Heterodimerization of Mdm2 and Mdm4 Is Critical for Regulating P53 Activity During Embryogenesis but Dispensable for P53 and Mdm2 Stability

Heterodimerization of Mdm2 and Mdm4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability

Vinod Panta, Shunbin Xionga, Tomoo Iwakumab, Alfonso Quintás-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, Louisiana State 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-containing To examine the physiological consequences of deletion of the ΔRING/+ proteins that negatively regulate the tumor suppressor p53 under Mdm4 RING domain, we intercrossed Mdm4 mice to ΔRING/ΔRING physiological and stress conditions. The RING domain of Mdm2 generate Mdm4 mice. Surprisingly, we did not ob- encodes an E3-ubiquitin ligase that promotes p53 degradation. In serve any homozygous pups from multiple crosses (Fig. 1B). The addition, Mdm2 and Mdm4 interact through their respective RING time point of this embryonic lethality was very similar to Mdm4 domains. The in vivo significance of Mdm2-Mdm4 heterodimeriza- null lethality (6, 17). PCR genotyping did not reveal any homo- tion in regulation of p53 function is unknown. In this study, we zygous embryos at embryonic day (E) 9.5. Genotyping analysis of Mdm4 generated an conditional allele lacking the RING domain to the corresponding deciduas, however, revealed homozygosity of ΔRING investigate its role in Mdm2 and p53 regulation. Our results dem- Mdm4 in the unresorped deciduas, suggesting recent de- onstrate that homozygous deletion of the Mdm4 RING domain mise of the conceptuses (Fig. 1C). Concomitant deletion of p53 results in prenatal lethality. Mechanistically, Mdm2-Mdm4 hetero- rescued the embryo lethal phenotype, implicating unrestrained dimerization is critical for inhibiting lethal p53 activation during ΔRING/ΔRING p53 activation in homozygous Mdm4 knockin mice early embryogenesis. However, Mdm2-Mdm4 interaction is dis- ΔRING/ΔRING (Fig. 1D). In addition, rescue of the Mdm4 lethality pensable for regulating p53 activity as well as the stability of was also accomplished by crossing these mice onto a homozygous Mdm2 and p53 at later stages of development. We propose that p53515A/515A Mdm4 is a key cofactor of Mdm2 that inhibits p53 activity primar- allele, which encodes the transcriptionally null mu- ily during early embryogenesis but is dispensable for regulating tant p53R172H protein (18). fi – p53 and Mdm2 stability in the adult mouse. To con rm correct RNA splicing at the intron exon junctions surrounding the RING deletion, we isolated RNA from ΔRING/ΔRING −/− Mdm4 p53 mouse spleen and carried out RT-PCR Mdmx | mouse models | p53 stability | ubiquitination ΔRING sequencing of Mdm4 (Fig. S1). Mdm4 transcribed a trun- urine double minute 2 (Mdm2) and its homolog Mdm4 cated mRNA lacking the sequence corresponding to the 49 RING-encoding amino acids. We further confirmed presence (also known as Mdmx) are two major negative regulators ΔRING M of the smaller protein encoded by the Mdm4 allele by of p53 (1–3). Genetic ablation of either in mice is embryonic −/− immunoprecipitating and Western blotting Mdm4 from p53 lethal and rescued by concomitant deletion of p53 (4–8). Both ΔRING/ΔRING −/− and Mdm4 p53 mouse embryonic fibroblasts proteins share considerable homology in their N-terminal p53 A binding and C-terminal really interesting new gene (RING) (MEFs) (Fig. 2 ). We next examined the interaction of endog- enously expressed Mdm4ΔRING protein with its two major domains (3). Although both proteins inhibit p53 activity by binding partners, p53 and Mdm2. Mdm4 interacts with p53 via its binding and masking its transactivation domain, the RING do- N-terminal domain, whereas it uses the C-terminal RING domain of Mdm2 also functions as an E3-ubiquitin ligase that main to dimerize with Mdm2 (3). We performed this analysis in promotes p53 degradation (9–12). However, similar analysis of ΔRING/ΔRING 515A/515A the mutant Mdm4 p53 background because the Mdm4 RING domain has remained inconclusive. Impor- p53R172H, although transcriptionally inactive, retains its in- tantly, these homologous proteins also interact with each other teraction with and regulation by Mdm2 (1, 19). As expected, through their respective RING domains (13). In vitro studies 515A/515A coimmunoprecipitation analysis of cell lysates from p53 have suggested that Mdm2-Mdm4 heterodimerization is essen- ΔRING/ΔRING 515A/515A and Mdm4 p53 MEFs with a p53 antibody tial for stabilizing Mdm2 and directing its ubiquitin ligase activity confirmed an intact albeit weaker interaction between mutant toward p53 (14–16). However, the in vivo significance of this Mdm4ΔRING and p53R172H under both normal and DNA- interaction in p53 regulation has not been investigated. Here we flxRING damaging conditions (Fig. 2B). Similar analysis with an Mdm2 generated and characterized a conditional Mdm4 knockin antibody revealed binding between Mdm2 and Mdm4 but loss of allele to specifically address these questions in vivo. the interaction between Mdm2 and Mdm4ΔRING. A slight de- Results and Discussion crease in Mdm4 levels after immunoprecipitation with an Mdm2

