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The Human p53 Negative Regulatory Domain Mediates Inhibition of Reporter Gene Transactivation in Yeast Lacking Thioredoxin Reductase¹

Department of Biochemistry and Biophysics and the Center for Gene Research and Biotechnology [G. F. M., G. D. P.], and Molecular and Cellular Biology Program [P. D.], Oregon State University, Corvallis, Oregon 97331

	ABSTRACT

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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 trr1 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 trr1 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.

	INTRODUCTION

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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.

	MATERIALS AND METHODS

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Yeast Strains.
Yeast strains MY322 (mat-a ade2 leu2 his3 trp1 can1 ura3:pSH-34SpeI:URA3) and MY323 (isogenic to MY322 except trr1:HIS3) were derived by transforming W303-1a (17) and MY301 (16) , respectively, with the LexOP-LacZ reporter gene plasmid pSH18-24SpeI 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 trr1: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 LexAp53364 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 LexAp53138 and LexAp53347 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-p53364 were constructed by digesting the plasmids encoding LexAp53 and LexAp53364 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 pSH18–34SpeI 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-ß-gal (19) with a ScaI/NotI-cleaved, LEU2-containing fragment from the integrative vector pRS305 (20) .

ß-Galactosidase and Immunoblot Assays.
ß-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).

	RESULTS

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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.

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 trr1 yeast (Fig. 1) . LexAp53138 (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 trr1 cells. The result suggests that the trr1 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. LexAp53347 (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 trr1 yeast. The result suggests that the restoration of the cysteine-containing core domain of p53 does not in itself restore thioredoxin reductase dependence. LexAp53364, 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 trr1 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 LexAp53364 fusion protein was inhibited by the trr1 mutation indicates that it is the negative regulatory domain of p53 that creates the dependence on thioredoxin reductase.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Structure and thioredoxin reductase-dependence of LexA/p53 fusion proteins. The full-length LexAp53 protein consists of the LexA protein fused to the NH2 terminus of the entire 393-residue p53 protein. The positions of four p53 structural domains are shown: residues 1–47 contain the proline-rich transactivation domain (Act); residues 100–300 contain all of the cysteines in the protein and comprise the DNA binding domain; residues 320–360 contain nuclear localization signals and the oligomerization domain (Oligo); residues 363–393 comprise the negative regulatory domain (Neg). The COOH-terminally truncated fusion proteins were generated by replacing the indicated residue with a stop codon. For example, in LexAp53364, residue 364 was replaced with a stop codon. Plasmids encoding the fusion proteins were transformed into TRR1 wild-type (MY322) or trr1 null (MY323) strains containing the integrated LexOP-LacZ reporter. At least six independent transformants were assayed in duplicate to arrive at the indicated ß-galactosidase activity values (mean ± SD).

p53364

View this table: [in this window] [in a new window]   Table 3 Reporter gene transactivation by p53 and p53364 depends on thioredoxin reductase

	DISCUSSION

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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 trr1 yeast (see data for LexAp53364, LexAp53347, and LexAp53138 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. 2A) . 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. 2B) . 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.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Model for the inhibition of p53 transactivating activity by thiol oxidation. The use of LexA/p53 fusion proteins allows the measurement of effects of oxidation on p53 functions other than DNA binding. A, thiol oxidation results in the masking of the acidic NH2-terminal activation domain by the basic COOH-terminal negative regulatory domain. B, the removal of the negative regulatory domain rescues p53 transactivation domain activity even in the presence of thiol oxidation. Although the diagram shows the formation of intramolecular disulfides, the formation of disulfide bonds with other p53 monomers, other proteins, or other thiol compounds is equally plausible.

	ACKNOWLEDGMENTS

 
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.

	FOOTNOTES

 
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.).

² To whom requests for reprints should be addressed, at Department of Biochemistry and Biophysics, ALS Building, Room 2011, Oregon State University, Corvallis, Oregon 97331. Phone: (541) 737-3119; Fax: (541) 737-0481;

³ The abbreviation used is: ONP, o-nitrophenol.

Received 1/25/99. Accepted 4/30/99.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Merrill, G. F. Articles by Pearson, G. D. Search for Related Content PubMed PubMed Citation Articles by Merrill, G. F. Articles by Pearson, G. D.