Trim24 targets endogenous p53 for degradation

Abstract

Numerous studies focus on the tumor suppressor p53 as a protector of genomic stability, mediator of cell cycle arrest and apoptosis, and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and regulation, comes from studies of tumor-derived, cultured cells where p53 and its regulatory controls may be mutated or dysfunctional. To address regulation of endogenous p53 in normal cells, we created a mouse and stem cell model by knock-in (KI) of a tandem-affinity-purification (TAP) epitope at the endogenous Trp-53 locus. Mass spectrometry of TAP-purified p53-complexes from embryonic stem cells revealed Tripartite-motif protein 24 (Trim24), a previously unknown partner of p53. Mutation of TRIM24 homolog, bonus, in Drosophila led to apoptosis, which could be rescued by p53-depletion. These in vivo analyses establish TRIM24/bonus as a pathway that negatively regulates p53 in Drosophila. The Trim24-p53 link is evolutionarily conserved, as TRIM24 depletion in human breast cancer cells caused p53-dependent, spontaneous apoptosis. We found that Trim24 ubiquitylates and negatively regulates p53 levels, suggesting Trim24 as a therapeutic target to restore tumor suppression by p53.

Fig. 1.

Knock-in of a C-terminal TAP-tag does not disrupt regulatory functions of p53. (A) Targeting strategy: Homologous recombination and targeting, as shown, generated a C-terminal TAP tag fusion of p53 by inserting a loxP-flanked PGK-neo cassette into intron 10 of p53 and a TAP-tag cassette in 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 create p53^TAP-KI/+ ES cells (see SI Text for details). (B) Apoptosis in vivo: IHC of e13.5 CNS (WT, p53^TAP-KI/+, p53^{TAP-KI/TAP-KI}) for cleaved caspase 3 to indicate apoptosis, without (no IR) or with exposure to γ-IR (5 Gy IR).

Fig. 2.

Trim24 is a negative regulator of p53. (A) RNAi-mediated depletion of Trim24 led to increased p53 protein in parallel with decreased Trim24 protein in ES cells (shTrim24 and shControl). (B) TRIM24 interacts with p53 in human cells. Cell lysates of U2OS, HEK293T, or MCF7 cells (Input) were used for IP of endogenous TRIM24, Flag-TRIM24 (Flag) and/or p53 after Dox (0.5 μg/mL) or without Dox induction of stress for the indicated time period (hr). Flag-TRIM24 was ectopically expressed (+ Flag-TRIM24) in U2OS and HEK293T cells and analyses performed 24 h later. Immunoblotting was conducted with indicated antibodies after SDS/PAGE to detect interacting proteins, as indicated. (C) Loss of Trim24 led to activation of specific p53-target genes in ES cells. Significant changes in RNA expression, determined by real-time RT-PCR, are indicated by P values less than 0.05 (Student's t test). Values are expressed as fold-change in comparison to siControl-treated cells at t = 0.

Fig. 3.

Loss of Trim24 causes apoptosis in Drosophila. (A) bonus mutant clones [marked by GFP (A)] in Drosophila wing imaginal discs are small due to apoptosis (Cas3*-labeling). (B) RNAi-mediated depletion of D-p53 in bonus mutant clones (marked by GFP) suppresses apoptosis (lack of Cas3*-labeling) and leads to significant increase in clone size. Aiii and Biii show the outline of the disc. (Scale bar, 20 μm.)

Fig. 4.

TRIM24 negatively regulates p53 protein stability. (A) Depletion of TRIM24 in MCF7 cells increases apoptosis and decreases mitosis. Antibodies recognizing cleaved lamin-A (top) to detect apoptosis (green), or phospho-histone H3 (bottom) to detect cells in mitosis (blue), were used after transfection with siControl (left) and siTRIM24 (right) oligos. Cells were additionally stained with Hoescht followed by automated image acquisition and analysis. (B) p53-half-life is decreased by TRIM24 expression. MCF7 cells were treated with cycloheximide for 0, 15, 30, 60, 120, and 240 min (upper, vector only transfected) without DNA damage (–Dox) or with DNA damage (+Dox); and (lower, Flag-TRIM24) transfected with Flag-TRIM24 followed by similar treatment. p53, TRIM24 and Flag-TRIM24 protein levels were analyzed by immunoblotting, quantified by densitometry, and plotted against time to determine p53-half-lives. (C) Endogenous p53-ubiquitination assay: MCF7 cells were transfected with Mdm2, Flag-TRIM24, Flag-TRIM24ΔRing, siTRIM24, or vector control, as indicated. p53 was immunoprecipitated from lysates of transfected MCF7 cells, treated with MG132 (+) or untreated (-), and immunoblotted with anti-ubiquitin antibody to capture polyubiquitinated p53 and anti-p53 antibody to indicate levels of p53. (D) In vitro ubiquitination of p53: In vitro ubiquitination reactions were performed on in vitro translated ³⁵S-p53 bound to hTRIM24 (+:20 μg, and ++:40 μg, estimated by staining) in the presence or absence of UbE1, UbcH8, and 25 μM zinc as indicated. Reactions were analyzed by autoradiogram.