Heterodimerization of Mdm2 and Mdm4 is critical forregulating p53 activity during embryogenesis butdispensable for p53 and Mdm2 stabilityVinod Panta, Shunbin Xionga, Tomoo Iwakumab, Alfonso Quints-Cardamaa,c, and Guillermina Lozanoa,1

Departments of aGenetics and cLeukemia, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; and bDepartment of Genetics, LouisianaState University Health Sciences Center, New Orleans, LA 70112

Edited by Karen H. Vousden, The Beatson Institute for Cancer Research, Glasgow, United Kingdom, and accepted by the Editorial Board June 10, 2011(received for review February 10, 2011)

Mdm2 and Mdm4 are homologous RING domain-containingproteins that negatively regulate the tumor suppressor p53 underphysiological and stress conditions. The RING domain of Mdm2encodes an E3-ubiquitin ligase that promotes p53 degradation. Inaddition, Mdm2 and Mdm4 interact through their respective RINGdomains. The in vivo signicance of Mdm2-Mdm4 heterodimeriza-tion in regulation of p53 function is unknown. In this study, wegenerated an Mdm4 conditional allele lacking the RING domain toinvestigate its role in Mdm2 and p53 regulation. Our results dem-onstrate that homozygous deletion of the Mdm4 RING domainresults in prenatal lethality. Mechanistically, Mdm2-Mdm4 hetero-dimerization is critical for inhibiting lethal p53 activation duringearly embryogenesis. However, Mdm2-Mdm4 interaction is dis-pensable for regulating p53 activity as well as the stability ofMdm2 and p53 at later stages of development. We propose thatMdm4 is a key cofactor of Mdm2 that inhibits p53 activity primar-ily during early embryogenesis but is dispensable for regulatingp53 and Mdm2 stability in the adult mouse.

Mdmx | mouse models | p53 stability | ubiquitination

Murine double minute 2 (Mdm2) and its homolog Mdm4(also known as Mdmx) are two major negative regulatorsof p53 (13). Genetic ablation of either in mice is embryoniclethal and rescued by concomitant deletion of p53 (48). Bothproteins share considerable homology in their N-terminal p53binding and C-terminal really interesting new gene (RING)domains (3). Although both proteins inhibit p53 activity bybinding and masking its transactivation domain, the RING do-main of Mdm2 also functions as an E3-ubiquitin ligase thatpromotes p53 degradation (912). However, similar analysis ofthe Mdm4 RING domain has remained inconclusive. Impor-tantly, these homologous proteins also interact with each otherthrough their respective RING domains (13). In vitro studieshave suggested that Mdm2-Mdm4 heterodimerization is essen-tial for stabilizing Mdm2 and directing its ubiquitin ligase activitytoward p53 (1416). However, the in vivo signicance of thisinteraction in p53 regulation has not been investigated. Here wegenerated and characterized a conditional Mdm4xRING knockinallele to specically address these questions in vivo.

Results and DiscussionTo generate a conditional allele of Mdm4 without the RINGdomain, we inserted a mutant copy of exon 11 lacking the RINGdomain between the endogenous exon 11 and 3 UTR of theMdm4 gene. The mutant exon 11 carried an in-frame deletionof the 49 RING-encoding amino acids. Before cre-mediated de-letion, the targeted allele expressed the full-length wild-typeMdm4 (Mdm4xRING). After cre-loxPmediated recombinationthe endogenous RING domain containing exon 11 could be pre-cisely replaced with the mutant version to generateMdm4RING/+

mice (Fig. 1A).

To examine the physiological consequences of deletion of theMdm4 RING domain, we intercrossed Mdm4RING/+ mice togenerate Mdm4RING/RING mice. Surprisingly, we did not ob-serve any homozygous pups from multiple crosses (Fig. 1B). Thetime point of this embryonic lethality was very similar to Mdm4null lethality (6, 17). PCR genotyping did not reveal any homo-zygous embryos at embryonic day (E) 9.5. Genotyping analysis ofthe corresponding deciduas, however, revealed homozygosity ofMdm4RING in the unresorped deciduas, suggesting recent de-mise of the conceptuses (Fig. 1C). Concomitant deletion of p53rescued the embryo lethal phenotype, implicating unrestrainedp53 activation in homozygous Mdm4RING/RING knockin mice(Fig. 1D). In addition, rescue of the Mdm4RING/RING lethalitywas also accomplished by crossing these mice onto a homozygousp53515A/515A allele, which encodes the transcriptionally null mu-tant p53R172H protein (18).To conrm correct RNA splicing at the intronexon junctions