To generate a conditional allele of Mdm4 without the RING antibody is likely due to masking of the Mdm2 epitope or due to GENETICS domain, we inserted a mutant copy of exon 11 lacking the RING domain between the endogenous exon 11 and 3′ UTR of the Mdm4 Author contributions: V.P., T.I., and G.L. designed research; V.P. and S.X. performed re- gene. The mutant exon 11 carried an in-frame deletion search; V.P. and A.Q.-C. analyzed data; and V.P. and G.L. wrote the paper. cre of the 49 RING-encoding amino acids. Before -mediated de- The authors declare no conﬂict of interest. letion, the targeted allele expressed the full-length wild-type ﬂxRING This article is a PNAS Direct Submission. K.H.V. is a guest editor invited by the Editorial Mdm4 (Mdm4 ). After cre-loxP–mediated recombination Board. the endogenous RING domain containing exon 11 could be pre- 1To whom correspondence should be addressed. E-mail: [email protected]. Mdm4ΔRING/+ cisely replaced with the mutant version to generate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mice (Fig. 1A). 1073/pnas.1102241108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1102241108 PNAS | July 19, 2011 | vol. 108 | no. 29 | 11995–12000 Downloaded by guest on September 30, 2021 A K K Wt Wt Mut Mut Wt. Allele 9 10 11

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B C M 1 4 5 6 7 9 10 11 12 13 15 + _ ∆RING/+ ∆RING/+ Mdm4 XMdm4 Wt Mut +/+ ∆RING/+ ∆RING/∆RING Mdm4 Mdm4 Mdm4 ** * _ Expected 80 160 80 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + Wt Observed 85 141 0 Mut _ _ ∆RING / D Mdm4∆RING/ p53 Expected Observed Ratio Mdm4∆RING/+X Mdm4∆RING/+ _ _ 1/16 3/55 p53+/ p53+/ ∆RING/+ ∆RING/∆RING Mdm4_ _ X Mdm4_ _ p53 / p53 / 1/2 18/35

ΔRING ΔRING Fig. 1. Generation of Mdm4 mice. (A) Targeting and screening strategy for generation of Mdm4 mice. Southern blots with 5′ and 3′ external probes were performed to conﬁrm homologous recombination. Wt, wild type; T.Vector, targeting vector; Mut, mutant. (B) Table shows the expected and observed frequencies of homozygous mutant mice obtained. (C) Upper: PCR genotyping for the embryos recovered at E9.5. Lower: Corresponding PCR genotyping of ΔRING/ΔRING ΔRING/ΔRING all deciduas, including ones with the homozygous Mdm4 genotype. Asterisks (*) indicate empty deciduas homozygous for the Mdm4 genotype. (D) Table shows the expected and observed frequencies of viable homozygous mutant mice obtained on a p53-null background.

