the p53qs transactivation-deficient mutant shows stress-specific apoptotic activity and induces...

The p53QS transactivation-deficient mutant showsstress-specific apoptotic activity and inducesembryonic lethalityThomas M Johnson1, Ester M Hammond1, Amato Giaccia1 & Laura D Attardi1,2

The role of transcriptional activation in p53 function is highly controversial. To define this role in vivo, we generated a Trp53knock-in construct encoding a protein carrying mutations of two residues that are crucial for transactivation (L25Q,W26S). Herewe show that these mutations have selective effects on the biological functions of p53. Although its ability to activate various p53target genes is largely compromised, the p53QS protein retains the ability to transactivate the gene Bax. The ability of the p53QS

mutant protein to elicit a DNA damage–induced G1 cell cycle–arrest response is also partially impaired. p53QS has selectivedefects in its ability to induce apoptosis: it is completely unable to activate apoptosis in response to DNA damage, is partiallyunable to do so when subjected to serum deprivation and retains substantial apoptotic activity upon exposure to hypoxia. Thesefindings suggest that p53 acts through distinct, stimulus-specific pathways to induce apoptosis. The importance of the biologicalactivity of p53QS in vivo is underscored by our finding that expression of p53QS, which cannot bind mdm2, induces embryoniclethality. Taken together, these results suggest that p53 has different mechanisms of action depending on specific contextual cues,which may help to clarify the function of p53 in preventing cancer.

The p53 tumor suppressor is a key component of the human defenseagainst neoplasia, a fact that is underscored by the high incidence ofmutations in TP53 in human cancers, as well as the finding that micelacking Trp53 are predisposed to develop tumors early in life1,2. p53acts as a cellular stress sensor, preventing the expansion of hyperpro-liferative or damaged cells by inducing G1 cell-cycle arrest, cellularsenescence or apoptosis. The best-characterized molecular function ofp53 is transcriptional activation, which is important in the G1 arrestcheckpoint response to DNA damage3–5. In contrast, the mechanismby which p53 induces apoptosis is controversial. p53 upregulatesvarious target genes that have proapoptotic activities, including Bax,Pmaip1 (also called Noxa), Perp and Bbc3 (also called Puma)6, andtranscriptional activation by p53 is important to its ability to induceapoptosis7,8. Consistent with this role for transactivation in apoptosis,knockout mice lacking any of these p53 target genes have compro-mised p53-dependent cell-death responses9–12. Other studies havesuggested that p53-dependent apoptosis can also proceed throughtransactivation-independent mechanisms, but such mechanisms havenot yet been well-defined13–16. One proposed mechanism is transcrip-tional repression, but the requirements of genes known to be repressedby p53 for apoptosis in vivo are not well understood. In addition,p53 can function at the mitochondria to initiate mitochondrialmembrane dysfunction and cytochrome c release, leading directly toapoptosis17–19. The diversity of molecular mechanisms for p53 action

proposed from in vitro studies highlights the need to dissect p53function in vivo, where it can be studied in a physiological context andin a wide range of tissues.

To define broadly the relevance of transcriptional activation in p53function, we constructed a Trp53 knock-in construct with a mutatedallele of Trp53 that has compromised transactivation. This constructcontains two amino acid substitutions in the transactivation domain,L25Q and W26S. The analogous mutations in human p53 confer themost severe transactivation deficiency of any mutations analyzedin vitro, retaining 5–10% of wild-type p53 activity, but still maintainthe structural integrity of the DNA-binding domain20. By analyzingthese knock-in mice and cells derived from them, we characterized thefunction of p53QS in transactivation, cell-cycle arrest and apoptosis.We determined that these mutations have selective effects on differentactivities of p53, suggesting that, in this setting of physiological p53expression, both transactivation-dependent and transactivation-independent modes of p53 action may contribute to its function asa tumor suppressor.

RESULTSGeneration of Trp53LSL-QS knock-in miceTo dissect the role of transcriptional activation in p53 function in vivo,we generated a Trp53 knock-in mouse strain carrying mutationsresulting in the amino acid substitutions L25Q and W26S. In addition

Published online 16 January 2005; doi:10.1038/ng1498

1Division of Radiation and Cancer Biology, Department of Radiation Oncology and 2Department of Genetics, Stanford University School of Medicine, Stanford,California 94305 USA. Correspondence should be addressed to L.D.A. ([email protected]).

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to severely compromising p53 transactivation, mutation of theseresidues inhibits binding of mdm2, an essential negative regulator ofp53 activity in vivo, because of an indistinguishable overlap in therecognition sites for mdm2 and components of the transcriptionalmachinery21. We anticipated the possibility that this mutant mayresult in embryonic lethality because of its inability to bind mdm2 andengineered the mutated locus such that expression of p53QS isconditional20,22. We constructed a targeting vector carrying both theamino acid substitutions L25Q and W26S and a transcriptional stopelement flanked by loxP recombination sites (loxP-Stop-loxP, LSL)upstream of the Trp53 coding region (Trp53LSL-QS) to silence expres-sion of the allele until introduction of Cre recombinase (Fig. 1a). We

introduced this vector into embryonic stem (ES) cells, identifiedcorrectly targeted ES clones and used them to generate mice(Fig. 1b). As a control for our experiments, we used mice carryingan LSL element upstream of the wild-type Trp53 gene (Trp53LSL-LW;A. Ventura, D. Tuveson and T. Jacks, unpublished data).

