Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in Δtrr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and Δtrr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.

The p53 tumor suppressor protein is a sequence-specific DNA-binding protein that is activated by DNA damage and stimulates transcription of genes that delay the cell cycle (1). Four functional domains within the protein have been identified:

(a) the NH2-terminal activation domain (residues 1–47) is required for transactivation (2) and has been shown to interact with P300 and CBP, coactivator proteins with histone acetylase activity (3);

(b) the central core domain (residues 100–300) is necessary and sufficient for sequence-specific DNA binding and is the location of almost all of the p53 gene mutations detected in human tumors (4);

(c) the oligomerization domain (residues 345–363) is required for p53 monomers to form tetramers (5), which is the form in which p53 binds to adjacent halfsites in its target genes (6); and

(d) the COOH-terminal negative regulatory domain (residues 364–393) is thought to inhibit the binding of latent p53 to its target sequence unless it is counteracted by specific covalent modifications (7, 8).

The possibility that the negative regulatory domain affects aspects of p53 functionality other than DNA binding has not been investigated.

Several lines of experimentation suggest that p53 may be subject to redox regulation. Binding of p53 to its DNA target sequence in vitro is stimulated by the presence of reductant and antagonized by the presence of oxidant in the binding buffer (9, 10, 11, 12, 13). Also, redox active compounds have been shown to affect p53 activation of reporter genes in transfected cells (12, 13). Additionally, the redox active protein Ref-1, which catalyzes the reduction of a conserved cysteine on Fos and Jun and enhances Fos/Jun binding to AP-1 sites (14), has been shown to strongly increase p53 binding to target DNA in vitro and moderately increase p53 activation of reporter genes in transfected cells (11). Finally, loss of function and null mutations in the gene encoding thioredoxin reductase have been shown to inhibit p53 activation of reporter genes in both Schizosaccharomyces pombe and Saccharomyces cerevisiae(15, 16).

To better understand the mechanism responsible for thioredoxin reductase-dependent p53 activity and to localize the p53 domain creating the requirement for thioredoxin reductase, LexA/p53 fusion proteins were assayed for their ability to transactivate a Lex operator-containing reporter gene in wild-type and thioredoxin reductase-deficient budding yeast. The results showed that a LexA fusion protein containing full-length p53 continued to require thioredoxin reductase to stimulate transcription, but a fusion protein lacking the COOH-terminal negative regulatory domain of p53 did not. The results suggested that, under oxidizing conditions, the negative regulatory domain of p53 inhibits target gene transcription even when the p53 activation domain is tethered to DNA by a heterologous DNA-binding protein. Deletion of the negative regulatory domain did not relieve the requirement of non-LexA-containing p53 for thioredoxin reductase. Viewed in the context of earlier studies (9, 10, 11, 12, 13), the current results suggest that oxidation inhibits both DNA binding and transactivation by p53, and that inhibition of transactivation, but not DNA binding, is dependent on the negative regulatory domain.

Yeast Strains.

Yeast strains MY322 (mat-a ade2 leu2 his3 trp1 can1 ura3:pSH-34ΔSpeI:URA3) and MY323 (isogenic to MY322 except Δtrr1:HIS3) were derived by transforming W303-1a (17) and MY301 (16), respectively, with the LexOP-LacZ reporter gene plasmid pSH18-24ΔSpeI that had been linearized by cleavage at the unique ApaI site in the URA3 gene. Strains MY320 (mat-a ade2 leu2 his3 trp1 can1 leu2:p53RE-LacZ:LEU2) and MY321 (isogenic to MY320 except Δtrr1:HIS3) were derived by transforming W303-1a and MY301, respectively, with the integrative p53RE-LacZ reporter gene plasmid that had been linearized by cleavage at the unique BstEII site in the LEU2 gene.

Plasmids.