increased degradation of the interacting proteins after ionizing developed in our laboratory (26). In this model, normal wild- radiation (IR) (20–22). type p53 expression can be restored by either tamoxifen injec- The prevailing view suggests that subcellular localization of tion in conjunction with a Cre-ER transgene in mice or by ade- p53, Mdm2, and Mdm4 is due to the interactions between them novirus-encoded cre recombinase (adeno-cre) infection in cells. Neo/Neo (7, 23). We therefore tested whether a defect in Mdm2–Mdm4 p53 mice express only ≈15% of normal wild-type p53 ΔRING interaction could alter the subcellular localization of these p53 levels and could rescue lethality of Mdm4 mice. We gen- Neo/Neo pathway proteins. We also examined whether stress-induced erated MEFs from p53 (wild type for Mdm4) and ﬁ ΔRING/ΔRING Neo/Neo modi cations alter the subcellular distribution of these proteins. Mdm4 p53 for this analysis. A functional p53 – To that end, we carried out nuclear cytoplasmic fractionation of allele (>90% recombination) was restored by overnight adeno- p53515A/515A Mdm4ΔRING/ΔRINGp53515A/515A and MEF cell lysates cre infection in these cells (Fig. 3A). Surprisingly, no difference ﬁ under both normal and DNA damage conditions. We con rmed in p53 and Mdm2 half-lives was observed in cycloheximide- the purity of the nuclear and cytoplasmic fractions by immuno- treated MEFs of both genotypes (Fig. 3B). Both proteins blotting with Parp and Gapdh antibodies, respectively. Inter- maintained ≈20-min half-lives in either genotype. However, the estingly, no differences in subcellular localization of p53R172H, truncated Mdm4ΔRING protein was not degraded in compari- Mdm2, Mdm4, or Mdm4ΔRING under both normal and DNA son with the full-length wild-type Mdm4 (Fig. 3B). These data damage conditions were observed (Fig. 2C). Whereas p53 and suggest that the interaction between Mdm4 and Mdm2 is nec- Mdm2 remained distributed in the nucleus and cytoplasm, Mdm4 and Mdm4ΔRING proteins maintained primarily cytoplasmic essary for Mdm4 degradation but not Mdm2 stability or p53 localization. Of note, the slightly higher levels of p53R172H ob- degradation under homeostatic conditions. We next analyzed the served in the cytoplasm could be either due to the p53 mutation role of the Mdm4 RING domain on DNA damage-induced p53Neo/Neo itself affecting localization or due to increased cytoplasmic re- degradation of p53 and Mdm2. We treated and Mdm4ΔRING/ΔRINGp53Neo/Neo tention of p53R172H by Mdm4ΔRING. MEFs with 10 Gy IR and after 4 h Mdm2-Mdm4 heterodimerization is perceived to stabilize measured the protein stability of the accumulated proteins. Mdm2 by inhibiting its autoubiquitination and in turn promoting Again, no differences in degradation kinetics were observed (Fig. p53 ubiquitination (16, 24, 25). However, because p53R172H 3C). Whereas p53 and Mdm4ΔRING remained stable during is easily stabilized under tissue culture conditions it could not the course of the experiment, the half-life of Mdm2 was short- be used for these experiments (19). To examine the effects of ened to ≈10 min in MEFs of both genotypes after IR. Notably, Mdm4ΔRING on wild-type p53 stability and activity, we there- Mdm4 was undetectable owing to its degradation after IR (21, Neo/Neo fore used a conditional hypomorphic p53 allele recently 22). These data further corroborate that Mdm2-Mdm4 hetero-