We examined the biological activity of p53QS in mouse embryonicfibroblasts (MEFs), a cell type in which p53 activity is well-character-ized. To confirm that the wild-type and mutated Trp53 allelesare expressed specifically upon Cre introduction, we generatedMEFs homozygous with respect to either wild-type Trp53LSL-LW ormutated Trp53LSL-QS and examined p53 RNA and protein expression.We used Cre-expressing adenoviruses (adeno-Cre) to introduce Cre

Figure 2 p53QS binds p53 response elements.

EMSAs were done on protein extracts generated

from either Trp53LSL-QS/LSL-QS or Trp53LSL-LW/LSL-LW

MEFs infected with empty adenovirus or

adeno-Cre and then either left untreated or

treated with doxorubicin (Dox). Extracts were

incubated with both pAb421 and radioactively

labeled oligonucleotides corresponding tothe p53 responsive elements in either the

Cdkn1a promoter (a) or the Perp first intron (b).

Free probe and the shifted p53-antibody-DNA

complex are indicated. Binding is specific, as

seen by the reduction of shifted complex upon

incubation with increasing concentrations of unlabeled binding site oligonucleotide (Spc. comp; at 20� and 100� excess) but not with increasing

concentrations of oligonucleotides containing point mutations at key consensus site nucleotides (Mut. comp; at 20� and 100� excess).

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Figure 1 Generation and characterization of Trp53LSL-QS mice. (a) A targeting vector containing intron 1–exon 6 of Trp53 with mutated sequence resulting

in the L25Q and W26S mutations (indicated by an asterisk) and an LSL transcriptional stop element was introduced into ES cells. Diagram not to scale.

(b) Correctly targeted ES cells were identified by Southern blotting using EcoRI-digested DNA and an EcoRI–BamHI fragment as probe (indicated in a).

PCR amplification of genomic DNA from Trp53LSL-QS/+ cells using a mutation-specific primer set (primers a and b; top panel) and an LSL element–specific

set (primers c and e; bottom panel) confirmed the presence of the L25Q and W26S mutations and LSL element, respectively. WT, wild-type Trp53 locus;

Mut, Trp53LSL-QS locus. (c) Northern-blot analysis of MEFs infected with empty adenovirus or adeno-Cre, with and without doxorubicin (dox) treatment.

Two separate lines of Trp53LSL-QS/LSL-QS MEFs were used. Gapd serves as a loading control. (d) Western-blot analysis of MEFs infected with empty

adenovirus or adeno-Cre, with and without doxorubicin (dox) treatment. Gapd serves as a loading control. (e) p53 immunofluorescence staining

was done on Trp53LSL-QS/LSL-QS MEFs infected with adeno-Cre.

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recombinase efficiently. In all experiments, we also infected cells withan empty adenovirus as an effective Trp53 null control and to confirmthat the phenotypes observed after infection with adeno-Cre were dueto Cre expression and not to nonspecific effects of adenoviral infec-tion. We did not detect Trp53 mRNA in Trp53LSL-LW/LSL-LW orTrp53LSL-QS/LSL-QS MEFs by northern blotting until they were infectedwith adeno-Cre, indicating that the stop element effectively silencesgene transcription (Fig. 1c). Examination of p53 protein by westernblotting verified that it was not expressed in Trp53LSL-LW/LSL-LW

or Trp53LSL-QS/LSL-QS MEFs without Cre introduction (Fig. 1d).Trp53LSL-LW/LSL-LW MEFs infected with adeno-Cre produced undetect-able levels of wild-type p53 in the absence of a DNA damage signal,but levels of wild-type p53 stabilized after treatment with doxorubicin.In contrast, p53QS was stable even in the absence of DNA damage,confirming previous observations that this p53 mutant is unableto interact with mdm2 (ref. 20). We also examined the efficiency ofstop-element deletion on a single-cell basis. On average, 495% ofTrp53LSL-LW/LSL-LW or Trp53LSL-QS/LSL-QS MEFs infected with adeno-Cre expressed p53 protein when examined by immunofluorescence

(Fig. 1e and data not shown), indicating that infection of MEFs withadeno-Cre yields an essentially homogenous population of cells inwhich the biological activity of p53 can be evaluated reliably. Inaddition, p53QS had a nuclear localization pattern (Fig. 1e), consistentwith reports that an inability to bind mdm2 renders p53 resistant tonuclear export23.