surrounding the RING deletion, we isolated RNA fromMdm4RING/RINGp53/ mouse spleen and carried out RT-PCRsequencing of Mdm4 (Fig. S1). Mdm4RING transcribed a trun-cated mRNA lacking the sequence corresponding to the 49RING-encoding amino acids. We further conrmed presenceof the smaller protein encoded by the Mdm4RING allele byimmunoprecipitating and Western blotting Mdm4 from p53/

and Mdm4RING/RINGp53/ mouse embryonic broblasts(MEFs) (Fig. 2A). We next examined the interaction of endog-enously expressed Mdm4RING protein with its two majorbinding partners, p53 and Mdm2. Mdm4 interacts with p53 via itsN-terminal domain, whereas it uses the C-terminal RING do-main to dimerize with Mdm2 (3). We performed this analysis inthe mutant Mdm4RING/RINGp53515A/515A background becausep53R172H, although transcriptionally inactive, retains its in-teraction with and regulation by Mdm2 (1, 19). As expected,coimmunoprecipitation analysis of cell lysates from p53515A/515A

and Mdm4RING/RINGp53515A/515A MEFs with a p53 antibodyconrmed an intact albeit weaker interaction between mutantMdm4RING and p53R172H under both normal and DNA-damaging conditions (Fig. 2B). Similar analysis with an Mdm2antibody revealed binding between Mdm2 and Mdm4 but loss ofthe interaction between Mdm2 and Mdm4RING. A slight de-crease in Mdm4 levels after immunoprecipitation with an Mdm2antibody is likely due to masking of the Mdm2 epitope or due to

Author contributions: V.P., T.I., and G.L. designed research; V.P. and S.X. performed re-search; V.P. and A.Q.-C. analyzed data; and V.P. and G.L. wrote the paper.

The authors declare no conict of interest.

This article is a PNAS Direct Submission. K.H.V. is a guest editor invited by the EditorialBoard.1To whom correspondence should be addressed. E-mail: gglozano@mdanderson.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102241108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102241108 PNAS | July 19, 2011 | vol. 108 | no. 29 | 1199512000

GEN

ETICS

increased degradation of the interacting proteins after ionizingradiation (IR) (2022).The prevailing view suggests that subcellular localization of

p53, Mdm2, and Mdm4 is due to the interactions between them(7, 23). We therefore tested whether a defect in Mdm2Mdm4interaction could alter the subcellular localization of these p53pathway proteins. We also examined whether stress-inducedmodications alter the subcellular distribution of these proteins.To that end, we carried out nuclearcytoplasmic fractionation ofp53515A/515A and Mdm4RING/RINGp53515A/515A MEF cell lysatesunder both normal and DNA damage conditions. We conrmedthe purity of the nuclear and cytoplasmic fractions by immuno-blotting with Parp and Gapdh antibodies, respectively. Inter-estingly, no differences in subcellular localization of p53R172H,Mdm2, Mdm4, or Mdm4RING under both normal and DNAdamage conditions were observed (Fig. 2C). Whereas p53 andMdm2 remained distributed in the nucleus and cytoplasm, Mdm4and Mdm4RING proteins maintained primarily cytoplasmiclocalization. Of note, the slightly higher levels of p53R172H ob-served in the cytoplasm could be either due to the p53 mutationitself affecting localization or due to increased cytoplasmic re-tention of p53R172H by Mdm4RING.Mdm2-Mdm4 heterodimerization is perceived to stabilize

Mdm2 by inhibiting its autoubiquitination and in turn promotingp53 ubiquitination (16, 24, 25). However, because p53R172His easily stabilized under tissue culture conditions it could notbe used for these experiments (19). To examine the effects ofMdm4RING on wild-type p53 stability and activity, we there-fore used a conditional hypomorphic p53Neo/Neo allele recently

developed in our laboratory (26). In this model, normal wild-type p53 expression can be restored by either tamoxifen injec-tion in conjunction with a Cre-ER transgene in mice or by ade-novirus-encoded cre recombinase (adeno-cre) infection in cells.p53Neo/Neo mice express only 15% of normal wild-type p53levels and could rescue lethality of Mdm4RING mice. We gen-erated MEFs from p53Neo/Neo (wild type for Mdm4) andMdm4RING/RINGp53Neo/Neo for this analysis. A functional p53allele (>90% recombination) was restored by overnight adeno-cre infection in these cells (Fig. 3A). Surprisingly, no differencein p53 and Mdm2 half-lives was observed in cycloheximide-treated MEFs of both genotypes (Fig. 3B). Both proteinsmaintained 20-min half-lives in either genotype. However, thetruncated Mdm4RING protein was not degraded in compari-son with the full-length wild-type Mdm4 (Fig. 3B). These datasuggest that the interaction between Mdm4 and Mdm2 is nec-essary for Mdm4 degradation but not Mdm2 stability or p53degradation under homeostatic conditions. We next analyzed therole of the Mdm4 RING domain on DNA damage-induceddegradation of p53 and Mdm2. We treated p53Neo/Neo andMdm4RING/RINGp53Neo/Neo MEFs with 10 Gy IR and after 4 hmeasured the protein stability of the accumulated proteins.Again, no differences in degradation kinetics were observed (Fig.3C). Whereas p53 and Mdm4RING remained stable duringthe course of the experiment, the half-life of Mdm2 was short-ened to 10 min in MEFs of both genotypes after IR. Notably,Mdm4 was undetectable owing to its degradation after IR (21,22). These data further corroborate that Mdm2-Mdm4 hetero-