11996 | www.pnas.org/cgi/doi/10.1073/pnas.1102241108 Pant et al. Downloaded by guest on September 30, 2021 Mdm4ΔRING/ΔRINGp53Neo/Neo A Mdm4 +/+ +/+ ∆R/∆R –/– approximately twofold in cells. None- p53 –/– +/+ –/– –/– M4Tg theless, the fold induction was similar between the genotypes, Mdm4 suggesting that the enhancement observed was due to increased ΔRING/ΔRING Neo/Neo Mdm4∆R basal p53 activity in Mdm4 p53 MEFs under tissue culture conditions (Fig. 4A). Corresponding Western blot B Mdm4 +/+ ∆R /∆R +/+ ∆R /∆R+/+ ∆R /∆R +/+ ∆R /∆R analyses also revealed relatively higher levels of phospho-p53 IPAb TT_ _ α _53 α _53 TTα53α 53 IR + + + + and the p53 targets p21 and Puma in adeno-cre–treated ΔRING/ΔRING Neo/Neo Mdm4 Mdm4 p53 MEFs (Fig. 4B). We subsequently Mdm4∆R ΔRING/ΔRING Neo/Neo generated viable homozygous Mdm4 p53 pups. WB : Mdm4 Neo/Neo This rescue is in contrast to the observation that p53 does p53 not similarly rescue the Mdm2 null lethality (26). This suggests WB : p53 that deletion of the Mdm4 RING domain results in a relatively Mdm2 Mdm4 +/+ ∆R /∆R +/+ ∆R /∆R +/+ ∆R /∆R +/+ ∆R /∆R weaker phenotype compared with -null and does not lead to IP Ab TTα2α2 TTα2α2 the analogous lethal effects of p53 activation. IR _ _ _ _ + + + + We next examined whether the Mdm4 RING deletion elicited Mdm4 Mdm4∆R a similar p53 response in adult mice. We injected adult condi- flxRING/ΔRING WB : Mdm4 tional Mdm4 CreER mice with tamoxifen to generate ΔRING/ΔRING recombined Mdm4 mice. Recombination (89%) was Mdm2 analyzed by PCR amplification of genomic DNA from spleen (Fig. ΔRING/ΔRING WB : Mdm2 4C). Notably, mice with recombined Mdm4 allele did not appear sick and were viable. RT-qPCR analysis on RNA from C Mdm4 +/+ ∆R /∆R +/+ ∆R /∆R _CNCNCNCN _ _ _ spleen of the recombined mice revealed slight up-regulation of IR + + + + p21 but no significant changes to other canonical p53 targets (Fig. C Mdm4 4 ). These data established that Mdm2-Mdm4 heterodimerization is dispensable for p53 inhibition in adult mouse tissues. p53 Because Mdm4ΔRING remained stable after IR, whereas p53 and Mdm2 degraded with similar kinetics in MEFs, we further evaluated the long-term effects of Mdm4ΔRING expression on Mdm2 +/− p53 regulation and survival after IR exposure in vivo. Mdm4 mice are radiosensitive and die after exposure to 6 Gy IR (27). Parp ΔRING/+ We similarly irradiated Mdm4 mice with 6 Gy IR and +/− monitored them for 50 d (Fig. 4D). Whereas 65% of Mdm4 ΔRING/+ Gapdh mice died after IR, only 1 of 17 Mdm4 mice succumbed Δ A to radiation. This result may be attributed to a partial function of Fig. 2. Characterization of Mdm4 RING protein. ( ) Immunoprecipitation Δ (IP) results from MEFs of different genotypes expressing the Mdm4 and the Mdm4 RING because it retains the p53 interaction domain Δ and is refractory to post-IR degradation. We also repeated this Mdm4 RING protein. Protein extract from an Mdm4 transgenic mouse spleen flxRING/ΔRING (M4Tg) is used as positive control. ΔR, ΔRING. (B) Coimmunoprecipitation of analysis in Mdm4 CreER mice, which express wild- 515A/515A ΔRING/ΔRING 515A/515A flxRING/ΔRING Mdm4 from p53 (wild type for Mdm4) and Mdm4 p53 type p53. We injected Mdm4 CreER mice once per (mutant for Mdm4) MEFs, treated with or without IR. Upper: Coimmunopreci- week for 3 wk with tamoxifen to initiate