Transcriptional regulation of the mdm2 oncogene by p53 requires TRRAP acetyltransferase complexes

Abstract

The p53 tumor suppressor regulates the cellular response to genetic damage through its function as a sequence-specific transcription factor. Among the most well-characterized transcriptional targets of p53 is the mdm2 oncogene. Activation of mdm2 is critical in the p53 pathway because the mdm2 protein marks p53 for proteosome-mediated degradation, thereby providing a negative-feedback loop. Here we show that the ATM-related TRRAP protein functionally cooperates with p53 to activate mdm2 transcription. TRRAP is a component of several multiprotein acetyltransferase complexes implicated in both transcriptional regulation and DNA repair. In support of a role for these complexes in mdm2 expression, we show that transactivation of the mdm2 gene is augmented by pharmacological inhibition of cellular deacetylases. In vitro analysis demonstrates that p53 directly binds to a TRRAP domain previously shown to be an activator docking site. Furthermore, transfection of cells with antisense TRRAP blocks p53-dependent transcription of mdm2. Finally, using chromatin immunoprecipitation, we demonstrate direct p53-dependent recruitment of TRRAP to the mdm2 promoter, followed by increased histone acetylation. These findings suggest a model in which p53 directly recruits a TRRAP/acetyltransferase complex to the mdm2 gene to activate transcription. In addition, this study defines a novel biochemical mechanism utilized by the p53 tumor suppressor to regulate gene expression.

FIG. 1.

Expression of the TRRAP cofactor stimulates transcription of the mdm2 promoter. (a) 293 cells were transiently transfected with a reporter plasmid in which the 840 bp flanking the murine mdm2 intron 1 promoter drives expression of the luciferase gene. In addition, cells were cotransfected with a CMV-driven expression vector encoding acetyltransferase complex subunit TRRAP (dark bars). Control transfections included the empty CMV expression vector (light bars). Where indicated, cells were transfected with a CMV-driven p53 expression vector or its empty vector control. Cells were harvested 24 h posttransfection, and luciferase activity was determined and expressed as relative light units (RLU). Transfections included 300 ng of mdm2 promoter-driven reporter plasmid and the indicated amounts of TRRAP expression vector. In addition, a parallel transfection included 5 μg of p53 expression vector. (b) 293 cells were transfected with expression vectors for either TRRAP (15 μg) or p53 (4 μg) as indicated. Cells were harvested as in panel a, and lysates were resolved by SDS-PAGE, blotted, and probed for mdm2. Migration of a size marker is indicated at left in kilodaltons. (c) Luciferase reporters driven by the promoters of the indicated cellular genes (500 ng) were cotransfected with 4 μg of the CMV-driven TRRAP expression vector (dark bars) or its empty vector control (light bars). Paired bars represent data from duplicate transfections.

FIG. 2.

Activation of mdm2 transcription by TRRAP requires p53. (a) A luciferase reporter (500 ng) driven by a fragment of the human mdm2 promoter encompassing the intron 1 p53 response element was transfected into 293 cells (left). Parallel transfections were performed with an isogenic reporter in which the p53 response element was deleted by a 5′ truncation (right). Transfections also included the TRRAP expression vector or its empty vector control as indicated. Transfections were performed in duplicate and included increasing amounts of plasmid (1, 3, and 7 μg). Positive-control transfections included the mdm2 reporter and a p53 expression vector (5 μg). Luciferase activity was assayed and expressed as relative light units (RLU). (b) p53-negative cell line H1299 was transfected as indicated. Cells were transfected with the murine mdm2 promoter-luciferase reporter (300 ng) used in Fig. 1a and CMV-driven expression vectors for either TRRAP (3 μg) or p53 (1 μg). Control transfections included appropriate amounts of empty CMV vector. Duplicate transfections were performed, and cells were harvested 24 h posttransfection.

FIG. 3.