9

Mdm4

Wt. Allele

TKT. Vector

8 Kb 11.5 Kb

5 Probe 3 Probe

10 11

9 10 11 pGKNeobpA

119 10

9 10 11-R

5

3

A

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 +

M 1 4 5 6 7 9 10 11 12 13 15 +

WtMut

WtMut

Mdm4RING/+ X Mdm4

Expected

Observed

Mdm4+/+

Mdm4RING/RING

85 141 0

80 160 80

B C

MutMutWt Wt

MutMutWt Wt

Mdm4p53+/

X Mdm4p53+/

Expected Observed

1/2 18/35

1/16 3/55

Mdm4p53

X Mdm4RING

_ _

/_

Mdm4RING/RING p53D

16Kb8Kb

16Kb11.5Kb

Mdm4

K K

K K

K K

K

KflxRING

Mdm4 RING

_p53

_/_

_

_* * *

/RING

pGKNeobpA 11-R

11-R

_/_

RING/+

RING/+

RING/+ RING/+

RING/+

Probe

Probe

Ratio

Fig. 1. Generation ofMdm4RINGmice. (A) Targeting and screening strategy for generation ofMdm4RINGmice. Southern blots with 5 and 3 external probeswere performed to conrm homologous recombination. Wt, wild type; T.Vector, targeting vector; Mut, mutant. (B) Table shows the expected and observedfrequencies of homozygous mutant mice obtained. (C) Upper: PCR genotyping for the embryos recovered at E9.5. Lower: Corresponding PCR genotyping ofall deciduas, including ones with the homozygous Mdm4RING/RING genotype. Asterisks (*) indicate empty deciduas homozygous for the Mdm4RING/RING

genotype. (D) Table shows the expected and observed frequencies of viable homozygous mutant mice obtained on a p53-null background.

11996 | www.pnas.org/cgi/doi/10.1073/pnas.1102241108 Pant et al.

dimerization is also dispensable for Mdm2 and p53 degradationunder genotoxic conditions.We next analyzed p53 and Mdm2 ubiquitination in the

Mdm4RING background. We transfected a hemagglutinin-tagged ubiquitin (HA-ub)expressing plasmid in p53Neo/Neo andMdm4RING/RINGp53Neo/Neo MEFs and analyzed p53 and Mdm2ubiquitination status after adeno-cremediated p53 restoration.In agreement with the above observations, we found no differ-ences in p53 and Mdm2 ubiquitination between the two geno-types. Similar high molecular weight smears, characteristic ofubiquitination, were observed in the proteasome inhibitor MG132-treated MEFs of both genotypes (Fig. 3D).Although Mdm2 and p53 stability was not altered in the

Mdm4RING background, the embryonic lethality at E9.5 sug-gested enhanced p53 activity during early development. Wetherefore next investigated whether lack of the Mdm4 RINGdomain also led to similar augmentation of p53 activity in MEFsgenerated from E13.5 Mdm4RING/RINGp53Neo/Neo embryos. Weinduced the expression of wild-type p53 in p53Neo/Neo andMdm4RING/RINGp53Neo/Neo MEFs by infecting with adeno-creand performed RTquantitative PCR (qPCR) for p53 targets.Indeed, Mdm2, p21, and Puma transcripts were up-regulated

approximately twofold in Mdm4RING/RINGp53Neo/Neo cells. None-theless, the fold induction was similar between the genotypes,suggesting that the enhancement observed was due to increasedbasal p53 activity in Mdm4RING/RINGp53Neo/Neo MEFs undertissue culture conditions (Fig. 4A). Corresponding Western blotanalyses also revealed relatively higher levels of phospho-p53and the p53 targets p21 and Puma in adeno-cretreatedMdm4RING/RINGp53Neo/Neo MEFs (Fig. 4B). We subsequentlygenerated viable homozygous Mdm4RING/RINGp53Neo/Neo pups.This rescue is in contrast to the observation that p53Neo/Neo doesnot similarly rescue the Mdm2 null lethality (26). This suggeststhat deletion of the Mdm4 RING domain results in a relativelyweaker phenotype compared withMdm2-null and does not lead tothe analogous lethal effects of p53 activation.We next examined whether the Mdm4 RING deletion elicited

