hADA3 is required for p53 activity - PubMed (original) (raw)

hADA3 is required for p53 activity

T Wang et al. EMBO J. 2001.

Abstract

The tumor suppressor protein p53 is a transcription factor that is frequently mutated in human cancers. In response to DNA damage, p53 protein is stabilized and activated by post-translational modifications that enable it to induce either apoptosis or cell cycle arrest. Using a novel yeast p53 dissociator assay, we identify hADA3, a part of histone acetyltransferase complexes, as an important cofactor for p53 activity. p53 and hADA3 physically interact in human cells. This interaction is enhanced dramatically after DNA damage due to phosphorylation event(s) in the p53 N-terminus. Proper hADA3 function is essential for full transcriptional activity of p53 and p53-mediated apoptosis.

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Figures

Fig. 1. The p53 dissociator assay in S.cerevisiae and p53 dissociators. (A) SV40 large T antigen functions as a p53 dissociator. Independent transformants of RBy41 (expressing wild-type p53), containing either a plasmid for SV40 large T antigen (pTD1-1) or a control plasmid, were replica plated from SC-His-Leu to SC-His-Leu + 0.1% FOA at 30°C and evaluated after 3 days. (B) p53 dissociator screens using the improved diploid p53 reporter strain. The use of a diploid p53 reporter strain with two genomic copies each of the p53 expression cassette and the URA3 reporter gene (RBy99) eliminated 96% of the background of false-positives encountered with RBy41. For library screens, cDNA expression library plasmids were transformed into RBy99, transformants were placed on plates selecting for the plasmids (SC-His) and replica plated (‘RP’) after 3–5 days to plates containing FOA (SC-His + 0.1% FOA). Over the next 2–7 days, FoaR clones emerged which were then analyzed further as described in the text. (C) Mdm2 isolated as a p53 dissociator in a p53 dissociator screen. A p53 dissociator screen with a murine pre-B-cell cDNA expression library isolated four identical plasmids coding for Mdm2. RBy99 containing either the Mdm2 expression plasmid or control plasmid pPC86 was replica plated from SC-Trp to SC-Trp + 0.1% FOA at 30°C, and growth was evaluated after 3 days. (D) hADA3 and 53BP1 isolated as p53 dissociators in a p53 dissociator screen. RBy99 containing HeLa cDNA library clones for 53BP1 and hADA3 were replica plated from SC-His to SC-His + 0.075% FOA at 30°C, and growth was evaluated after 3 days. p2.5 was used as vector control.

Fig. 2. hADA3 interacts with components of HAT complexes. (A) Comparison of hADA3 with yAda3p and versions of hADA3 used in this study. Based on studies for yAda3p, the N-terminal half of hADA3 is predicted to interact with transcription factors, while the C-terminal half is predicted to interact with hADA2 and other members of HAT complexes. Besides full-length hADA3 and N-terminally truncated hADA3 from the HeLa library, the N- and C-terminal halves alone were also used in this study. (B) The C-terminal half of hADA3 interacts with hADA2 in yeast two-hybrid assays. PJ69-4A containing the indicated plasmid combinations were grown on SC-Leu-Trp plates and replica plated to SC-His at 30°C to evaluate activation of the reporter gene HIS3. Vector controls contained the GAL4 DNA-binding domain (GAL4-DBD) or GAL4 transactivation domain (GAL4-TAD) alone. Not all combinations of hADA2 and hADA3 could be evaluated, since full-length hADA3 and hADA3(aa1–214) fused to the GAL4-DBD activated the HIS3 reporter gene (data not shown). (C) The C-terminal half of hADA3 is in complexes with p300. FLAG-tagged full-length hADA3, the N-terminal half of hADA3 or the C-terminal half with NLS were co-expressed with p300 (CMVβ-p300) in 293 cells. After in vivo cross-linking with DTBP, anti-FLAG immunoprecipitation was followed by anti-p300 immunoblotting. ‘αp300 (before Co-IP)’ and ‘αFLAG (before Co-IP)’ show the amount of p300 and FLAG-tagged proteins in 4% of the cell lysate prior to co-immunoprecipitation.

Fig. 3. The physical interaction of hADA3 and p53 in human cells requires the N-terminal half of hADA3 and is enhanced by DNA damage. (A) The N-terminus of hADA3 interacts with p53. FLAG-tagged full-length hADA3, the N-terminal half of hADA3 or the C-terminal half with NLS were expressed in 293 cells. Co-immunoprecipitation and immunoblotting of cell lysates were performed as in Figure 2C, except for the indicated antibodies. SV40 large T antigen and N-terminally truncated 53BP1 served as positive controls; the vector control was pFLAG-CMV2. (B) γ-irradiation markedly increases the amount of p53 co-immunoprecipitated with hADA3. Experiments were performed as in (A), except for the exposure to 80 Gy of γ-irradiation and lysis 1.5 h later. SV40 large T antigen was used as a control that did not show enhanced interaction with p53 after γ-irradiation. (C) UV-irradiation markedly increases the amount of p53 co-immunoprecipitated with hADA3. Experiments were performed as in (A) and (B), except for the exposure to 50 J/m2 UV-irradiation and lysis of cells 3 h later. _p53_-negative H1299 cells with transiently transfected p53 (pC53-SN3) do not show an increase of p53 protein after UV-irradiation due to high baseline p53 levels, while U2OS cells with endogenous wild-type p53 do. For H1299 cells, the enhanced p53–hADA3 interaction is seen despite unequal levels of FLAG-tagged hADA3 favoring the lane without UV-irradiation. (D and E) Endogenous hADA3 and p53 physically interact in human cells. To induce DNA damage, U2OS cells were exposed to 50 J/m2 UV-irradiation prior to lysis 3 h later (D) and A549 cells to 0.4 µg/ml doxorubicin for 12 h (E). After lysis, a mixture of five monoclonal anti-p53 antibodies, cross-linked to protein A– or protein G–agarose, was used to immunprecipitate p53, followed by detection of hADA3 by immunoblotting.