p53QS is selectively compromised in transactivationTo characterize the ability of p53QS to regulate gene expression, weexamined both its DNA-binding capacity and its transactivationpotential. We assessed the ability of p53QS to bind DNA in asequence-specific manner by electromobility shift assays (EMSAs)using oligonucleotides corresponding to the p53-responsive elementsderived from either the promoter of Cdkn1a (also called p21) or thefirst intron of Perp (Fig. 2). We chose these two p53 target genesbecause they represent transcriptional targets involved in cell-cyclearrest and apoptosis, respectively, and p53 has been proposedto interact with response elements in arrest and apoptosis genesdifferentially24,25. We observed robust binding of wild-type p53 to

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Figure 3 p53QS is impaired for transactivation. (a) Northern-blot analysis

was done on RNA extracted from MEFs infected with empty adenovirus or

adeno-Cre and then left untreated or treated with doxorubicin (dox). The p53

target genes Cdkn1a, Mdm2, Ccng1, Perp, Pmaip1 and Bax were examined.

Gapd serves as a loading control. (b) The same northern blot as in a with

an overexposure of the Cdkn1a target gene shows that p53QS retains some

transactivation capacity.

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Figure 4 p53QS has compromised cell-cycle checkpoint function. (a) Wild-

type, Trp53LSL-LW/LSL-LW or Trp53LSL-QS/LSL-QS MEFs were infected with empty

adenovirus or adeno-Cre, with and without doxorubicin (dox) treatment and

were pulsed with BrdU. BrdU immunofluorescence analysis was done to

determine the percentages of cells in S phase. Cells were costained with

DAPI to assess total cell number. (b) Graphical representation showing

percentages of BrdU-positive cells from immunofluorescence analysis.

Comparison of Trp53+/+ MEFs infected with empty adenovirus and adeno-

Cre shows that expression of Cre has no detectable genotoxic effects leading

to cell cycle arrest. Trp53LSL-QS/LSL-QS MEFs infected with empty adenovirus

and treated with doxorubicin were also examined to show that doxorubicin

has minimal p53-independent effects on the cell cycle. Data are the average

7 s.e.m. of three independent experiments.


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these sites only in the presence of a DNA damage signal. In contrast,p53QS was able to bind both these sites effectively, even in the absenceof DNA damage, consistent with its greater stability. The amount ofshifted complex observed with both p53QS and wild-type p53 mir-rored the protein levels observed by western-blot analysis (Fig. 1d).These results indicate that p53QS retains DNA-binding activity ondifferent p53 response elements.

To determine the transactivation capacity of p53QS, we examined itsability to stimulate expression of a variety of p53 target genes (Fig. 3a).We infected Trp53LSL-QS/LSL-QS, Trp53LSL-LW/LSL-LW and wild-type(Trp53+/+) MEFs with empty adenovirus or adeno-Cre and then eithertreated them with doxorubicin or left them untreated. In Trp53+/+

MEFs, the mRNA levels of p53 target genes were similar in cellsinfected with empty adenovirus and with adeno-Cre but were substan-tially induced after exposure to DNA damage. These data indicate that,in this system, Cre recombinase does not produce genotoxic signalsthat inappropriately activate p53. Similarly, in Trp53LSL-LW/LSL-LW

MEFs, we did not observe high levels of target gene activation unlessdoxorubicin treatment was given along with adeno-Cre infection. Incontrast, the induction of most target genes was markedly impaired inTrp53LSL-QS/LSL-QS MEFs, even in the presence of DNA damage.Though compromised, the transactivation ability of p53QS is notcompletely defective, as visualized in longer exposures of the blotafter hybridization with the Cdkn1a probe (Fig. 3b) or other probes(data not shown). Quantitative analysis of mRNA levels showed thatp53QS was able to activate transcription of various target genes to levelsB5–30% of those in MEFs expressing wild-type p53 after adeno-Creinfection and doxorubicin treatment (data not shown). In contrast, theapoptotic target gene Bax was activated in MEFs expressing p53QS tolevels equivalent to those observed in either Trp53LSL-LW/LSL-LW orTrp53+/+ MEFs infected with adeno-Cre. Taken together, these resultsindicate that the ability of p53QS to activate many p53 target genes isgreatly, but not completely, compromised, and that activation of sometargets, such as Bax, is not considerably affected by these mutations.These findings suggest that the ability of p53 to activate transcriptionrelies on distinct, target gene–specific mechanisms.