Expression of antisense TRRAP blocks p53-mediated transcription of mdm2. 293 cells were transiently transfected with the murine mdm2 promoter-luciferase reporter described for Fig. 1 (500 ng). Transfections also included 1 μg of a CMV-driven expression vector for p53 or an empty vector control as indicated. The p53 expression vector was cotransfected with increasing amounts of a CMV-driven expression vector for antisense TRRAP or the empty vector control (1 and 3 μg), as indicated. At 24 h posttransfection, cells were lysed and luciferase activity was determined.

FIG. 4.

The ATM homology domains of TRRAP are required for optimal mdm2 transcription. (a) The TRRAP protein shares three carboxy-terminal domains of homology with the other ATM family proteins, FAT, PIK-like, and FATC. Carboxy-terminal truncation TRRAP mutants were created from a CMV expression vector. Numbers at left indicate the TRRAP amino acids encoded by each construct, with the 1-3830 construct representing full-length TRRAP. (b) Cotransfection of 293 cells with the mdm2 luciferase reporter (300 ng) and the TRRAP expression vectors (5 μg) diagrammed in panel a. A control transfection included the mdm2 reporter and a CMV-driven p53 vector (1 μg). Transfections were performed in duplicate, and bars represent averaged luciferase activity expressed as relative light units (RLU). (c) Western blot of protein expression levels for the TRRAP truncation mutants used in panel b. After transfection, 293 cells were lysed and immunoprecipitations were performed with anti-FLAG antibodies. Precipitates were resolved by SDS-4 to 12% PAGE. After being transferred, the blot was probed for the FLAG epitope common to all the TRRAP mutants. Migration of size standards is indicated at left in kilodaltons. The position of the heavy chain from the immunoprecipitating antibody is also indicated (IgH).

FIG. 5.

Inhibition of cellular deactylases augments mdm2 transcription. H1299 cells were cotransfected with the mdm2 luciferase reporter (500 ng) and a p53 expression vector (3 μg) or empty vector as indicated. Following transfection, cells were treated for 16 h with sodium butyrate (100 μM or 1 mM) or left untreated. Transfections were performed in duplicate as represented by each pair of bars. Cells were lysed 16 h posttransfection, and luciferase activity was determined.

FIG. 6.

Carboxy-terminal acetylation of p53 is not required for synergy with TRRAP. (a) The p53 protein contains multiple carboxy-terminal lysines which are targets for acetylation by HAT PCAF/hGCN5 or p300/CBP (asterisks). To eliminate potential effects of acetylation, these and adjacent lysines were mutated to arginine, resulting in the compound mutant designated 9KR. The lysines mutated represent amino acids 319, 320, 321, 370, 372, 373, 381, 382, and 386 in human p53. (b) p53-negative H1299 cells were cotransfected with the mdm2 luciferase reporter (300 ng) and expression vectors for TRRAP (5 μg) and either wild-type (wt) or mutant (9KR) p53 (1.5 μg) as indicated. Paired bars represent data from duplicate transfections, and control transfections included equivalent amounts of appropriate empty vectors. (Inset) Western blots of H1299 lysates from parallel transfections were probed for p53 to determine protein expression levels. Migration of size markers is indicated at right in kilodaltons. (c) ChIP analysis was performed on 293 cells after transfection with the murine mdm2 promoter-luciferase construct and expression vectors for wild-type or 9KR versions of p53, as indicated. DNA/protein complexes were immunoprecipitated with antibodies against p53 (lanes 3, 6, and 9) or control antibodies (lanes 2, 5, and 8). Precipitates were washed, cross-linking was reversed, and DNA was purified. PCR was then performed using primers that flank the p53 binding sites in the transfected mdm2 promoter. A parallel PCR was performed using purified DNA from each of the three transfection groups that was not subjected to immunoprecipitation (lanes 1, 4, and 7). Migration of a 564-bp size marker is shown for reference (marker). (d) H1299 cells were transfected with a p53 expression vector (5 μg) and two concentrations of vectors encoding FLAG epitope-tagged versions of either CBP (lanes 2 and 3) or TRRAP (lanes 4 and 5). A control transfection included the empty expression vector at the higher dose (lane 1). Cell lysates were produced and either resolved directly or subjected to immunoprecipitation with antisera directed against acetylated lysine residues. After Western blotting, lysates were probed for the FLAG epitope shared by CBP and TRRAP (top) or for p53 (middle). Antiacetyllysine immunoprecipitates were also probed for p53 (bottom).