The effector plasmid encoding LexAp53 was constructed by inserting a PCR-generated fragment extending from a primer-derived XhoI site 12 bp upstream of the human p53 start codon to a primer-derived BamHI site immediately downstream from the p53 stop codon into a derivative of the 2 μ TRP1 vector pBTM116 (18). The effector plasmid encoding the LexAp53Δ364 fusion protein was derived by inserting a PCR-generated fragment extending from the p53 StuI site to a primer-derived stop codon and BamHI site immediately downstream from p53 codon 363 into a derivative of the LexAp53 plasmid from which the internal StuI/BamHI fragment was removed. Effector plasmids encoding the LexAp53Δ138 and LexAp53Δ347 fusion proteins were derived by cleaving the LexAp53 plasmid with BamHI endonuclease, filling with Klenow polymerase, cleaving with BalI or StuI endonuclease, respectively, and recircularizing with DNA ligase. The effector plasmids p414GPD-p53 and p414GPD-p53Δ364 were constructed by digesting the plasmids encoding LexAp53 and LexAp53Δ364 with SmaI and BamHI, and cloning the released p53 insert into a p414GPD vector that was prepared by cutting with SpeI, filling with Klenow polymerase, and cutting with BamHI.

In an earlier study of p53 activity in yeast (16), we used an episomal reporter gene. In the present study, we used strains containing integrated reporter genes. Integrated reporter genes were considered more representative of true chromatin structure and were expected to give greater reproducibility, because of absolute uniformity in gene copy number between transformants. Also, because the reporter genes were already resident in the host cell, effector plasmids could be introduced via a single transformation.

The integrative LexOP-LacZ reporter gene plasmid pSH18–34ΔSpeI was derived by cutting pSH18–34 (obtained from Roger Brent, Massachusetts General Hospital, Boston, MA) with SpeI, filling with Klenow polymerase, and recircularizing the 2 μ-deleted plasmid fragment with DNA ligase. The integrative p53RE-LacZ reporter gene plasmid was derived by fusing a ScaI/NotI-cleaved, LacZ-containing fragment from pRS315-PG-β-gal(19) with a ScaI/NotI-cleaved, LEU2-containing fragment from the integrative vector pRS305 (20).

β-Galactosidase and Immunoblot Assays.

β-galactosidase assays were performed on exponentially growing cells (107cell/ml) as described by Pearson and Merrill (16), and activity was expressed as nmol ONP/min/107 cells3. LexAp53 fusion protein levels in yeast transformants were determined by immunoblotting as described by Pearson and Merrill (16) using mouse monoclonal antibody DO-1, which recognizes an epitope near the p53 NH2 terminus (Santa Cruz Biotechnology).

Reporter gene activation by human p53 is compromised in yeast lacking the TRR1 gene encoding thioredoxin reductase. Although several functional domains on the p53 polypeptide have been defined, localization of the domain creating thioredoxin reductase dependence is complicated by the fact that reporter gene activation in vivo requires that transactivation, DNA binding, and oligomerization domains all be present. For this reason the approach of adding a heterologous DNA binding domain to the p53 polypeptide was investigated.

Table 1 shows that a fusion protein consisting of the bacterial repressor protein LexA joined to the NH2-terminal residue of full-length p53 (LexAp53) efficiently stimulated expression of an integrated LacZ reporter gene containing an upstream Lex operator (LexOP-LacZ) but was incapable of stimulating an integrated LacZ reporter gene containing an upstream p53 response element (p53RE-LacZ). Apparently, the presence of LexA residues at the NH2 terminus of the LexAp53 fusion protein prevented the p53 DNA binding domain of the protein from binding the p53 response element. The alternative explanation—that the presence of LexA residues at the NH2 terminus of the fusion protein prevented the p53 activation domain from recruiting coactivator—seems untenable in that the p53 activation domain of the LexAp53 fusion protein efficiently recruited the coactivator to the LexOP-LacZ reporter gene promoter. The pBTM116 vector plasmid, encoding only LexA, did not stimulate either reporter gene. Table 1 also shows that native p53 efficiently stimulated the integrated p53RE-LacZ reporter gene but was incapable of stimulating the integrated LexOP-LacZ reporter gene. The pRS314 vector plasmid did not stimulate either reporter gene. The results indicate that the LexAp53 protein specifically binds and activates the Lex operator-equipped reporter gene.