p53QS is impaired in G1 checkpoint activationp53 can arrest cells in G1 in response to DNA damage to prevent thepropagation of oncogenic mutations26,27. To determine the biologicalconsequences of impaired p53 transactivation potential, we examined

the activity of p53QS in regulating this G1 checkpoint. We measuredthe G1 arrest response by determining the percentages of cells inS phase, through 5-bromodeoxyuridine (BrdU) labeling, after treat-ment with doxorubicin. As expected, the percentage of BrdU-positivewild-type cells decreased greatly in the presence of doxorubicin,indicative of a G1 arrest response (Fig. 4b). Trp53LSL-LW/LSL-LW andTrp53LSL-QS/LSL-QS MEFs infected with empty adenovirus, which arefunctionally Trp53 null, both showed very high levels of BrdUincorporation (Fig. 4). In Trp53LSL-LW/LSL-LW MEFs infected withadeno-Cre, BrdU incorporation was reduced to levels comparableto those seen in wild-type MEFs with no damage (Fig. 4b). Aftertreatment with doxorubicin, MEFs expressing wild-type p53 withthe LSL element deleted had markedly fewer cells incorporatingBrdU, like doxorubicin-treated wild-type cells. In contrast, infectionof Trp53LSL-QS/LSL-QS MEFs with adeno-Cre produced only a slightreduction in BrdU incorporation relative to Trp53LSL-QS/LSL-QS MEFsinfected with empty adenovirus (Fig. 4). Furthermore, this decreasewas only moderately enhanced by treatment with doxorubicin, indi-cating that this mutant is only partially competent for G1 arrest.Fluorescence-activated cell-sorting analysis of gamma-irradiated MEFsexpressing p53QS verified that the observed partial arrest was in the G1phase of the cell cycle (data not shown). These results suggest that theability of p53QS to arrest cells in response to DNA damage iscompromised, but this impairment is not as severe as that observedin Trp53-deficient cells, correlating well with its hypomorphic trans-activation potential. In particular, the ability of p53QS to mildlyupregulate expression of Cdkn1a, a crucial component of the G1arrest response (Fig. 3), suggests an explanation for the observedpartial arrest activity4,5.

Apoptosis induction by p53QS is stress-dependentThe induction of apoptosis is another central mechanism by which p53limits tumorigenesis. For example, hyperproliferative MEFs expressingoncoproteins such as adenovirus E1A are sensitized to undergo p53-dependent apoptosis in response to various stresses, including DNAdamage, hypoxia, growth-factor deprivation and matrix detach-ment28,29. To assess the ability of p53QS to induce cell death in responseto diverse stimuli, we subjected MEFs expressing E1A to treatmentwith the DNA-damaging agent doxorubicin, to serum deprivation orto hypoxia and assessed apoptosis by annexin V–propidium iodidestaining followed by fluorescence-activated cell-sorting analysis.

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Figure 5 p53QS is defective in inducing apoptosis in response to DNA damage. (a) E1A-expressing Trp53LSL-QS/LSL-QS MEFs infected with adeno-Cre were

immunostained to verify p53 expression. (b) The indicated E1A-expressing MEFs were treated with doxorubicin and collected at the indicated times to

assess apoptosis. The graph shows the average percentages 7 s.e.m. of viable (annexin V–negative and propidium iodide–negative) cells in threeindependent experiments. Cells lacking p53 expression are represented by green lines, cells expressing wild-type p53 by blue lines and cells expressing

p53QS by a red line. (c) Non-E1A-expressing MEFs of the indicated genotypes were infected with adenoviruses and irradiated with 20 J m–2 UV-C, and

apoptosis was assessed 24 h later. The graph shows the average percentages 7 s.e.m. of viable cells in three independent experiments.


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In response to doxorubicin, Trp53+/+ cells expressing E1A under-went apoptosis as expected, with most cells losing viability within36 h (Fig. 5b). We observed that E1A-expressing Trp53LSL-LW/LSL-LW

MEFs infected with adeno-Cre also underwent cell death in responseto doxorubicin treatment, with kinetics similar to those observedin E1A-expressing wild-type MEFs. In contrast, E1A-expressingTrp53LSL-QS/LSL-QS MEFs infected with adeno-Cre were refractoryto apoptosis in response to doxorubicin, with levels of cell deathequivalent to those observed in cells lacking p53, including E1A-expressing Trp53LSL-QS/LSL-QS or Trp53LSL-LW/LSL-LW MEFs infected withempty adenovirus and E1A-expressing Trp53–/– MEFs. In all experi-ments, wild-type p53 or p53QS was expressed in 495% of cells,as shown by immunofluorescence staining (Fig. 5a and data notshown). To extend these results to another DNA-damaging agentthat does not induce double-strand breaks, we examined the apoptoticresponse of non-E1A-expressing MEFs to ultraviolet-C (UV-C) irra-diation, which has a p53-dependent component at early time points(Supplementary Fig. 1 online). Upon UV-C irradiation, both wild-type and Trp53LSL-LW/LSL-LW MEFs infected with adeno-Cre underwentsubstantial apoptosis at 24 h (Fig. 5c). In contrast, Trp53LSL-QS/LSL-QS

MEFs infected with adeno-Cre showed levels of apoptosis that wereindistinguishable from those observed in Trp53LSL-QS/LSL-QS MEFsinfected with empty adenovirus. These findings indicate that p53QS isdefective in inducing apoptosis in response to multiple forms of DNAdamage, despite the fact that it retains slight transactivation functionon certain genes, as well as the capacity to activate Bax efficiently.