recombination of the pitation with p53 antibody (α53). Lower: Coimmunoprecipitation with Mdm2 Mdm4 conditional allele. Forty-eight hours after the final in- antibody (α2). T, 10% of input. (C) Subcellular localization of Mdm2, Mdm4, jection, mice were irradiated with 6 Gy IR. Again, no mortality p53515A/515A Mdm4ΔRING/ΔRINGp53515A/515A and p53 in protein lysates from and was observed during the subsequent 50-d observation period. MEFs treated with or without IR. Parp and Gapdh were used as nuclear (N) and Because sublethal doses of IR primarily cause death by hema- cytoplasmic (C) fraction markers, respectively. +/− topoietic failure in Mdm4 mice, these results suggest that the Mdm4ΔRING protein inhibits the lethal activation of p53 dimerization is also dispensable for Mdm2 and p53 degradation in the radiosensitive hematopoietic compartment under stress under genotoxic conditions. conditions. We next analyzed p53 and Mdm2 ubiquitination in the Previous data suggest that an Mdm2 RING mutant that lacks Mdm4ΔRING background. We transfected a hemagglutinin- the E3-ubiquitin ligase activity and the ability to heterodimerize Neo/Neo tagged ubiquitin (HA-ub)–expressing plasmid in p53 and with Mdm4 leads to p53 stabilization (28). Our results here ΔRING/ΔRING Neo/Neo Mdm4 p53 MEFs and analyzed p53 and Mdm2 demonstrate that in the absence of Mdm2-Mdm4 heterodimerization, p53 and Mdm2 degradation rates are not altered. ubiquitination status after adeno-cre–mediated p53 restoration. Taken together, these data imply that p53 degradation by Mdm2 In agreement with the above observations, we found no differ- ubiquitin ligase activity is independent of its association with ences in p53 and Mdm2 ubiquitination between the two geno- Mdm4. Additionally, Mdm2-Mdm4 heterodimerization is nec- types. Similar high molecular weight smears, characteristic of essary for Mdm4 but not Mdm2 degradation. ubiquitination, were observed in the proteasome inhibitor MG132- Interestingly, Mdm4ΔRING protein was stable under DNA- D treated MEFs of both genotypes (Fig. 3 ). damaging conditions. This phenomenon is similar to that observed GENETICS Although Mdm2 and p53 stability was not altered in the with the Mdm4-3SA phosphorylation mutant protein, which Mdm4ΔRING background, the embryonic lethality at E9.5 sug- resists degradation after radiation damage (29). This could explain ΔRING/+ gested enhanced p53 activity during early development. We the lack of radiosensitivity of Mdm4 and tamoxifen-injected flxRING/ΔRING therefore next investigated whether lack of the Mdm4 RING Mdm4 CreER mice after 6 Gy IR exposure. A non- domain also led to similar augmentation of p53 activity in MEFs fi ΔRING/ΔRING Neo/Neo degradable, RING-de cient Mdm4 protein could remain bound generated from E13.5 Mdm4 p53 embryos. We Neo/Neo to p53 and effectively inhibit its transcriptional activity. induced the expression of wild-type p53 in p53 and A short form of Mdm4 (Mdmx-S) has been reported in human ΔRING/ΔRING Neo/Neo Mdm4 p53 MEFs by infecting with adeno-cre normal and cancer cells (30). This short form lacks the RING and performed RT–quantitative PCR (qPCR) for p53 targets. domain and thus resembles Mdm4ΔRING. Mdmx-S is amplified Indeed, Mdm2, p21, and Puma transcripts were up-regulated in human cancers such as glioblastoma and soft tissue sarcoma

