Trim24 Targets Endogenous P53 for Degradation
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Trim24 targets endogenous p53 for degradation Kendra Alltona,b,1, Abhinav K. Jaina,b,1, Hans-Martin Herza, Wen-Wei Tsaia,b, Sung Yun Jungc, Jun Qinc, Andreas Bergmanna, Randy L. Johnsona,b, and Michelle Craig Bartona,b,2 aDepartment of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences and bCenter for Stem Cell and Developmental Biology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; and cDepartment of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030 Edited by Carol L. Prives, Columbia University, New York, NY, and approved May 15, 2009 (received for review December 23, 2008) Numerous studies focus on the tumor suppressor p53 as a protector regulator of p53 suggests that potential therapeutic targets to of genomic stability, mediator of cell cycle arrest and apoptosis, restore p53 functions remain to be identified. and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and Results and Discussion regulation, comes from studies of tumor-derived, cultured cells Creation of a Mouse and Stem Cell Model for p53 Analysis. To where p53 and its regulatory controls may be mutated or dysfunc- facilitate analysis of endogenous p53 in normal cells, we per- tional. To address regulation of endogenous p53 in normal cells, formed gene targeting to create mouse embryonic stem (ES) we created a mouse and stem cell model by knock-in (KI) of a cells and mice (12), which express endogenously regulated p53 tandem-affinity-purification (TAP) epitope at the endogenous protein fused with a C-terminal TAP tag (p53-TAPKI, Fig. 1A) Trp-53 locus. Mass spectrometry of TAP-purified p53-complexes (13). Germline transmission of the p53-TAPKI allele was con- from embryonic stem cells revealed Tripartite-motif protein 24 firmed by Southern hybridization and allele-specific PCR ( Fig. (Trim24), a previously unknown partner of p53. Mutation of S1A). p53-TAPKI remains entirely responsive to exogenous and TRIM24 homolog, bonus,inDrosophila led to apoptosis, which endogenous stimuli in p53TAP-KI/ϩ ES cells and mice. The could be rescued by p53-depletion. These in vivo analyses establish induction of apoptosis (Fig. S1B) and expression levels of TRIM24/bonus as a pathway that negatively regulates p53 in p53-regulated genes, Cdkn1a (p21/CIP1), Mdm2, and Perp (Fig. Drosophila. The Trim24-p53 link is evolutionarily conserved, as S1C), were virtually identical over a time course of doxorubicin TRIM24 depletion in human breast cancer cells caused p53-depen- exposure in p53-TAPKI and wild-type ES cells. Chromatin dent, spontaneous apoptosis. We found that Trim24 ubiquitylates immunoprecipitation (ChIP) showed that p53-TAPKI and p53 and negatively regulates p53 levels, suggesting Trim24 as a ther- proteins are identical in their interactions with endogenous apeutic target to restore tumor suppression by p53. p53-response elements of specific genes (Fig. S1D). In ES cells, p53-mediated apoptosis is independent of tran- apoptosis ͉ bonus ͉ TAP ͉ ubiquitylation scription, as it is primarily driven by p53-mitochondrial interac- tions (14). Therefore, we performed analyses in vivo to assess lthough intensively studied in tumor cells, the mechanisms p53-TAPKI response to stress, where downstream functions Athat regulate tumor suppressor p53 in normal cells remain of p53 are primarily driven by p53-regulated transcription. poorly understood. Biochemical analysis of endogenous p53 p53TAP-KI/ϩ mice were intercrossed to obtain viable, homozygous poses a challenge, as p53 is held at low levels in non-transformed p53-TAPKI (p53TAP-KI/TAP-KI) mice, which were born at the cells in the absence of stress (1). Mutant p53 generally exhibits expected ratio of genotypes and show no tumor development 1 increased protein stability in tumor-derived cells (2, 3). This year after birth (Fig. S1E). We exposed embryonic day 13.5 offers opportunities for molecular analysis, although of a protein (e13.5) embryos, of wild-type, heterozygous, and homozygous that may display loss- or gain-of-function (4). Indeed, several p53/TAPKI genotype, to ␥-irradiation to induce an apoptotic assumptions about p53 regulation, including the relative impor- response that is p53-dependent (15). Sections of the central tance of specific post-translational modifications, sites of muta- nervous system (CNS) and immunohistochemistry (IHC) for tion, and interactions with regulatory proteins, have been chal- activated, cleaved caspase 3 showed that p53-TAPKI mounted an lenged in mouse models where in vivo functions of p53 can be apoptotic response in vivo, indistinguishable from wild-type p53 determined (3, 5–7). (Fig. 1B). In addition to its principle role in maintenance of genomic To regulate apoptosis and multiple downstream pathways, p53 stability and tumor suppression, p53 regulates specific functions forms a tetrameric