTTAGAAGGTACCG The putative YREs and 3bp flanking nucleotides on either side are indicated in bold.


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Table 4 Distribution of Yap1p, Yap8p and Acr3p orthologs in fungal genomes and identification of putative Yap1p/Yap8p response elements (YRE) in the promoters of putative ACR3 genes Organism Yap1 ortholog Yap8 ortholog Acr3 ortholog YRE position YRE sequence Saccharomyces cerevisiae YML007W YPR199C YPR201W -128 to -116 TGATTATTAATCA Saccharomyces paradoxus PORF17363 PORF9552 PORF24493 -129 to -117 TGATTATTAATCA Saccharomyces kudriavzevii Skud_Contig2043.2 Skud_Contig1995.3 Skud_Contig1995.1 -177 to -165 TGATTATTAATCA Kluyveromyces lactis KLLA0A01760g KLLA0E00220g KLLA0E00176g -181 to -169 TGATTATTAATCA KLLA0F00242g -181 to -169 TGATTATTAATCA Saccharomyces kluyveri SKLU-Cont10078 None SKLU-Cont10104 -48 to -36 TCATTACTAATTA Saccharomyces castellii Scas_Contig714.56 None Scas_Contig550.7 -221 to -209 ACCTTATTCATGT Candida albicans orf19.1623 None Ca019.10634 -331 to -319 TGATTACTAATAC -180 to -168 TGATTAGTCAGCC -93 to -81 ATTTTACAAATGT Candida tropicalis CTRG_02398.3 None CTRG_03764.3 -986 to -974 GTTTTAATAATGT -594 to -585 TTTTTATTAAAAT -212 to -200 TGATGAGTCAAGA Pichia guilliermondii PGUG_00664.1 None PGUG_05759.1 -425 to -413 TTATGACTAAACA -139 to -127 TACTGACTAATAA Pichia stipitis PICST_66589 None PICST_83261 -487 to -475 GAGTGACAAATAT Yarrowia lipolytica YALI0F03388g None YALI0C07590g -298 to -286 ACGTGACTAACTA Lodderomyces longisporus LELG_02745.1 None LELG_04862.1 -516 to -504 TTTTTACTAATCT Debaryomyces hansenii DEHA0G02904g None DEHA0D15488g -171 to -159 CTCTGAGAAACTA Magnaporthe grisea MGG_12814 None MGG_03399.5 -762 to -750 CATTTATTAACAT -128 to -116 TTTTGACAAATTC Aspergillus fumigatus AFUA_6G09930 None AFUA_1G05760 -178 to -166 TTTTCAGTAATCA -86 to -74 CATTTAGTCAGAG AFUA_1G16100 -731 to -719 AGGTGAGTAAGGA AFUA_5G15010 -151 to -139 TATTTAGTAACAT Aspergillus terreus ATEG_06717 None ATEG_03049 -803 to -791 TGATCACAAACAT Aspergillus nidulans AN7513.2 None AN4101.2 -785 to -773 TGCTCACTAATTG Coprinus cinereus CC1G_08635 None CC1G_07351 -588 to -576 GAGTTACTAATGA Cryptococcus neoformans CNA02300 None CNK00040 -371 to -359 GAATTATTCAATT


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-80 to -68 GTGTTAATAATCT CNBK3390 -653 -640 ATCTTATTCAGCT -80 to -68 GTGTTAATAATCT Ustilago maydis UM02191.1 None UM01862.1 -906 to -894 CGATCACAAAGCG


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FIGURE LEGENDS Figure 1 Identification of a putative Yap8p-binding site. (A) Cartoon of the ACR2/ACR3 promoter and truncations made. The ACR3-promoter-lacZ fusion constructs were transformed into wild type cells and the transformants were assayed for β-galactosidase activity in untreated controls and during As(III) exposure (0.1 mM, 6 hours). Cells transformed with the empty vector served as the control. The results are the average of at least three independent experiments and the error bars represent S.D. (B) Mutations within the ACR3 promoter affect As(III)-dependent ACR3 expression. β-galactosidase activity was measured in wild type, yap8Δ, yap1Δ and yap1Δ yap8Δ cells transformed with the indicated ACR3-lacZ promoter constructs in untreated controls and during As(III) exposure (0.1 mM, 6 hours). Nucleotide substitutions are underlined. (C) Mutations within the ACR3 promoter abolish Acr3p-mediated As(III)-resistance. The acr3Δ mutant was transformed with the indicated plasmids and growth of the transformants was monitored. Cells were grown in liquid medium and ten-fold serial dilutions of the cultures were spotted onto solid agar plates containing sodium arsenite. Growth was scored after 2 days at 30ºC