Pant et al. PNAS | July 19, 2011 | vol. 108 | no. 29 | 11997 Downloaded by guest on September 30, 2021 Mdm4 +/+ +/+ ∆R/∆ R ∆R/∆R +/+ +/+ ∆R/∆R ∆R/∆R A D MG132 _ ++_ _ ++_ ∆ R/∆R +/+ ∆ R/∆R +/+ ∆R/∆R Mdm4 +/+_ _ Ad - Cre ++ + + p53 Ub-Mdm2 Ub-p53 Mdm2 p53 Short Exp Short Exp

Neo/Neo ΔRING/ΔRING Neo/Neo B Time After p53 Mdm4 p53 ) ) )

CHX (Min) % 100 0 10 20 30 45 60 0 10 20 30 45 60 100 100 % % Mdm2 50 50 50 fp53( o Mdm4 10 10 10 mount of Mdm2 ( mount of Mdm4 ( ea p53 ea WT WT WT ative amount -IR -IR -IR l RING RING 1 1 RING 1 Relativ Re Actin 0 10 20 30 4045 0 10 20 30 4045 Relativ 01020304045 Chase (min) Chase (min) Chase (min)

Neo/Neo ΔRING/ΔRING Neo/Neo C Time After p53 Mdm4 p53 CHX (Min) 0 10 20 30 45 60 0 10 20 30 45 60 ) Mdm2 100 1% 00 50 50 fp53(%) Mdm4 o 10 10

p53 mount of Mdm2 ( WT +IR ea WT +IR 1 RING 1 RING

Relative amount 0 10 20 30 40 0 10 20 30 4045 45 Relativ Actin Chase (min) Chase (min)

Neo/Neo Fig. 3. Mdm2-Mdm4 heterodimerization is dispensable for p53 and Mdm2 degradation. (A) PCR genotyping shows recombination efﬁciency in p53 ΔRING/ΔRING Neo/Neo Neo/Neo ΔRING/ΔRING Neo/Neo and Mdm4 p53 MEFs after adeno-cre (Ad-Cre) virus infection. ΔR, ΔRING (B) Adeno-cre–infected p53 and Mdm4 p53 MEFs were treated with cycloheximide ((CHX) 20 μg/mL) and harvested at different time points. WT, wild type; RING, Mdm4ΔRING. (C). Adeno-cre–infected p53Neo/Neo and Mdm4ΔRING/ΔRINGp53Neo/Neo MEFs were treated with 10 Gy IR. Proteins were allowed to accumulate for 4 h, followed by cycloheximide treatment (20 μg/mL) and cells harvested at different time points. (D) p53Neo/Neo and Mdm4ΔRING/ΔRINGp53Neo/Neo MEFs were transfected with HA-ub, followed by infection with adeno-cre virus. Protein lysate was immunoprecipitated with HA-conjugated beads and immunoblotted with p53 and Mdm2 antibodies.