complex (16). Endogenous p53-TAPKI and in normal cells (1). These include regulation of gene expression wild-type p53 expressed in p53TAP-KI/ϩ ES cells formed mixed during development (8, 9), cellular progression to senescence hetero-tetramer p53-TAPKI/wild-type p53 protein complexes, and aging (10), and metabolic functions (6). Although a fractionating between 200 kDa and Ͼ1 MDa by size-exclusion p53ERTAM mouse model, where p53 function and response may chromatography (Fig. S1F). Taken together, these data show be temporally regulated (11), has been invaluable, questions that TAP-fusion to p53 and formation of heteromeric, p53 regarding endogenous regulation and control of p53, before protein complexes did not disrupt p53-functions in transcription stress stimuli, transformation, or tumorigenesis, remain unan- and apoptosis. swered. To elucidate mechanisms that regulate endogenous p53, we developed a mouse and stem cell model based on TAP-tag fusion with the ORF of Tpr53, which allows TAP-purification Author contributions: A.K.J., A.B., R.L.J., and M.C.B. designed research; K.A., A.K.J., H.-M.H., and analysis of p53 protein partners and post-translational W.-W.T., S.Y.J., and R.L.J. performed research; K.A., A.K.J., H.-M.H., J.Q., A.B., R.L.J., and modifications by mass spectrometry. Here, we describe a M.C.B. analyzed data; and A.K.J., A.B., and M.C.B. wrote the paper. protein-interaction partner of p53 that negatively regulates p53 The authors declare no conflict of interest. protein stability by ubiquitylation. We addressed the in vivo This article is a PNAS Direct Submission. significance of Trim24/bonus-p53 interaction in Drosophila, and See Commentary on page 11431. uncovered a negative regulator of p53 levels in this organism. 1K.A. and A.K.J. contributed equally to this work. Human tumor-derived cancer cells, which retain wild-type TP53, 2To whom correspondence should be addressed. E-mail: [email protected]. respond to siRNA-targeted depletion of TRIM24 by undergoing This article contains supporting information online at www.pnas.org/cgi/content/full/ apoptosis. This identification of a previously unknown, negative 0813177106/DCSupplemental. 11612–11616 ͉ PNAS ͉ July 14, 2009 ͉ vol. 106 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813177106 Downloaded by guest on September 23, 2021 S3A) or lacking (Fig. S2D) p53 did not show significant effects A of Trim24 depletion, supporting the p53-dependence of Trim24- mediated regulation. SEE COMMENTARY An In Vivo Analysis of the Trim24-p53 Regulatory Pathway. Mamma- lian Trim24 is 1 of 3 members of the TIF1 subfamily within the larger TRIM group of 70 family members (25). Protein-protein interactions between TRIM family members are known, and siRNA depletion of Trim24, Trim28, and Trim33 (TIF1␣, TIF1ß, and TIF1␥, respectively) revealed compensation, which may alter Trim24-regulated responses. Mammalian Trim28 (Kap1, TIF1ß) has known roles in epigenetic silencing, E3-SUMO- ligase activity and interacts with several proteins, including TAP-KI/+ TAP-KI/TAP-KI B WT p53 p53 Mdm2 and E2F1 (26–28). To determine if Trim24 specifically regulates p53 in vivo, we turned to Drosophila where the gene bonus is the single, evolutionarily conserved homologue of the TIF1 subfamily (29), and most similar to TRIM24 and TRIM33. Bonus is a member of the Polycomb group of genes, and is required for male viability and metamorphosis (29). By mosaic No IR analysis in imaginal discs, we found that clones of cells mutated in bonus, marked by green fluorescent protein (GFP, Fig. 3A), are small and highly apoptotic (Fig. 3Ai, cleaved caspase 3- positive). RNAi-mediated depletion of Drosophila p53 (D-p53) rescued the bonus apoptotic phenotype, resulting in considerably larger bonus clones and an absence of caspase 3 cleavage (Fig. 3B). Similar rescue was achieved by a dominant-negative form of 5 Gy IR mutant D-p53. These observations suggested that loss of bonus causes unrestrained D-p53 activity and induces apoptosis. Thus, bonus may be the principle negative regulator of D-p53 activity Fig. 1. Knock-in of a C-terminal TAP-tag does not disrupt regulatory func- and cell death in tissues of Drosophila, which has no ortholog of tions of p53. (A) Targeting strategy: Homologous recombination and target- Mdm2 or previously identified pathway regulating p53 protein ing, as shown, generated a C-terminal TAP tag fusion of p53 by inserting a stability (30). loxP-flanked PGK-neo cassette into intron 10 of p53 and a TAP-tag cassette in The link between TRIM24/bonus and p53-dependent cell frame with full-length p53 into exon 11. After selection of targeted ES cells, the neo cassette was removed by introduction of a cre-expressing plasmid to death is conserved through evolution. Loss of TRIM24 in the create p53TAP-KI/ϩ ES cells (see SI Text for details). (B) Apoptosis in vivo: IHC of human breast cancer cell line MCF7 (Fig.