Fig. 4. The enhanced interaction of hADA3 and p53 after DNA damage depends on N-terminal phosphorylation of p53. (A) γ-irradiation does not increase the amount of the transcriptionally inactive transactivation domain mutant p53 22Q23S co-immunoprecipitated with hADA3. Wild-type p53 and p53 22Q23S were transiently expressed in H1299 cells with hADA3 (or SV40 large T antigen as control), and co-immunoprecipitation was performed as in Figure 3A–C with and without 50 Gy of γ-irradiation. It is unclear why SV40 large T antigen co-immunoprecipitates p53 22Q23S as three bands. (B) One or several phosphorylation events in the p53 N-terminus govern the interaction with hADA3. Wild-type p53 and p53 with alanines instead of serines at positions 6, 9, 15, 20, 33, 37 and 46 (p53-7A) were evaluated with hADA3 as in (A) with and without 30 Gy of γ-irradiation.

Fig. 5. Full-length hADA3 increases and the N-terminal half of hADA3 inhibits p53 transcriptional activity. (A) p53 reporter gene assays with WWP-Luc. Increasing amounts of an expression plasmid for FLAG-tagged full-length hADA3 (50, 100 and 500 ng) were co-transfected into _p53_-negative H1299 cells with the p53 expression plasmid pC53-SN3 (100 ng), pIC400 expressing β-galactosidase (250 ng) and the reporter plasmid WWP-Luc containing luciferase under the control of the _p21_WAF1/CIP1/SDI1 promoter (500 ng). The cells were lysed after 24 h, and the luciferase activity, adjusted for β-galactosidase, was determined. Error bars represent the standard deviation for three independent experiments. p53 protein levels were assessed by anti-p53 immunoblotting after adjustment of the cell lysates from the shown reporter gene assays for β-galactosidase activity. (B) p53 reporter gene assays with PG13-Luc. The experiments were performed as described for (A), except that PG13-Luc containing a tandem array of 13 p53-binding sites and 100, 500 and 900 ng of FLAG-tagged hADA3 were used. (C) hADA3(aa1–214) inhibits p53 transcriptional activity. Increasing amounts of expression plasmids (100, 500 and 800 ng) for hADA3(aa1–214) with and without an NLS were co-transfected with 500 ng of PG13-Luc and 500 ng of pIC400 into 293 cells with endogenous wild-type p53. Luciferase activity was determined, and anti-FLAG immunoblotting for cell lysates with 800 ng of hADA3 expression plasmid was performed similarly to (A) for p53.

Fig. 6. The N-terminal half of hADA3 prevents p53-mediated apoptosis. (A) U2OS cells with endogenous p53 were transfected with either vector control, SV40 large T antigen or hADA3(aa1–214) and membrane-bound GFP (pPCMVEGFPspectrin), and exposed to UV-irradiation (20 J/m2). GFP-positive cells were analyzed 24 h later for changes in the sub-G1 fraction of apoptotic cells. (C) SW480 cells were co-transfected with p53, pPCMVEGFPspectrin and vector control, SV40 large T antigen or hADA3(aa1–214). After 24 h, the sub-G1 fraction was determined as above. (B and D) The results were normalized to the percentage of apoptotic cells above background (expressed as 100%) that were induced by UV-irradiation in U2OS cells (B) or p53 transfection in SW480 cells (D) alone. Error bars represent the standard deviation for three independent experiments.

Fig. 7. Reduction of hADA3 protein results in decreased p53 transcriptional activity. (A) Doxorubicin increases p53 transcriptional activity in A549 cells. Reporter gene assays were performed as in Figure 5B, except that the transcriptional activity of endogenous p53 was evaluated in the presence of increasing amounts of doxorubicin (0.05, 0.1, 0.2 and 0.5 µg/ml). (B) Two antisense oligomers specific for hADA3 mRNA markedly reduce p53 transcriptional activity. Experiments were performed similarly to (A) with 0.5 µg/ml doxorubicin, except for the co-transfection of antisense oligomers. The results for the hADA3 oligomers were compared with specific control oligomers and oligomers for luciferase and p53 mRNA. p53 transcriptional activity in the absence of any antisense oligomers is also shown. (C) Two antisense oligomers specific for hADA3 mRNA reduce hADA3, but not p53 protein levels. Experiments were performed similarly to (B), except for anti-hADA3 and anti-p53 immunoblotting after cell lysis.

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hADA3 is required for p53 activity - PubMed (original) (raw)