and correlates with poor prognosis (31, 32). The consequences cassette was included for negative selection (Fig. 1A). The targeting con- of increased levels of Mdmx-S in these patients after DNA- struct was sequenced completely and electroporated into TC1 mouse em- damaging chemo/radiotherapeutic agents may include inhibition bryonic stem cells. G418 resistant clones were analyzed for correct ′ ′ of p53 activity and/or p53-independent activities and warrant homologous recombination by Southern blotting using 5 and 3 external probes (Fig. 1A). Two independent correctly targeted clones were expanded further investigation. ΔRING/+ and injected into C57BL/6 blastocysts to generate Mdm4 chimeras. Our results clearly show that RING domain-mediated Mdm2- Male chimeras were backcrossed to C57BL/6 mice to obtain germline trans- Mdm4 heterodimerization is critical for regulating p53 activity mission of the mutant allele. during early embryogenesis. However, during later develop- mental stages and adult life this interaction becomes dispensable Mouse Breeding, Maintenance, and Genotyping. All mice were maintained in or redundant. In stark contrast to Mdm2 deletion, the relatively >90% C57BL/6 background. All mouse studies were conducted in compliance minor nonlethal phenotypes observed with deletion of Mdm4 in with Institutional Animal Care and Use Committee protocols. Before neomycin adult animal tissues also support this notion (33). In contrast to cassette deletion, mouse genotyping was done by PCR amplification using loss of Mdm4 function as studied in these animal models, am- primer sets 5′-cta gtg aga cgt gct act tc-3′ and 5′-gga gag atg tac acc tgt gt-3′. plification of Mdm4 occurs in multiple human tumors, and many The neomycin selection cassette was deleted by crossing germline transmitted Zp3-Cre of these retain wild-type p53 (27, 34–36). These data support the F1 generation mice with deleter mice (37). Subsequently, genotyping was carried out by PCR amplification with primer sets 5′-ggc aac tcc aga taa importance of Mdm4 in regulating p53 under tumor and non- cta cc-3′ and 5′-cag tac ctc ttg ctt gga g-3′ and resolved on an agarose gel. physiological stress conditions. Collectively, these results suggest that Mdm4 primarily acts as MEF Preparation and Cell Culture. Embryonic day 13.5 embryos were used to a cofactor with Mdm2 to inhibit p53 during embryogenesis but is generate MEFs, which were maintained in DMEM (Invitrogen) supplemented dispensable for regulating p53 and Mdm2 stability in the adult with 10% FBS, penicillin (100 IU/mL), and streptomycin (100 μg/mL). Early- under homeostatic conditions. passage MEFs (P2–P3) were used for analysis.

Materials and Methods Protein Analysis. MEFs were lysed in Nonidet P-40 buffer. Protein estimation Generation of Mdm4ΔRING/+ Mice. A loxP ﬂanked neomycin cassette and an was carried out with Bicinchoninic Acid (BCA Protein Assay Kit; Pierce). Ap- additional copy of exon 11 lacking the RING domain was inserted between proximately 100 μg of lysate was resolved on 8% SDS/PAGE and Western blotted the endogenous exon 11 and the 3′ UTR of the Mdm4 gene. Additionally, with antibodies against p53 (CM5; Vector Laboratories, 1:1,000), Mdm2 (2A10; a loxP sequence was placed upstream of the endogenous Mdm4. Hsv-Tk1 Calbiochem, 1:500), Mdm4 (MX82; Sigma, 1:500), S15-p53 (9284; Cell Signaling

11998 | www.pnas.org/cgi/doi/10.1073/pnas.1102241108 Pant et al. Downloaded by guest on September 30, 2021 Mdm2 p21 Puma A 3.5 12.5 p53N/N 8 p53N/N + Ad Cre 7 3.0 10.0 N/N ΔRΔR p53 6 2.5 ΔRΔRp53N/N + Ad Cre 7.5 5 2.0 4 1.5 5.0 3 1.0 2 2.5 1 0.5 Fold mRNA Induction Fold mRNA Induction 0.0 Fold mRNA Induction 0 0.0

Neo/Neo ΔRING/ΔRING Neo/Neo flxNeo/ΔRING p53 Mdm4 p53 Mdm4 CreER B ______C _ D 100 + + + + + + Ad -Cre + + + Tam 2.5 RING p53 Neo 2.0 75 pS15-p53 1.5 50 p21 1.0 Mdm4 +/+ (15) Mdm4ΔRING/+ (17) 0.5 25 +/– Puma Mdm4 (15) Percent survival Percent

Relative mRNA Levels 0.0 Actin Oil Tam Oil Tam Oil Tam Oil Tam 0 10 20 30 40 50 Cyclin G1 Mdm2 p21 Puma Days Post 6Gy IR