We next examined the response of E1A-expressing MEFsexpressing p53QS to serum deprivation. E1A-expressing wild-typeand Trp53LSL-LW/LSL-LW MEFs infected with adeno-Cre showed highlevels of apoptosis 48 h after serum withdrawal (Fig. 6). In contrast,E1A-expressing Trp53LSL-LW/LSL-LW and Trp53LSL-QS/LSL-QS MEFsinfected with empty adenovirus and E1A-expressing Trp53�/� MEFshad a reduced apoptotic response. E1A-expressing Trp53LSL-QS/LSL-QS

MEFs infected with adeno-Cre showed a level of apoptotic activitythat was intermediate between those of wild-type and of p53-deficientcells. The fact that this mutant is ineffective in stimulating apoptosisupon treatment with DNA-damaging agents but retains activity uponserum deprivation indicates that there are stress-specific differences inthe mode of action of p53 in apoptosis.

To extend further our analysis of different apoptotic triggers, weevaluated the response of MEFs expressing p53QS to hypoxia, whichstimulates p53-mediated apoptosis through a different signalingcascade than does DNA damage30. E1A-expressing wild-type andTrp53LSL-LW/LSL-LW MEFs infected with adeno-Cre had high levelsof apoptosis under hypoxic conditions (Fig. 7a). As expected,E1A-expressing Trp53LSL-LW/LSL-LW and Trp53LSL-QS/LSL-QS MEFsinfected with empty adenovirus had low levels of apoptosis, similarto those of E1A-expressing Trp53�/� MEFs. Notably, E1A-expressingTrp53LSL-QS/LSL-QS MEFs infected with adeno-Cre had substantiallevels of apoptosis in response to hypoxia, with levels of cell deathapproaching those observed in E1A-expressing MEFs expressing wild-type p53. Examination of the RNA levels of the proapoptotic p53targets Bax, Perp and Pmaip1, as well as Cdkn1a, Ccng1 and Mdm2, inthese E1A-expressing MEFs showed that neither wild-type p53 norp53QS substantially activated the transcription of these genes underhypoxic conditions (Fig. 7b). These findings suggest that p53-induced

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Figure 7 p53QS has robust apoptotic, but not transactivation, activity under

hypoxic conditions. (a) The indicated E1A-expressing MEFs were exposed

to hypoxia (o0.2% oxygen) for 16 h and assayed for apoptosis. The

percentages of viable (annexin V–negative and propidium iodide–negative)

cells are shown, after normalization to apoptosis levels in E1A-expressing

Trp53�/� MEFs. The graph represents the average 7 s.e.m. in three

independent experiments. (b) p53 target genes are not activated in response

to hypoxia. Northern-blot analysis was done on RNA collected from E1A-

expressing MEFs of the indicated genotypes exposed to hypoxia for 6 h

or left untreated. Slc2a1 is a control to confirm the hypoxic response.Gapd serves as a loading control.

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The indicated E1A-expressing MEFs were subjected to growth factor

withdrawal (0.1% serum). The percentages of viable (annexin V–negative

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lacking p53 expression are represented by green lines, cells expressing

wild-type p53 by blue lines and cells expressing p53QS by a red line. The

graph represents the average percentages 7 s.e.m. of viable cells in three

independent experiments.


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cell death in response to hypoxia does not rely on the transactivationof canonical p53 proapoptotic target genes, but instead relates to someother activity of p53. The results that p53QS has no activity intriggering DNA damage–induced apoptosis, has partial apoptoticactivity upon serum deprivation and has a considerable capacity toinduce cell death in response to hypoxia strongly suggest that differentdownstream activities of p53 are important for the apoptotic responseto different stresses.

p53QS expression induces embryonic lethalityWe next assessed the apoptotic activity of p53QS in vivo. Mice lackingmdm2 undergo early embryonic lethality due to a failure to down-regulate p53 and consequent induction of apoptosis22. Because p53QS

is unable to bind mdm2 but retains selective apoptotic activity, wesought to determine whether expression of this mutant duringdevelopment would recapitulate the lethality observed in Mdm2 nullmice. To test this idea, we bred Trp53LSL-LW/+ and Trp53LSL-QS/+ miceto deleter-Cre mice, a strain that expresses Cre in germ cells, andexamined the frequency of progeny of various genotypes at 3–4weeks of age. We obtained a normal mendelian ratio of Cre+

Trp53LW/+ mice lacking the LSL element but observed no viable Cre+

Trp53QS/+ adults lacking the LSL element (Table 1; P o 0.0001). Thesedata, coupled with the fact that we observed no obvious postnatallethality in the progeny from these crosses, suggest that expression ofp53QS during development results in embryonic lethality. Preliminaryresults from timed pregnancies showed that at embryonic day (E)10.5, Cre+ Trp53QS/+ embryos lacking the LSL element were comple-tely resorbed, further supporting the idea that death occurs early inembryogenesis (data not shown). Future studies with these mice,comparing them with Mdm2�/� mice, will provide insight into themechanism by which p53 induces lethality when inappropriatelyactivated during development.

