Article Figures & Data
Figures
- FIG 1
TRIM24 ubiquitinates p53 independently of MDM2. (A and B) Endogenous p53 ubiquitination. MCF7 cells transiently transfected with TRIM24 and MDM2 plasmids (A) or transiently transfected with control, TRIM24, and MDM2 siRNA (B) were treated with MG132 (+) for 8 h. Total lysates were immunoprecipitated (IP) with p53 antibody and probed for ubiquitin (Ub). Inputs are shown in the bottom panels. WB, Western blotting. (C) TRIM24-MDM2 interaction. MCF7 cells were pretreated with MG132 for 4 h, followed by treatment with 250 ng/ml adriamycin (D) for 1 h or exposed to 10 Gy ionizing radiation (IR) and harvested after 1 h. Total cell lysates were immunoprecipitated with either TRIM24 or MDM2 antibody and immunoblotted. IgG served as a negative control for the immunoprecipitation reactions. (D) TRIM24-MDM2 interaction. Total lysates from U2OS cells transfected with pCMV-MDM2 were immunoprecipitated with MDM2 antibody and immunoblotted for MDM2, TRIM24, and p53.
- FIG 2
DNA damage induces degradation of TRIM24 in the nucleus. (A and B) MCF7 cells were treated with actinomycin D (10 ng/ml) (A) or high-dose adriamycin (Adr) (500 ng/ml) (B) for the indicated lengths of time, and total TRIM24, MDM2, and p53 were analyzed by Western blotting. (C) MCF7 cells were treated with adriamycin (250 ng/ml) for 4 h, followed by washing the medium and harvesting the cells at the indicated times, and total TRIM24, and p53 were analyzed by Western blotting. Blots were quantitated, and fold changes in total protein levels normalized to actin are shown in parentheses under the blots in panels A to C. (D and E) TRIM24 nuclear localization. Nuclear extracts (Nuc. Ext) prepared from MCF7 cells treated with adriamycin for different time points (0, 15 or 30 min, up to 8 h) in the absence (D) or presence (E) of the nuclear export inhibitor leptomycin B (LMB) were analyzed by Western blotting. Blots were quantitated, and fold changes in nuclear protein levels were plotted in the graphs to the right of the blots. (F and G) TRIM24 nuclear localization. Nuclear extracts prepared from HEK293T cells treated with Adr for different time points in the presence of LMB (F) or the protein synthesis inhibitor cycloheximide (CHX) (G) were analyzed by Western blotting. Blots were quantitated, and fold changes in nuclear protein levels were plotted.
- FIG 3
TRIM24 undergoes ubiquitination-mediated degradation in response to DNA damage. (A) MCF7 cells stably integrated with nontarget shRNA (shControl) or shRNA specific to TRIM24 (shTRIM24) were treated with MG132 for 8 h. Total p53 and TRIM24 were analyzed by Western blotting. (B) TRIM24 and p53 half-lives. MCF7 cells were treated with CHX for the indicated time points without (Blank) or with (Adr) DNA damage. p53 and TRIM24 protein levels were analyzed by immunoblotting, quantified by densitometry, and plotted against time to determine TRIM24 and p53 half-lives. (C) TRIM24 ubiquitination. MCF7 cells transiently transfected with Flag-TRIM24 and His-Ub plasmids were pretreated with MG132 for 6 h followed by treatment with Adr plus MG132. Total lysates were prepared under denaturing conditions. Equal amounts of lysates were purified on a Ni column and Western blotted with Flag antibody to detect ubiquitinated Flag-TRIM24. The positions of molecular size markers (in kilodaltons) are shown to the left of the gel. (D and E) TRIM24 autoubiquitination. MCF7 cells transiently transfected with Flag-TRIM24, Flag-TRIM24ΔRING, or CMV-MDM2 plus His-Xpress-Ub were treated with MG132 for 8 h. (D) Total cell lysates prepared under denaturing conditions were subjected to Ni-purification followed by Western blotting with Flag antibody to detect ubiquitinated Flag-TRIM24. (E) In a similar experiment, total lysates were immunoprecipitated with Flag antibody, followed by immunoblotting with Xpress antibody to detect ubiquitinated Flag-TRIM24. (F) TRIM24ΔRING half-life. MCF7 cells transfected with Flag-TRIM24ΔRING were treated with CHX for different time points without (Blank) or with (Adr) DNA damage. TRIM24 protein levels were analyzed by immunoblotting with Flag antibody, quantified by densitometry, and plotted against time to determine TRIM24 half-lives.