Having established that the LexAp53 fusion protein specifically stimulated expression of an integrated LexOP-LacZ reporter gene in wild-type yeast, the ability of the fusion protein to stimulate reporter gene expression in Δtrr1 yeast was investigated. As shown in Table 2, and similar to previously published results using an episomal reporter gene (16), stimulation of the p53RE-LacZ reporter gene by unfused p53 was reduced 7-fold by the Δtrr1 mutation. Significantly, stimulation of the LexOP-LacZ reporter gene by the LexAp53 fusion protein was also reduced by the Δtrr1 mutation. Thus, although p53 was now tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene activation remained thioredoxin reductase-dependent. As previously demonstrated for native p53 (16), immunoblots confirmed that the levels of LexAp53 protein in wild-type and Δtrr1 yeast were equal and of the expected size (data not shown). Therefore, the Δtrr1 mutation is affecting the activity, and not the level, of the LexAp53 fusion protein.

The finding that reporter-gene activation remained thioredoxin reductase-dependent even when p53 was tethered to DNA by a heterologous DNA-binding protein suggested that the inhibition of DNA binding cannot be the sole basis for the inhibition of reporter gene expression. The Δtrr1 mutation may also inhibit the ability of DNA-bound p53 to transactivate transcription. Several mechanisms can be envisioned. The Δtrr1 mutation may inhibit the activity of a downstream effector, such as a coactivator, that is required for p53 to stimulate transcription. Alternatively, the Δtrr1 mutation may inactivate an upstream affector, such as an activating kinase, that is required to activate latent p53. Finally, rather than acting through a downstream effector or upstream affector, the absence of thioredoxin reductase may inhibit the activity of p53 directly. The p53 polypeptide contains several cysteines, the oxidation of which may prevent p53 from recruiting downstream effectors to the promoter.

To begin to distinguish between the above possibilities, and to localize the region on p53 that creates dependence on thioredoxin reductase, COOH-terminally deleted LexA/p53 fusion protein genes were constructed and assayed for reporter gene activation in wild-type and Δtrr1 yeast (Fig. 1). LexAp53Δ138 (which contains the p53 activation domain but lacks the DNA-binding, oligomerization, and negative regulatory domains) was equally effective in stimulating reporter gene expression in wild-type and Δtrr1 cells. The result suggests that the Δtrr1 mutation is not inhibiting a downstream effector of p53 because such an effector should continue to show inhibition even if COOH-terminal portions of p53 were missing. LexAp53Δ347 (which contains the activation and DNA-binding domains of p53 but lacks the oligomerization and negative regulatory domains) was also equally effective in stimulating reporter gene expression in wild-type and Δtrr1 yeast. The result suggests that the restoration of the cysteine-containing core domain of p53 does not in itself restore thioredoxin reductase dependence. LexAp53Δ364, which contains all of the characterized p53 domains except the negative regulatory domain, was also equally effective in stimulating reporter gene expression in wild-type and Δtrr1 yeast. The result indicates that the restoration of the p53 oligomerization domain does not restore thioredoxin reductase dependence. The observation that transactivation by the full-length LexAp53 fusion protein but not the LexAp53Δ364 fusion protein was inhibited by the Δtrr1 mutation indicates that it is the negative regulatory domain of p53 that creates the dependence on thioredoxin reductase.