a similar p53 response in adult mice. We injected adult condi-tional Mdm4xRING/RINGCreER mice with tamoxifen to generaterecombined Mdm4RING/RING mice. Recombination (89%) wasanalyzed by PCR amplication of genomic DNA from spleen (Fig.4C). Notably, mice with recombined Mdm4RING/RING allele didnot appear sick and were viable. RT-qPCR analysis on RNA fromspleen of the recombined mice revealed slight up-regulation ofp21 but no signicant changes to other canonical p53 targets (Fig.4C). These data established that Mdm2-Mdm4 heterodimeriza-tion is dispensable for p53 inhibition in adult mouse tissues.Because Mdm4RING remained stable after IR, whereas p53

and Mdm2 degraded with similar kinetics in MEFs, we furtherevaluated the long-term effects of Mdm4RING expression onp53 regulation and survival after IR exposure in vivo. Mdm4+/

mice are radiosensitive and die after exposure to 6 Gy IR (27).We similarly irradiated Mdm4RING/+ mice with 6 Gy IR andmonitored them for 50 d (Fig. 4D). Whereas 65% of Mdm4+/

mice died after IR, only 1 of 17 Mdm4RING/+ mice succumbedto radiation. This result may be attributed to a partial function ofthe Mdm4RING because it retains the p53 interaction domainand is refractory to post-IR degradation. We also repeated thisanalysis in Mdm4xRING/RINGCreER mice, which express wild-type p53. We injected Mdm4xRING/RINGCreER mice once perweek for 3 wk with tamoxifen to initiate recombination of theMdm4 conditional allele. Forty-eight hours after the nal in-jection, mice were irradiated with 6 Gy IR. Again, no mortalitywas observed during the subsequent 50-d observation period.Because sublethal doses of IR primarily cause death by hema-topoietic failure in Mdm4+/ mice, these results suggest thatthe Mdm4RING protein inhibits the lethal activation of p53in the radiosensitive hematopoietic compartment under stressconditions.Previous data suggest that an Mdm2 RING mutant that lacks

the E3-ubiquitin ligase activity and the ability to heterodimerizewith Mdm4 leads to p53 stabilization (28). Our results heredemonstrate that in the absence of Mdm2-Mdm4 hetero-dimerization, p53 and Mdm2 degradation rates are not altered.Taken together, these data imply that p53 degradation by Mdm2ubiquitin ligase activity is independent of its association withMdm4. Additionally, Mdm2-Mdm4 heterodimerization is nec-essary for Mdm4 but not Mdm2 degradation.Interestingly, Mdm4RING protein was stable under DNA-

damaging conditions. This phenomenon is similar to that observedwith the Mdm4-3SA phosphorylation mutant protein, whichresists degradation after radiation damage (29). This could explainthe lack of radiosensitivity ofMdm4RING/+ and tamoxifen-injectedMdm4xRING/RINGCreER mice after 6 Gy IR exposure. A non-degradable, RING-decient Mdm4 protein could remain boundto p53 and effectively inhibit its transcriptional activity.A short form of Mdm4 (Mdmx-S) has been reported in human

normal and cancer cells (30). This short form lacks the RINGdomain and thus resembles Mdm4RING. Mdmx-S is ampliedin human cancers such as glioblastoma and soft tissue sarcoma

Mdm4

WB : Mdm4

WB : Mdm2

WB : Mdm4

WB : p53

IR _ _ _ _ + +

IR _ _ _ _ + +

A

B

CC N C N C N C N

p53

Mdm2

Parp

Gapdh

Mdm4

IR _ _ _ _ + + + +

IPAb

M4Tg

Mdm4Mdm4R

Mdm4Mdm4R

Mdm2

p53

Mdm4 +/+ R /R +/+ R /R

Mdm4 +/+ +/+ R/R / p53 / +/+ / /

Mdm4 +/+ R /R +/+ R /RT T 2 2 IP Ab

Mdm4Mdm4R

+/+ R /R +/+ R /RT T 2 2

+ +

+/+ R /R +/+ R /R +/+ R /R +/+ R /RT T 53 53 T T 53 53

+ +

Fig. 2. Characterization of Mdm4RING protein. (A) Immunoprecipitation(IP) results from MEFs of different genotypes expressing the Mdm4 andMdm4RING protein. Protein extract from an Mdm4 transgenic mouse spleen(M4Tg) is used as positive control. R, RING. (B) Coimmunoprecipitation ofMdm4 from p53515A/515A (wild type forMdm4) andMdm4RING/RINGp53515A/515A

(mutant for Mdm4) MEFs, treated with or without IR. Upper: Coimmunopreci-pitation with p53 antibody (53). Lower: Coimmunoprecipitation with Mdm2antibody (2). T, 10% of input. (C) Subcellular localization of Mdm2, Mdm4,and p53 in protein lysates from p53515A/515A and Mdm4RING/RINGp53515A/515A

MEFs treated with or without IR. Parp and Gapdh were used as nuclear (N) andcytoplasmic (C) fraction markers, respectively.