Neo/Neo N/N Fig. 4. RING domain deletion does not promote lethal p53 activation in adult mouse tissues. (A) Real-time PCR for p53 targets in p53 (p53 )and Mdm4ΔRING/ΔRINGp53Neo/Neo (ΔRΔRp53N/N) MEFs after adeno-cre (Ad-Cre) infection. Data were normalized to expression in untreated p53Neo/Neo controls and represent a mean of three independent experiments ± SEM. (B) Western blot analysis from adeno-cre–treated p53Neo/Neo and Mdm4ΔRING/ΔRINGp53Neo/Neo ﬂxRING/ΔRING MEFs. (C) Five-week-old Mdm4 :CreER mice were injected with either corn oil or tamoxifen. RNA from spleen was used for RT-qPCR analyses for p53 targets. Data were normalized to expression levels in oil-injected controls, n =3,±SEM. Inset: Homologous recombination after tamoxifen (Tam) injection. (D) +/+ +/− ΔRING/+ Kaplan-Meyer survival curve of Mdm4 , Mdm4 , and Mdm4 mice after 6 Gy irradiation.

Technology, 1:1,000), vinculin (V9131; Sigma, 1:1,000), and β-actin (AC15; Sigma, was determined by PCR ampliﬁcation of genomic DNA and subsequent 1:5,000). Two milligrams of protein was used for immunoprecipitation with the densitometric ratio analysis of bands resolved on agarose gels. same antibodies. Subcellular fractionation was carried out using a nuclear extract kit (Active Motif, #40010) according to the manufacturer’sprotocol. Quantitative RT-PCR. RNA was isolated from tissues/MEFs using TRIzol (Invi- trogen). One microgram of RNA was reverse-transcribed to cDNA (First Strand p53Neo/Neo Mdm4ΔRING/ΔRINGp53Neo/Neo Ubiquitination Assay. and MEFs were Synthesis Kit; GE Healthcare). First-strand reaction was diluted 10-fold, and transfected with HA-ub plasmid (kind gift from Dr. M. H. Lee, M.D. Anderson 2 μL of the diluted reaction was used in qPCR reaction for p53 targets in an Cancer Center, Houston, TX). After 48 h cells were infected with adeno-cre ABI 7900HT real-time PCR machine. Primer sequences have been previously virus [100 multiplicity of infection (MOI)] and harvested 24 h later. Protein was described (38). immunoprecipitated with anti-hemagglutinin antibody-tagged protein-A agarose beads (Sigma, #E6779), resolved on 8% SDS/PAGE, and immuno- IR Studies. Mice were irradiated at 6 Gy in a cesium-137 irradiator and blotted with p53 or Mdm2 antibody. monitored for 50 d. MEFs were cultured in a 100-mm tissue culture dish, Neo/Neo ΔRING/ΔRING Neo/Neo irradiated with 10 Gy IR, and incubated at 37 °C before harvesting at dif- Protein Stability Assay. p53 and Mdm4 p53 MEFs were infected with adeno-cre for 24 h before treatment with cycloheximide (20 ferent time points for experimental analyses. μg/mL). Cells were harvested at different time points and protein analyzed by Western blotting. ACKNOWLEDGMENTS. We thank Drs. Sean Post and James Jackson for critical reading of the manuscript. Mice were made by the Genetically Engineered Mouse Facility at The M. D. Anderson Cancer Center. Studies Conditional Recombination of Allele. Conditional recombination of the mouse were supported by National Institutes of Health Grant CA47296 (to G.L.). allele was carried out by immunoprecipitation. Tamoxifen injection was as V.P. has been supported in part by Molecular Genetics of Cancer Training previously described (26). Recombination in MEFs was achieved by overnight Grant CA009299 and is a recipient of fellowships from the Dodie P. Hawn adeno-cre virus infection (100 MOI) to the cells. The rate of recombination and Lupe C. Garcia Foundations.

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