FIG. 7.

p53 directly binds to TRRAP in vitro and in vivo. (a) Fragments of TRRAP spanning the entire protein were expressed as GST fusions in Escherichia coli. The p53 binding domain defined in panel c is indicated. (b) Purified GST-TRRAP fusion proteins were resolved by SDS-PAGE, blotted, and probed for GST to document relative expression levels and confirm predicted molecular weights. The TRRAP amino acids encompassed in each fusion protein are indicated. (c) After purification, GST-TRRAP fusion proteins were used as bait in in vitro pull-down reactions. FLAG-tagged p53 expressed and affinity purified from baculovirus-infected cells served as the target protein in these reactions. After a washing, binding reaction mixtures were resolved by SDS-PAGE, blotted, and probed for p53. (d) Nondenaturing lysates of 293 cells were subjected to immunoprecipitation using antibodies directed against TRRAP or a control antibody. Precipitates were resolved, blotted, and probed for both TRRAP (top) and p53 (bottom). Migration of size markers is indicated at left in kilodaltons.

FIG. 8.

p53 mediates recruitment of TRRAP to the endogenous mdm2 promoter in vivo. (a) ChIP analysis was performed on 293 cells after formaldehyde cross-linking. DNA/protein complexes were immunoprecipitated with antibodies against TRRAP (lane 3) or control antibodies (lane 2). Precipitates were washed, cross-linking was reversed, and DNA was purified. PCR was then performed using primers which flank the p53 binding sites in the endogenous human mdm2 promoter. A parallel PCR was performed using purified DNA that was not subjected to immunoprecipitation (lane 1). The position of the 397-bp mdm2 promoter PCR product is indicated at the right. IgG, immunoglobulin G. (b) H1299 cells were rescued for p53 expression by infection with a p53-expressing adenovirus (lane 2). As a control, a mock rescue was performed by infection with a GFP-expressing adenovirus (lane 1). Whole-cell lysates from infected H1299 cells were resolved by SDS-PAGE and transferred to membranes. Blots were probed with antibodies against p53 or the mdm2 protein. As a loading control, blots were probed for the constitutively expressed MAX protein. (c) ChIP analysis was performed on H1299 cells in which p53 expression was rescued by adenovirus infection as in panel b. Again, mock-rescued cells served as a control. Immunoprecipitation of DNA/protein complexes was performed by using an anti-p53 antibody or a control antibody. As above, precipitated DNA was purified and subjected to PCR to amplify the mdm2 promoter region containing the p53 binding sites. DNA purified (but not immunoprecipitated) from mock- and p53-rescued cells (input; bottom) was subjected to PCR for the same region of the mdm2 promoter. (d) ChIP and PCR were performed as in panel a using antibodies against TRRAP or a control antibody. Analysis was performed on p53- (lanes 1 and 2) and mock-rescued cells (lanes 3 and 4). The position of the amplified mdm2 promoter fragment is indicated at right, and the migration of the 564-bp size marker is indicated at the left.

FIG. 9.

TRRAP expression results in increased acetylation of histones H3 and H4 at the mdm2 promoter. 293 cells were transfected with the murine mdm2 promoter luciferase construct and a CMV-driven TRRAP expression vector (or empty vector), as indicated. ChIP analysis was then performed as for Fig. 6c using antibodies against TRRAP (right) or acetylated forms of histone H3 (left) or H4 (middle). The ChIP PCR products generated from the transfected mdm2 promoter were resolved by agarose gel electrophoresis, stained with ethidium bromide, quantitated, and normalized to input DNA. The resulting values were expressed graphically.