Pant et al. PNAS | July 19, 2011 | vol. 108 | no. 29 | 11997

GEN

ETICS

and correlates with poor prognosis (31, 32). The consequencesof increased levels of Mdmx-S in these patients after DNA-damaging chemo/radiotherapeutic agents may include inhibitionof p53 activity and/or p53-independent activities and warrantfurther investigation.Our results clearly show that RING domain-mediated Mdm2-

Mdm4 heterodimerization is critical for regulating p53 activityduring early embryogenesis. However, during later develop-mental stages and adult life this interaction becomes dispensableor redundant. In stark contrast to Mdm2 deletion, the relativelyminor nonlethal phenotypes observed with deletion of Mdm4 inadult animal tissues also support this notion (33). In contrast toloss of Mdm4 function as studied in these animal models, am-plication of Mdm4 occurs in multiple human tumors, and manyof these retain wild-type p53 (27, 3436). These data support theimportance of Mdm4 in regulating p53 under tumor and non-physiological stress conditions.Collectively, these results suggest that Mdm4 primarily acts as

a cofactor with Mdm2 to inhibit p53 during embryogenesis but isdispensable for regulating p53 and Mdm2 stability in the adultunder homeostatic conditions.

Materials and MethodsGeneration of Mdm4RING/+ Mice. A loxP anked neomycin cassette and anadditional copy of exon 11 lacking the RING domain was inserted betweenthe endogenous exon 11 and the 3 UTR of the Mdm4 gene. Additionally,a loxP sequence was placed upstream of the endogenous Mdm4. Hsv-Tk1

cassette was included for negative selection (Fig. 1A). The targeting con-struct was sequenced completely and electroporated into TC1 mouse em-bryonic stem cells. G418 resistant clones were analyzed for correcthomologous recombination by Southern blotting using 5 and 3 externalprobes (Fig. 1A). Two independent correctly targeted clones were expandedand injected into C57BL/6 blastocysts to generate Mdm4RING/+ chimeras.Male chimeras were backcrossed to C57BL/6 mice to obtain germline trans-mission of the mutant allele.

Mouse Breeding, Maintenance, and Genotyping. All mice were maintained in>90% C57BL/6 background. All mouse studies were conducted in compliancewith Institutional Animal Care andUse Committee protocols. Before neomycincassette deletion, mouse genotyping was done by PCR amplication usingprimer sets 5-cta gtg aga cgt gct act tc-3 and 5-gga gag atg tac acc tgt gt-3.The neomycin selection cassette was deleted by crossing germline transmittedF1 generation mice with Zp3-Cre deleter mice (37). Subsequently, genotypingwas carried out by PCR amplication with primer sets 5-ggc aac tcc aga taacta cc-3 and 5-cag tac ctc ttg ctt gga g-3 and resolved on an agarose gel.

MEF Preparation and Cell Culture. Embryonic day 13.5 embryos were used togenerate MEFs, which were maintained in DMEM (Invitrogen) supplementedwith 10% FBS, penicillin (100 IU/mL), and streptomycin (100 g/mL). Early-passage MEFs (P2P3) were used for analysis.

Protein Analysis. MEFs were lysed in Nonidet P-40 buffer. Protein estimationwas carried out with Bicinchoninic Acid (BCA Protein Assay Kit; Pierce). Ap-proximately100gof lysatewas resolvedon8%SDS/PAGEandWesternblottedwith antibodies against p53 (CM5; Vector Laboratories, 1:1,000), Mdm2 (2A10;Calbiochem, 1:500), Mdm4 (MX82; Sigma, 1:500), S15-p53 (9284; Cell Signaling

0 10 20 30 45 60 0 10 20 30 45 60Mdm2

p53

Mdm4

Actin

0 10 20 30 45 60 0 10 20 30 45 60p53Neo/Neo Mdm4RING/RINGp53Neo/NeoTime Aft er

CHX (Mi n)

p53 Neo/Neo Mdm4RING/RINGp53 Neo/Neo

Ad- Cre + + + +p53

_ _

A

B

MG132 + +

C

D

p53Mdm2

Ub-p53

Ub-Mdm2

0 10 20 30 401

10

100

WTRING -IR

45

50

Chase (min)