DISCUSSIONBy generating and analyzing mice expressing p53QS, a transactivation-deficient mutant, we identified the existence of distinct pathways ofp53 action in vivo. Though largely compromised for transactivation,p53QS retains p53 biological activity in selective settings, indicatingthat the pathway activated by p53 is dictated by contextual signals,such as stress stimulus and cell type. The findings that this mutant

shows activity in hypoxia-induced apoptosis and that its expressionduring development results in embryonic lethality underscore the factthat p53QS has physiological activity and suggest that some of theactivities of p53 rely on transactivation-independent functions.

Consistent with previous studies20,31, we observed that p53QS couldbind to p53 response elements but was deficient in transactivating anumber of p53 target genes. One notable exception, however, is thep53 target gene Bax, whose induction was largely uncompromised inMEFs expressing p53QS, suggesting that its activation by p53 mayproceed through a distinct mechanism from those of other genes weexamined. p53 stimulates transcription by attracting both histone-modifying enzymes to convert chromatin to an open conformationand coactivator proteins to stimulate formation of the preinitiationcomplex. Our data suggest that p53 could use different activationsurfaces to recruit different cofactors depending on the gene at whichit is acting. In the activation of Bax, our findings specifically implicatethe participation of a cofactor whose binding to p53 is not affected bythe L25Q and W26S mutations. Consistent with this idea are reportsof a second critical region of p53 involved in transactivation, in whichamino acids 53 and 54 are central32,33. Future genome-level expressionprofiling analysis will help to identify other p53-inducible genes whoseexpression is unperturbed by the L25Q and W26S mutations and,together with biochemical analyses, will ultimately help to provide anunderstanding of the different molecular mechanisms by which p53regulates its target genes.

Our results show that the p53-dependent apoptotic response toDNA damage in MEFs relies on robust transactivation activity. Multi-ple p53 target genes, including Bax, Pmaip1, Perp and Bbc3, areimportant for a complete apoptotic response to DNA damage9,10,12,34.Despite the facts that p53QS can activate minimal transcription of theapoptosis-inducing genes Pmaip1 and Perp and can stimulate sub-stantial transcription of the proapoptotic gene Bax under conditionsof DNA damage, p53QS is completely defective in inducing apoptosisin response to doxorubicin and UV-C. One possible explanation forthese findings is that effective engagement of the apoptotic cascade inresponse to DNA damage requires expression of a cohort of p53 targetgenes to reach a particular threshold level. Thus, although p53QS caninduce low-level expression of certain transcripts, this expression isinadequate to direct cells to an apoptotic fate. This contrasts with itscell cycle–arrest activity, where limited activation of arrest genes suchas Cdkn1a by p53QS suffices to promote a partial G1 arrest response.

Notably, p53QS has partial apoptotic activity in response to serumdeprivation. The pathway by which p53 stimulates this response hasnot been well-characterized, but our results suggest that the transcrip-tional activation of p53 target genes is crucial to achieve maximallevels of apoptosis. The finding that p53QS retains some apoptoticactivity in this setting could relate to a requirement for bothtranscriptional activation–dependent and –independent activities toinitiate cell death upon serum withdrawal. Alternatively, serumstarvation–induced, p53-dependent apoptosis may rely strictly onthe transactivation of target genes, and p53QS may be able to efficientlyinduce some, but not all, of the target genes involved in this response,consistent with our observation that p53QS can upregulate p53 targetgenes differentially. Further study of the global transcriptional activityof p53QS under conditions of serum deprivation, along with theexamination of apoptosis in MEFs expressing p53QS but lacking p53target genes such as Perp or Bax, will help to elucidate the mechanismby which p53 induces cell death in response to low serum levels.

In contrast to its response to DNA damage, p53QS retains sub-stantial apoptotic activity under hypoxic conditions. Our results andprevious studies indicate that p53 does not activate canonical p53

Table 1 Expression of p53QS during embryogenesis induces lethality

Trp53LSL-QS/+ � Trp53+/+ deleter-Cre Observed progeny (expected progeny)

Trp53QS/+ deleter-Cre 0 (19)

Trp53+/+ deleter-Cre 17 (19)

Trp53LSL-QS/+ 29 (19)

Trp53+/+ 30 (19)

n ¼ 76, P o 0.0001

Trp53LSL-LW/+ � Trp53+/+ deleter-Cre

Trp53LW/+ deleter-Cre 12 (8.5)

Trp53+/+ deleter-Cre 6 (8.5)

Trp53LSL-LW/+ 8 (8.5)

Trp53+/+ 8 (8.5)

n ¼ 34, P o 0.526

Breeding of Trp53LSL-QS/+ mice to mice carrying the deleter-Cre transgene showed thatp53QS induced embryonic lethality. Trp53LSL-LW/+ mice bred to mice carrying the deleter-Cre transgene produced a normal mendelian ratio of viable mice with a deleted LSLelement, as assessed by a w2 test. In contrast, upon crossing Trp53LSL-QS/+ mice withdeleter-Cre transgenic mice, no progeny were obtained with a deleted LSL element. Thegenotypes of mice carrying a deleter-Cre transgene lack the LSL designation because theLSL element has been removed from the genome.