Unlike other regions of the p53 protein, the negative regulatory domain is not essential for p53 activity. Thus, the thioredoxin reductase-dependence of a non-LexA-tethered p53 deletion mutant lacking the negative regulatory domain could be investigated. Table 3 shows the level of expression of an integrated LacZ reporter gene (p53RE-LacZ) in wild-type and Δtrr1 cells transformed with effector plasmids encoding either full-length p53 (p53) or p53 truncated at codon 364 (p53Δ364). In contrast to the thioredoxin reductase-independent activity of LexAp53Δ364 (see Fig. 1), the nontethered p53Δ364 protein was as dependent on thioredoxin reductase as full-length p53. The results suggest that, in the absence of a heterologous DNA binding domain, when p53 must rely on its own DNA-binding domain to bind its response element, deletion of the COOH-terminus is not sufficient to overcome the requirement for thioredoxin reductase. This conclusion is consistent with the results of electrophoretic mobility shift experiments that show that the deletion of the p53 negative regulatory domain does not relieve the requirement for DTT in DNA binding assays (11).

Previous studies (12, 13, 21, 22) have indicated that p53 functionality is impaired by conditions expected to promote thiol oxidation. Exposure to pharmacological oxidants has been shown to inhibit p53 activation of reporter genes in transfected cells. Omission of the reductant DTT or inclusion of an oxidant such as diamide results in poor or aberrant binding of purified p53 to its DNA target in bandshift assays (11, 12, 13, 21, 23). The bifunctional protein Ref-1/APE1, which has been shown to affect the redox state of a specific cysteine residue in Fos and Jun and to boost Fos/Jun binding to AP-1 sites (14), has also been shown to enhance the binding of p53 to its target DNA (11). The effects of redox active compounds and proteins on p53 binding to DNA in vitro have led least parsimoniously to the idea that oxidizing conditions inhibit p53 functionality by inhibiting p53 binding to its target. However, our results, using a heterologous DNA binding domain to tether p53 to a reporter gene, indicate that the inhibition of DNA binding may not be the sole basis for impaired p53 functionality under oxidizing conditions. The observation that the LexAp53 fusion protein still requires thioredoxin reductase to activate reporter gene transcription in yeast suggests that oxidizing conditions either inhibit the LexAp53 fusion protein from binding to LexA operator sites or inhibit DNA-bound LexAp53 protein from activating transcription.

Interestingly, the requirement of the LexAp53 fusion protein for thioredoxin reductase is alleviated if the last 30 residues of p53 are deleted. These COOH-terminal residues, termed the negative regulatory domain, reduce sequence-specific DNA binding in vitro. A number of reagents that counteract the negative regulatory domain have been identified, including single-stranded DNA that nonspecifically binds the domain (24), an antibody that recognizes the domain (7), COOH-terminal peptides that mimic the domain (25), and cellular proteins that interact with the domain (26). In addition, in vitro phosphorylation or acetylation of residues within the domain, at sites that undergo phosphorylation or acetylation in vivo, has been shown to enhance sequence-specific binding of full-length p53 to DNA (7, 8, 27) and to increase the activity of p53 in in vitro transcription assays (28). In contrast, none of these reagents or covalent modifications are required for efficient binding of a COOH-terminally truncated p53 protein to its target DNA. These results have led to the idea that the COOH-terminal domain prevents latent p53 from activating target gene transcription, and that the domain must be counteracted by processes such as phosphorylation, acetylation, or interaction with other proteins in order for p53 to bind and activate its target genes. Our results suggest that the COOH-terminal domain also creates a requirement for thioredoxin reductase. Involvement of the COOH-terminus in redox control of p53 is similarly suggested by the results of Jayaraman et al.(11), who showed that full-length p53 but not COOH-terminally truncated p53, requires Ref-1 to bind DNA sequence-specifically in vitro and to efficiently activate reporter gene transcription in transfected cells. However, importantly, deletion of the COOH-terminus did not relieve the requirement for DTT in p53 DNA binding assays (11).