Rel

ative

amou

ntof

p53

(%)

0 10 20 30 401

10

100

WTRING -IR

45

50

Chase (min)

Rela

tive

amou

ntof

Mdm

2(%

)0 10 20 30 40

1

10

100

WTRING +IR

45

50

Chase (min)0 10 20 30 40

1

10

100

WTRING +IR

45

50

Chase (min)

Time AfterCHX (Min)

+ +_ _ _ _

Mdm4

Mdm4 +/+ R/R+/+ R/R +/+ R/R+/+ R/R

+/+ R/R +/+ R/R +/+ R/R

Short ExpShort Exp

-IR

Rela

tive

amou

ntof

Mdm

4(%

)

Chase (min)

1

10

100

50

0 10 20 30 4045

WTRING

Rela

tive

amou

ntof

Mdm

2(%

)

Rel

ative

amou

ntof

p53

(%)

Mdm2

Mdm4

p53

Actin

Fig. 3. Mdm2-Mdm4 heterodimerization is dispensable for p53 and Mdm2 degradation. (A) PCR genotyping shows recombination efciency in p53Neo/Neo

and Mdm4RING/RINGp53Neo/Neo MEFs after adeno-cre (Ad-Cre) virus infection. R, RING (B) Adeno-creinfected p53Neo/Neo and Mdm4RING/RINGp53Neo/Neo

MEFs were treated with cycloheximide ((CHX) 20 g/mL) and harvested at different time points. WT, wild type; RING, Mdm4RING. (C). Adeno-creinfectedp53Neo/Neo and Mdm4RING/RINGp53Neo/Neo MEFs were treated with 10 Gy IR. Proteins were allowed to accumulate for 4 h, followed by cycloheximidetreatment (20 g/mL) and cells harvested at different time points. (D) p53Neo/Neo andMdm4RING/RINGp53Neo/Neo MEFs were transfected with HA-ub, followedby infection with adeno-cre virus. Protein lysate was immunoprecipitated with HA-conjugated beads and immunoblotted with p53 and Mdm2 antibodies.

11998 | www.pnas.org/cgi/doi/10.1073/pnas.1102241108 Pant et al.

Technology, 1:1,000), vinculin (V9131; Sigma, 1:1,000), and -actin (AC15; Sigma,1:5,000). Twomilligrams of protein was used for immunoprecipitation with thesame antibodies. Subcellular fractionation was carried out using a nuclear ex-tract kit (Active Motif, #40010) according to the manufacturers protocol.

Ubiquitination Assay. p53Neo/Neo and Mdm4RING/RINGp53Neo/Neo MEFs weretransfected with HA-ub plasmid (kind gift from Dr. M. H. Lee, M.D. AndersonCancer Center, Houston, TX). After 48 h cells were infected with adeno-crevirus [100 multiplicity of infection (MOI)] and harvested 24 h later. Protein wasimmunoprecipitated with anti-hemagglutinin antibody-tagged protein-Aagarose beads (Sigma, #E6779), resolved on 8% SDS/PAGE, and immuno-blotted with p53 or Mdm2 antibody.

Protein Stability Assay. p53Neo/Neo and Mdm4RING/RINGp53Neo/Neo MEFs wereinfected with adeno-cre for 24 h before treatment with cycloheximide (20g/mL). Cells were harvested at different time points and protein analyzedby Western blotting.

Conditional Recombination of Allele. Conditional recombination of the mouseallele was carried out by immunoprecipitation. Tamoxifen injection was aspreviously described (26). Recombination in MEFs was achieved by overnightadeno-cre virus infection (100 MOI) to the cells. The rate of recombination

was determined by PCR amplication of genomic DNA and subsequentdensitometric ratio analysis of bands resolved on agarose gels.

Quantitative RT-PCR. RNA was isolated from tissues/MEFs using TRIzol (Invi-trogen). Onemicrogram of RNAwas reverse-transcribed to cDNA (First StrandSynthesis Kit; GE Healthcare). First-strand reaction was diluted 10-fold, and2 L of the diluted reaction was used in qPCR reaction for p53 targets in anABI 7900HT real-time PCR machine. Primer sequences have been previouslydescribed (38).

IR Studies. Mice were irradiated at 6 Gy in a cesium-137 irradiator andmonitored for 50 d. MEFs were cultured in a 100-mm tissue culture dish,irradiated with 10 Gy IR, and incubated at 37 C before harvesting at dif-ferent time points for experimental analyses.