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target genes during hypoxia-induced apoptosis, suggesting that trans-activation may not have a key role in this context35. Instead, it hasbeen proposed that transcriptional repression may be crucial for theability of p53 to respond to this stress. In agreement with this idea,microarray analysis showed that well-characterized p53 target genesare not induced by hypoxia and that p53 specifically represses manygenes in response to hypoxia (E.M.H. and A.G., unpublished data).Therefore, the observed apoptotic activity of p53QS under hypoxicconditions may reflect its function as a transcriptional repressor.Although the transcriptional repression activity of p53QS has notbeen examined extensively, it is known to be competent for repressionof the gene Bcl2 (ref. 36). Determining whether p53QS retains repres-sion activity at a global genomic level will help to determine whetherp53-mediated repression indeed provides the molecular rationale forhypoxia-induced apoptosis or whether some other function of p53,such as the transactivation of a small set of target genes unaffected bythe L25Q and W26S mutations, must be invoked.

p53 must be tightly regulated during development, because micelacking the mdm2 ubiquitin ligase responsible for signaling p53degradation die early in embryogenesis owing to inappropriateapoptosis22,37. In contrast, compound Mdm2 Trp53 mutant miceare viable, indicating that the lethality observed in the absence ofmdm2 results from ectopic p53 activity. Like Mdm2�/� mice, miceexpressing p53QS, which is not subject to mdm2 binding andsubsequent downregulation, die during embryogenesis. Our findingshelp to elucidate the mechanism by which p53, in the absence ofmdm2, causes lethality during development. As we have shown, p53QS

has only a partial capacity to arrest MEFs after exposure to DNAdamage and is ineffective at promoting an apoptotic response toDNA damage in hyperproliferative MEFs. Therefore, it seems un-likely that a DNA-damage stress is the initiating factor in inducinglethality. Instead, we hypothesize that p53QS induces apoptosis inresponse to the hypoxic environment in which the early embryoexists38–40. In a normal setting during embryogenesis, mdm2 restrainsp53 activity by provoking its degradation. When mdm2 is eitherabsent or unable to bind p53, however, the hypoxic signal maystimulate p53 to induce death. Further study of the developmentaldefect in Trp53QS mutant mice will determine whether lethality is aresult of inappropriate cell death and whether hypoxia is the triggerfor the observed apoptosis.

Our results contrast with previous reports on knock-in miceconstitutively expressing p53QS, which showed that the behavior ofthe mutated Trp53QS allele was indistinguishable from that of a Trp53null allele41. Although the exact reason for the differences between ourphenotype and the published data is uncertain, one explanation couldbe that the p53 construct previously used41 inadvertently contained asecondary point mutation in the p53 DNA-binding domain (G. Wahl,personal communication). This mutation, A135V, renders the func-tion of p53 temperature-sensitive, inactive at 39 1C and active at 32 1C(ref. 42). This additional mutation probably rendered the proteinconsiderably more hypomorphic than the one we studied.

Further analysis of the p53QS mutant mouse will enhance ourunderstanding of the mechanisms of p53 action in apoptosis andtumor suppression. Moreover, because p53QS retains selective p53functions, such as substantial activity in hypoxia-induced apoptosis,but completely lacks other p53 functions, such as the ability to induceapoptosis in response to DNA damage, these mice provide a means todissect the relative contributions of these activities to p53-mediatedtumor suppression. Such experiments will better define which func-tions of p53 are inactivated during tumorigenesis in various contexts,thereby illuminating how p53 loss contributes to cancer development.

METHODSGeneration of Trp53LSL-QS mice. We constructed a targeting vector using a

genomic fragment of Trp53 that included intron 1–exon 6. We inserted a

cassette bearing a transcriptional stop element comprising four tandem poly-A

sequences and a PGK-puromycin selectable marker, both flanked by loxP

recombination sites43, into the second XhoI site in the first intron of the

Trp53 genomic fragment. We generated the L25Q and W26S codon changes by

site-directed mutagenesis to alter the nucleotides TTATGG to CAAUCG. We

inserted the hsv-tk cassette downstream of exon 6 (ref. 2). We introduced the

targeting vector into ES cells and then selected them with puromycin and

gancyclovir. We screened for positive clones by Southern-blot analysis using

both 5¢ and 3¢ external probes in Express Hyb, in accordance with the

manufacturer’s instructions (Clontech). We identified ES cells carrying the

L25Q and W26S mutations using a primer in intron 1 (primer a) and a

mutant-specific primer (primer b). Sequencing of an RT-PCR product derived

from MEFs after Cre introduction confirmed that no other mutations

were present (data not shown). We injected two independent, correctly targeted

ES cell clones into blastocysts to generate mice and characterized both lines in

our experiments. We genotyped mice by assessing the presence of the LSL

element using a primer in the first intron (primer c) and a primer in the stop

element (primer e), yielding a 270-bp band. In the absence of the LSL element,

primer c and a second primer in intron 1 (primer d) resulted in a 365-bp

band. Primer sequences are available on request. All animal work was done

in accordance with the Stanford University Administrative Panel for Laboratory

Animal Care.