- FIG 4
Phosphorylation at S768 by ATM kinase induces TRIM24 degradation during DNA damage. (A) ATM phosphorylation sites on TRIM24. Scheme representing two conserved ATM sites (S217 and S768) on TRIM24 proteins. I and II, B-box I and II; BBC, coiled-coil domain. (B) In vitro phosphatase assay. MCF7 cells were treated with Adr for 1 h or exposed to IR and allowed to rest for 1 h. Thirty micrograms of lysate was incubated with shrimp alkaline phosphatase (SAP) and analyzed by Western blotting. (C) TRIM24 phosphorylation. MCF7 cells transfected with Flag-TRIM24 were collected at 30 min and 2 h after IR with or without MG132 treatment. Total lysates were immunoprecipitated with anti-pS/T ATM substrate antibody and blotted with Flag to detect phosphorylated TRIM24. pChk2 was used as a control for ATM activation. Inputs are shown in bottom panels. (D) TRIM24 phosphorylation. MCF7 cells transfected with phosphosite mutants (S217A or S768A) of TRIM24 were treated and analyzed as described for panel C. The immunoprecipitated bands were quantitated, and the percent phosphorylation was plotted in the graph shown below the blots. (E) TRIM24 half-life. MCF7 cells transfected with wild-type or phosphomutants of Flag-TRIM24 were treated with CHX for different time points without (Blank) or with (Adr) DNA damage. TRIM24 protein levels were analyzed by immunoblotting with Flag antibody, quantified by densitometry, and plotted against time to determine TRIM24 half-lives. (F) TRIM24 ubiquitination. MCF7 cells transiently transfected with Flag-TRIM24 or Flag-TRIM24S768A plus His-Ub were treated with MG132. Total cell lysates were subjected to Flag immunoprecipitation followed by Western blotting with ubiquitin antibody to detect ubiquitinated Flag-TRIM24. The total Flag-TRIM24 immunoprecipitated is shown in the bottom blot. (G) TRIM24-S768D half-life. MCF7 cells were transfected with phosphomimic mutant Flag-TRIM24-S768D, and its half-life was assessed as described for panel E.
- FIG 5
DNA damage-induced degradation of TRIM24 is dependent on ATM. (A) Wild-type (ATM+/+) and ATM-null (ATM−/−) fibroblasts were collected 1 h after IR exposure with or without MG132 treatment. p53 and TRIM24 protein levels were analyzed by Western blotting and quantified by densitometry. (B) TRIM24 phosphorylation. Wild-type and ATM-null fibroblasts were exposed to IR in the absence or presence of MG132. Total lysates were immunoprecipitated by anti-pS/T ATM substrate antibody and blotted to detect phosphorylated TRIM24 and pSer15-p53. (C and D) TRIM24 protein levels. Wild-type (ATM+/+) and ATM-null (ATM−/−) fibroblasts were exposed to IR alone, CHX alone, or IR followed by CHX (IR+CHX) and harvested at different time points. TRIM24 and p53 protein were analyzed by Western blotting. The blots were quantified by densitometry, and the average values ± standard errors of the means (error bars) from two experiments are plotted against time.