ACKNOWLEDGMENTS. We thank Drs. Sean Post and James Jackson forcritical reading of the manuscript. Mice were made by the GeneticallyEngineered Mouse Facility at The M. D. Anderson Cancer Center. Studieswere supported by National Institutes of Health Grant CA47296 (to G.L.).V.P. has been supported in part by Molecular Genetics of Cancer TrainingGrant CA009299 and is a recipient of fellowships from the Dodie P. Hawnand Lupe C. Garcia Foundations.

1. Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogeneproduct forms a complex with the p53 protein and inhibits p53-mediated trans-activation. Cell 69:12371245.

2. Shvarts A, et al. (1996) MDMX: A novel p53-binding protein with some functionalproperties of MDM2. EMBO J 15:53495357.

3. Marine JC, Dyer MA, Jochemsen AG (2007) MDMX: From bench to bedside. J Cell Sci120:371378.

4. Montes de Oca Luna R, Wagner DS, Lozano G (1995) Rescue of early embryonic le-thality in mdm2-decient mice by deletion of p53. Nature 378:203206.

5. Jones SN, Roe AE, Donehower LA, Bradley A (1995) Rescue of embryonic lethality inMdm2-decient mice by absence of p53. Nature 378:206208.

6. Parant J, et al. (2001) Rescue of embryonic lethality in Mdm4-null mice by loss ofTrp53 suggests a nonoverlapping pathway with MDM2 to regulate p53. Nat Genet 29:9295.

7. Migliorini D, et al. (2002) Mdm4 (Mdmx) regulates p53-induced growth arrest andneuronal cell death during early embryonic mouse development. Mol Cell Biol 22:55275538.

8. Finch RA, et al. (2002) mdmx is a negative regulator of p53 activity in vivo. Cancer Res62:32213225.

9. Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of

p53. Nature 387:296299.10. Honda R, Tanaka H, Yasuda H (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for

tumor suppressor p53. FEBS Lett 420:2527.11. Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 stability by Mdm2.

Nature 387:299303.12. Haines DS, Landers JE, Engle LJ, George DL (1994) Physical and functional interaction

between wild-type p53 and mdm2 proteins. Mol Cell Biol 14:11711178.13. Tanimura S, et al. (1999) MDM2 interacts with MDMX through their RING nger

domains. FEBS Lett 447:59.14. Pan Y, Chen J (2003) MDM2 promotes ubiquitination and degradation of MDMX.Mol

Cell Biol 23:51135121.15. Kawai H, Lopez-Pajares V, Kim MM, Wiederschain D, Yuan ZM (2007) RING domain-

mediated interaction is a requirement for MDM2s E3 ligase activity. Cancer Res 67:

60266030.16. Okamoto K, Taya Y, Nakagama H (2009) Mdmx enhances p53 ubiquitination by al-

tering the substrate preference of the Mdm2 ubiquitin ligase. FEBS Lett 583:

27102714.

Puma

0.00.5

1.0

1.5

2.0

2.5

3.0

3.5p21

012345678

Fold

mR

NA

Induct

ion

Fold

mR

NA

Induct

ion

Fold

mR

NA

Induct

ion

Re

lativ

e m

RN

A L

eve

ls

Mdm2

0.0

2.5

5.0

7.5

10.0

12.5

+ Ad CreRR

+ Ad Cre

_ _ _ + + + _ _ _ + + +p53

Neo/NeoMdm4

RING/RINGp53Neo/Neo

p53

p21

Puma

Ad-Cre

Actin

C

Oil Tam Oil Tam Oil Tam Oil Tam0.0

0.5

1.0

1.5

2.0

2.5

Cyclin G1 Mdm2 p21 Puma

DRINGNeo

+ + + Tam

0 10 20 30 40 50

25

50

75

100

Mdm4RING/+ (17)

Mdm4+/+ (15)

Mdm4+/ (15)

Days Post 6Gy IR

Perc

ent surv

ival

p53N/N

flxNeo/RINGMdm4 CreER_

p53N/N

p53N/N

RRN/N

p53

pS15-p53

A

B

Fig. 4. RING domain deletion does not promote lethal p53 activation in adult mouse tissues. (A) Real-time PCR for p53 targets in p53Neo/Neo (p53N/N) andMdm4RING/RINGp53Neo/Neo (RRp53N/N) MEFs after adeno-cre (Ad-Cre) infection. Data were normalized to expression in untreated p53Neo/Neo controls andrepresent a mean of three independent experiments SEM. (B) Western blot analysis from adeno-cretreated p53Neo/Neo and Mdm4RING/RINGp53Neo/Neo

MEFs. (C) Five-week-oldMdm4xRING/RING:CreERmice were injected with either corn oil or tamoxifen. RNA from spleen was used for RT-qPCR analyses for p53targets. Data were normalized to expression levels in oil-injected controls, n = 3, SEM. Inset: Homologous recombination after tamoxifen (Tam) injection. (D)Kaplan-Meyer survival curve of Mdm4+/+, Mdm4+/, and Mdm4RING/+ mice after 6 Gy irradiation.