Thioredoxin reductase is a protein disulfide reductase. The observation that p53 requires thioredoxin reductase to transactivate reporter genes in yeast (15, 16) suggests that either the p53 protein itself or a protein required to activate p53 is prone to oxidative inactivation. To adopt an active conformation in yeast, p53 may require covalent modifications or specific protein-protein interactions. For example, high copy expression of a protein termed Pak1, which contains protein kinase motifs, has been shown to rescue p53 activity in yeast mutants that are defective in p53-dependent reporter gene activation (19). Oxidation of Pak1, an alternative activating kinase or acetylase or a protein with which p53 interacts, may indirectly inhibit p53 activity. Alternatively, given the effect of thiol active compounds on p53 binding to DNA in vitro, it is reasonable to suspect that p53 itself contains cysteines that are prone to oxidation. Wu and Momand (13) used thiol reactivity after N-ethylmaleimide and DTT treatment to show that a detectable fraction of the p53 in a rat embryo cell line contains oxidized cysteines, and that the fraction increases when cells are treated with pyrrolidine dithiocarbamate—a compound that inhibited the accumulation of Mdm2 mRNA, which normally occurs in response to p53 activating conditions. Although the COOH-terminal negative regulatory domain of p53 does not contain cysteine residues, it is possible that cysteine oxidation elsewhere in the molecule reinforces the effect of the negative regulatory domain on p53 functionality.

Fig. 2 shows a working model for how oxidizing conditions may affect p53 functionality. The model assumes that the LexA domain of LexA/p53 fusion proteins constitutively binds to Lex operator sites, regardless of the oxidation state of the cell. The assumption seems reasonable in that LexA does not contain cysteine residues and can efficiently tether the activation domain of p53 to DNA in wild-type and Δtrr1 yeast (see data for LexAp53Δ364, LexAp53Δ347, and LexAp53Δ138 in Fig. 1). Possibly, under oxidizing conditions, the negative regulatory domain may sequester the full-length LexAp53 fusion protein in a compartment or state in which it is unable to bind DNA sequence specifically. However, it is difficult to reconcile such a sequestration mechanism with the observation that deletion of the negative regulatory domain did not relieve the thioredoxin reductase requirement of non-LexA-containing p53. In the model shown in Fig. 2, p53 is depicted as having oxidation-prone cysteines, the oxidation of which inhibits the function of both the DNA binding and transactivation domains of p53. Thus, in vivo, even when p53 is tethered to DNA by a heterologous DNA-binding domain, reporter gene transactivation is inhibited by oxidizing conditions (Fig. 2,A). The inhibition of transactivation due to thiol oxidation is depicted as requiring the negative regulatory domain because a COOH-terminally truncated LexAp53 fusion does not require thioredoxin reductase to stimulate transcription in vivo (Fig. 2,B). The inhibition of DNA binding due to thiol oxidation may or may not require the negative regulatory domain. Although deletion of the negative regulatory domain enhances sequence-specific DNA binding in vitro, the COOH-terminally deleted protein still requires a reductant such as DTT to bind its target sequence (11). For the sake of simplicity, the model in Fig. 2 depicts oxidized p53 as forming an intramolecular disulfide. It is equally possible that p53 forms intermolecular disulfides between p53 monomers or between p53 and other cellular proteins. It is also possible that the inhibition of p53 transactivation under oxidizing conditions is an indirect consequence of oxidative inactivation of another protein required to counteract the negative regulatory domain. Regardless of the molecular details of the mechanism responsible for the thioredoxin reductase dependence of p53, the finding that the LexAp53 fusion protein is also dependent suggests that redox control of transactivation domain activity represents an additional level at which p53 function can be regulated.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

      
1

This work was supported by National Science Foundation Grant MCB-9728782, Medical Research Foundation of Oregon Grant MRF598, and Oregon Division of the American Cancer Society Grant ACS988 (to G. F. M.), and by a Pilot Project of National Institute of Environmental Health Sciences Grant ES00210 and Oregon State University Research Council Grant (to G. D. P.).

            
3

The abbreviation used is: ONP, o-nitrophenol.

We thank Chris Stoner, Jeff Hutchinson, John Wilson, and Jana Reitmajer for technical assistance; Mark Leid for advice concerning LexA fusion proteins; and Sam Thiagalingam (the Johns Hopkins University, Baltimore, MD) for supplying the plasmids used in constructing p53 reporter gene and effector gene plasmids.

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