Adenoviral infections. We generated MEFs from E13.5 embryos as described44.

We used an empty adenovirus and adeno-Cre (Ad5 CMV empty and Ad5 CMV

Cre, respectively; University of Iowa GTVR) to infect MEFs with an approx-

imate multiplicity of infection of 100. We incubated MEFs in medium

containing adenovirus for 24 h to allow for efficient infection and LSL excision.

Northern-blot analysis. We prepared RNA from MEFs using Trizol (Invitro-

gen) after adenoviral infections, with and without 8 h of treatment with

0.2 mg ml�1 doxorubicin (Sigma) or 6 h of exposure to hypoxic conditions

(o0.2% oxygen) and carried out northern blotting using standard procedures.

We probed blots with Trp53, Cdkn1a, Mdm2, Ccng1, Bax, Pmaip1, Perp, Slc2a1

and Gapd cDNAs45 and quantified them with a Storm phosphorimager using

ImageQuant software (Molecular Dynamics).

Western-blot analysis. We extracted protein from MEFs in 100 mM Tris

(pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol and 1� protease

inhibitor cocktail (Roche). For treatment with DNA-damaging agents, we

subjected cells to 8 h of treatment with 0.2 mg ml�1 doxorubicin. We separated

50 mg of lysate on a 10% SDS-PAGE gel and carried out western blotting

using standard methods. We used antibodies against p53 (Ab-1 and Ab-3,

Oncogene Science) and Gapd (Research Diagnostics, Inc.) at 1:500 and

1:10,000 dilutions, respectively.

EMSAs. We prepared protein extracts from MEFs as described above and

incubated them with pAb421 (Ab-1, Oncogene Science) and 4.5 mg of salmon

sperm DNA in 10 mM Tris-Cl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM

dithiothreitol, 10% glycerol and 0.05% Nonidet P-40 for 20 min on ice. We

added radioactively labeled oligonucleotide, incubated the samples for 20 min

at room temperature and then separated them by electrophoresis in a 4%

polyacrylamide, 0.25� Tris-borate-EDTA gel. Oligonucleotide sequences used

included the p53 response elements from the Cdkn1a promoter and the Perp

first intron46,47. Mutant competitor oligonucleotides contained alterations in

the conserved p53 consensus binding site nucleotides (cxxg was changed to txxt

in both half sites).

BrdU assays and immunofluorescence. We infected MEFs with either empty

adenovirus or adeno-Cre for 24 h, then left them untreated or treated them

with 0.2 mg ml�1 doxorubicin for an additional 24 h and then subjected them

to a 4-h pulse with 3 mg ml�1 BrdU. We immunostained cells to verify efficient

p53 expression. We immunostained cells for BrdU (1:50, BD Biosciences) and

p53 (CM5, 1:150, Novocastra) as described previously8. In each experiment, we

counted 200–300 cells for each sample.


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Apoptosis assays. We infected cells with adenovirus as described above. After

infection, we transduced cells with E1A retroviruses as described previously45.

After 3 d of selection, we plated cells for the different assays. Cells were either

treated with 0.2 mg ml�1 doxorubicin, washed with phosphate-buffered saline

and placed into 0.1% fetal calf serum, or placed in a hypoxia chamber (Bactron

Anaerobic Chamber, Sheldon Manufacturing Company). For the hypoxia

experiments, we plated all cells in glass dishes to achieve critically low oxygen

concentrations ([O2] o 0.2%). For the UV apoptosis assays, we infected MEFs

with empty adenovirus or adeno-Cre as described. We removed the growth

medium, irradiated cells with 20 J m�2 UV-C and then collected them at the

indicated time points. We assayed cell death by annexin V staining as described

by the manufacturer (Caltag Laboratories). We measured the percentage of

viable (annexin-negative and propidium iodide–negative) cells by flow

cytometry using CellQuest software (Becton-Dickinson).

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTSWe thank D. Tuveson for providing the transcriptional stop cassette; T. Jacksfor providing the Trp53LSL-LW mice; N. Denko for providing the Slc2a1 probe;R. Freiberg and S. Basak for technical assistance; and S. Artandi, A. Brunet,R. Ihrie and J. Sage for critical reading of the manuscript. This work wassupported by a Lucille P. Markey Biomedical Research Stanford GraduateFellowship and a National Science Foundation Graduate Research Fellowship toT.M.J., by the National Institutes of Health to A.G. and by the Damon RunyonCancer Research Foundation to L.D.A.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 17 August; accepted 13 December 2004

Published online at http://www.nature.com/naturegenetics/

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the p53qs transactivation-deficient mutant shows stress-specific apoptotic activity and induces...

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