Pant et al. PNAS | July 19, 2011 | vol. 108 | no. 29 | 11999

GEN

ETICS

17. Grier JD, Xiong S, Elizondo-Fraire AC, Parant JM, Lozano G (2006) Tissue-specicdifferences of p53 inhibition by Mdm2 and Mdm4. Mol Cell Biol 26:192198.

18. Lang GA, et al. (2004) Gain of function of a p53 hot spot mutation in a mouse modelof Li-Fraumeni syndrome. Cell 119:861872.

19. Terzian T, et al. (2008) The inherent instability of mutant p53 is alleviated by Mdm2 orp16INK4a loss. Genes Dev 22:13371344.

20. Cheng Q, Chen J (2011) The phenotype of MDM2 auto-degradation after DNAdamage is due to epitope masking by phosphorylation. Cell Cycle 10:11621166.

21. Chen L, Gilkes DM, Pan Y, Lane WS, Chen J (2005) ATM and Chk2-dependent phos-phorylation of MDMX contribute to p53 activation after DNA damage. EMBO J 24:34113422.

22. Kawai H, et al. (2003) DNA damage-induced MDMX degradation is mediated byMDM2. J Biol Chem 278:4594645953.

23. Li C, Chen L, Chen J (2002) DNA damage induces MDMX nuclear translocation by p53-dependent and -independent mechanisms. Mol Cell Biol 22:75627571.

24. Jackson MW, Berberich SJ (2000) MdmX protects p53 from Mdm2-mediated degra-dation. Mol Cell Biol 20:10011007.

25. Gu J, et al. (2002) Mutual dependence of MDM2 and MDMX in their functional in-activation of p53. J Biol Chem 277:1925119254.

26. Wang Y, et al. (2011) Restoring expression of wild-type p53 suppresses tumor growthbut does not cause tumor regression in mice with a p53 missense mutation. J ClinInvest 121:893904.

27. Terzian T, et al. (2007) Haploinsufciency of Mdm2 and Mdm4 in tumorigenesis anddevelopment. Mol Cell Biol 27:54795485.

28. Itahana K, et al. (2007) Targeted inactivation of Mdm2 RING nger E3 ubiquitin ligaseactivity in the mouse reveals mechanistic insights into p53 regulation. Cancer Cell 12:355366.

29. Wang YV, Leblanc M, Wade M, Jochemsen AG, Wahl GM (2009) Increased radio-resistance and accelerated B cell lymphomas in mice with Mdmx mutations thatprevent modications by DNA-damage-activated kinases. Cancer Cell 16:3343.

30. Rallapalli R, Strachan G, Cho B, Mercer WE, Hall DJ (1999) A novel MDMX transcriptexpressed in a variety of transformed cell lines encodes a truncated protein withpotent p53 repressive activity. J Biol Chem 274:82998308.

31. Riemenschneider MJ, et al. (1999) Amplication and overexpression of the MDM4(MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation orMDM2 amplication. Cancer Res 59:60916096.

32. Bartel F, et al. (2005) Signicance of HDMX-S (or MDM4) mRNA splice variant over-expression and HDMX gene amplication on primary soft tissue sarcoma prognosis.Int J Cancer 117:469475.

33. Xiong S, Van Pelt CS, Elizondo-Fraire AC, Fernandez-Garcia B, Lozano G (2007) Loss ofMdm4 results in p53-dependent dilated cardiomyopathy. Circulation 115:29252930.

34. Valentin-Vega YA, Barboza JA, Chau GP, El-Naggar AK, Lozano G (2007) High levelsof the p53 inhibitor MDM4 in head and neck squamous carcinomas. Hum Pathol 38:15531562.

35. Riemenschneider MJ, Knobbe CB, Reifenberger G (2003) Rened mapping of 1q32amplicons in malignant gliomas conrms MDM4 as the main amplication target. IntJ Cancer 104:752757.

36. Laurie NA, et al. (2006) Inactivation of the p53 pathway in retinoblastoma. Nature444:6166.

37. Lewandoski M, Wassarman KM, Martin GR (1997) Zp3-cre, a transgenic mouse line forthe activation or inactivation of loxP-anked target genes specically in the femalegerm line. Curr Biol 7:148151.

38. Post SM, et al. (2010) A high-frequency regulatory polymorphism in the p53 pathwayaccelerates tumor development. Cancer Cell 18:220230.

12000 | www.pnas.org/cgi/doi/10.1073/pnas.1102241108 Pant et al.