- FIG 6
p53 activates TRIM24 transcription after DNA damage. (A) Total lysates from MCF7 cells treated with Adr (250 ng/ml) for different lengths of time were immunoblotted to detect TRIM24, MDM2, and p53 protein levels. (B) Total lysates from MCF7 cells treated with Adr for the indicated lengths of time in the absence or presence of CHX were immunoblotted to detect TRIM24 and p53. (C) Quantitative real-time PCR (qRT-PCR) assay. MCF7 cells were transfected with nontarget siRNA (siControl) or siRNA specific to p53 (siTP53) and treated with Adr for different lengths of time. RNA prepared from these cells was subjected to qRT-PCR assay with primers specific for TP53, TRIM24, MDM2, and CDKN1A. (D and E) MCF7 cells stably expressing nontarget shRNA (shControl) or shRNAs specific to p53 (shTP53) (clones 2 and 3) were treated with different doses (0, 100, 250, and 500 ng/ml) of adriamycin for 24 h. Protein levels were analyzed by Western blotting (D), and RNA levels were analyzed by qRT-PCR (E). (F) Trim24 protein levels in Val5 cells 3 and 6 h after a shift to 32°C in the presence of absence of CHX.
- FIG 7
TRIM24 is a direct gene target of p53. (A) p53 ChIP analysis in human cells. p53-bound chromatin was immunoprecipitated from MCF7 cells treated with Adr for 12 h, and p53 enrichment on TRIM24, MDM2, and CDKN1A was analyzed by qPCR using primers encompassing p53REs and plotted as the fold increase in p53 enrichment compared to input. (B) p53 ChIP analysis in mouse ES cells. p53-bound chromatin was immunoprecipitated from mES cells treated with Adr for 5 or 12 h, and p53 enrichment on Trim24, Mdm2, and Cdkn1a was analyzed by qPCR using primers encompassing p53REs and plotted as the fold increase in p53 enrichment compared to input.
- FIG 8
TRIM24 preferentially targets phosphorylated p53. (A to D) TRIM24-p53 interaction. (A) MCF7 cells were transfected with Flag-TRIM24 plasmids, followed by treatment with Adr for different lengths of time. Total cell lysates were immunoprecipitated with Flag antibody and immunoblotted for Flag, p53, and actin. (B) MCF7 cells transfected with Flag-TRIM24 plasmid were exposed to IR. The cell lysates were incubated with calf intestinal phosphatase (CIP) for 1 h and subjected to immunoprecipitation with Flag antibody, followed by immunoblotting with Flag and p53. Inputs were also probed with pSer15-p53 and pCHK2 to confirm dephosphorylation (bottom blots). (C) Lysates from MCF7 cells treated with Adr for different lengths of time were immunoprecipitated with pSer15-p53 antibody and immunoblotted with TRIM24 and pSer15-p53. Inputs are shown in the bottom blots. (D) Lysates from MCF7 cells treated with Adr or nutlin for 24 h were immunoprecipitated with TRIM24 antibody and immunoblotted with TRIM24, p53, and TRIM33. Inputs were also probed with pSer15-p53 to determine p53 phosphorylation (bottom blots). (E) Model demonstrating the autoregulatory feedback loop between p53 and TRIM24. TRIM24 targets endogenous p53 for ubiquitin-mediated degradation. DNA damage activates ATM kinase that phosphorylates p53, resulting in its stabilization and activation. Active p53 then induces transcription of downstream genes, including TRIM24. In parallel, ATM kinase phosphorylates S768 of TRIM24, which dissociates TRIM24 from p53 and targets itself for autodestruction via its RING domain. Further, active p53 directly binds to p53RE on TRIM24, resulting in TRIM24 transcription activation in response to DNA damage. During the postdamage recovery phase, newly synthesized TRIM24 preferentially interacts with phosphorylated p53 and targets it for degradation in order to bring excess p53 back to normal threshold levels, thus completing a negative-feedback loop. P, phosphate group; TSS, transcription start site.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1 and S2 (DNA damage and degradation of TRIM24), S3 (Phosphorylation by ATM kinase and TRIM24 degradation), S4 (ATM and DNA damage-induced degradation of TRIM24), S5 (p53 and TRIM24 transcription after DNA damage), S6 (TRIM24 targeting by p53), and S7 (TRIM24 targeting of phosphorylated p53) and Tables S1 and S2 (Oligonucleotide sequences)
PDF, 8.0M
- Supplemental file 1 -