Figure 2 Differential regulation of the ACR3 expression by Yap1p and Yap8p. (A) β-galactosidase activity assays in wild type, yap8Δ, yap1Δ and yap1Δ yap8Δ cells transformed with the indicated ACR3-lacZ promoter constructs in untreated controls and during As(III) exposure (0.1 mM, 6 hours). Nucleotide substitutions are underlined. The results are the average of at least three independent experiments and the error bars represent S.D. (B) Time-course assay of ACR3 expression from the ACR3-lacZ (wild type) and ACR3-mut3-lacZ reporters. β-galactosidase activity was determined in wild type, yap8Δ, yap1Δ and yap1Δ yap8Δ cells transformed with the indicated ACR3-lacZ promoter constructs before and during As(III) exposure (0.1 mM).

Figure 3 Electrophoretic mobility shift assays confirm Yap8p-DNA interactions. (A) GST-Yap8p is functional since it restores As(III) resistance to the yap8Δ mutant. Cells were grown in liquid medium and ten-fold serial dilutions of the cultures were spotted onto solid agar plates containing sodium arsenite. Growth was scored after 2 days at 30ºC. (B) GST-Yap8p binds to the ACR3 promoter. The probe (lane 1), a 386bp 32P-labelled DNA fragment derived from the ACR3 promoter, was incubated with GST-Yap8p (lane 3) produced and purified from E. coli as described in Experimental. As a control, the probe was also incubated with purified GST (lane 2). The cold probe (lane 4) was added in ten-fold molar excess. (C) GST-Yap8p binds to the wild type and mut3 promoters. Purified GST-Yap8p was incubated with oligonucleotides corresponding to the wild type ACR3 promoter and the mutated mut2, mut3, mut4 and mut6 promoters. Upper panel: competition assays where either the wild type probe or those corresponding to the mutated promoters were added to the mixture of the wild type probe and GST-Yap8p. Lower panel: binding assay where GST-Yap8p was incubated with the probes corresponding to the mutated promoters mut3, mut4 and mut6. For experimental details, see Material and methods. The sequences of the oligonucleotides are provided in Table 3. (D) GST-Yap8p binds to the FRM2 promoter. Purified GST-Yap8p was incubated with oligonucleotides corresponding to the wild type ACR3 and FRM2 promoters and EMSA was performed as above.


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Figure 4 Amino acid substitutions within the bZIP domain of fungal AP-1 transcription factors. Alignment of the bZIP domain of fungal AP-1 transcription factors: Pap1p from Schizosaccharomyces pombe, Yap1p-Yap8p from S. cerevisiae, Yap8p orthologues from S. paradoxus (SpYap8p), S. kudriavzevii (SkYap8p) and Kluyveromyces lactis (KlYap8p). The amino acid residues that contact DNA as determined for the S. pombe protein Pap1p [32] are labeled in boldface. These residues are conserved in the Pap1p/Yap1p-like proteins whereas Yap8p-like proteins have amino acid substitutions at three key positions (underlined).


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Figure 1: Ilina et al

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Yap1p-subfamily

Yap8p-subfamily

Pap1p (81-94) KRKAQNRAAQRAFR Yap1p (69-82) KRTAQNRAAQRAFR Yap2p (48-61) RRTAQNRAAQRAFR Yap3p (149-162) KKKAQNRAAQKAFR Yap4p (242-255) KRAAQNRSAQKAFR Yap5p (63-76) KKKRQNRDAQRAYR Yap6p (226-239) RRAAQNRTAQKAFR Yap7p (130-143) KRRRQNRDAQRAYR

ScYap8p (21-34) KRAAQLRASQNAFR SpYap8p (21-34) KRAAQLRASQNAFR SkYap8p (37-50) KRTAQLRASQNAFR KlYap8p (21-34) KRTAQLRKSQKTYR - - -


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Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p

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Transcript of Characterization of the DNA-binding motif of the arsenic-responsive transcription factor Yap8p