1. Tip60-Dependent Acetylation of p53 Modulates the Decision between Cell-Cycle Arrest and Apoptosis 
Yi Tang, Jianyuan Luo, Wenzhu Zhang, Wei Gu 
Molecular Cell 
Volume 24, Issue 6, Pages (December 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of a Previously Unknown Acetylation Site, K120, within the Human p53 DNA-Binding Domain
(A) Schematic representation of the human p53 protein with known acetylation sites indicated. In this study, K120 was identified to be acetylated by Tip60. K320 was previously shown to be acetylated by PCAF; the other six lysines (K305, K370, K372, K373, K381, and K382) by p300/CBP.
(B) Mass-spectrometry analysis of the p53-derived peptides containing K120 or acetylated K120 (AcK120). The protein was prepared as described in the Experimental Procedures.
(C) Alignment of the K120 flanking regions from the human p53 protein with those from other species. The conserved lysines are underlined and marked bold.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
In Vitro and In Vivo Acetylation of p53 at K120 by the Tip60 Acetyltransferase
(A) K120 is specifically acetylated by Tip60, but not by p300 or PCAF, in vitro. The GST-p53 or GST-p53-K120R fusion protein was incubated with Tip60, p300, or PCAF as described in the Experimental Procedures. Reaction mixtures were analyzed by western blot using the site-specific polyclonal antibodies against acetylated K120 (anti-AcK120-p53), K320 (anti-AcK320-p53), or the C terminus (anti-Ac-p53 [CT]). The anti-Flag monoclonal antibody was used to detect Tip60, p300, and PCAF. The bottom panel shows the levels of the GST-p53 fusion protein stained by Coomassie blue.
(B) In vitro acetylation of p53 by Tip60. The GST-p53 (lanes 2, 4, and 7) or GST-p53-K120R (lanes 3, 5, and 8) recombinant protein was incubated without (lanes 4 and 5) or with Flag/HA-Tip60 (lanes 2 and 3) or with GST-Tip60 (lanes 7 and 8). The reaction products were resolved by SDS-PAGE and visualized by autoradiography (top). The levels of the GST fusion protein substrates are shown in the bottom panel stained by Coomassie blue.
(C) In vivo acetylation of p53 at K120 by Tip60. H1299 cells were transfected with the plasmid DNA expressing Flag-p53 (WT or mutants) or/and Tip60. Total cell extracts and the M2 immunoprecipitated materials were analyzed by western blot using the anti-Tip60 (CLHF) antibody, the anti-AcK120-p53, and anti-p53 (DO-1) antibodies, respectively.
(D) In vivo acetylation of p53 at K120 by a Tip60 mutant (Q377E/G380E) defective in acetyl-Co-A binding. The acetylation was detected as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Tip60 Interacts with p53 In Vitro and In Vivo
(A) Direct interaction of Tip60 with p53 in vitro. The full-length GST-p53 fusion protein (lane 3), its N-terminal fragment (1–73) (lane 5), middle fragment (100–290) (lane 6), C-terminal fragment (290–393) (lane 7), or the GST protein (lane 2) was used in the GST pull-down assay with in vitro-translated 35S-methionine-labeled Tip60 protein. The levels of the GST fusion proteins are shown in the rightmost panel (lanes 8–11).
(B) Coimmunoprecipitation of p53 with Tip60 in HCT116 cells treated without (lanes 1 and 5) or with 10 nM actinomycin D (lanes 2–4 and 6–8) for 2, 5, or 10 hr. The whole-cell extracts (bottom panels) and the immunoprecipitated materials by either the Tip60-specific antibodies (top right) or the control IgG (top left) were analyzed by western blot using the anti-p53 monoclonal antibody DO-1.
(C) The MEF p53−/− and MEF p53−/− mdm2−/− cells were transfected with plasmid DNA expressing Tip60 and/or Flag-p53, as indicated. The cell extracts and the M2 immunoprecipitates were analyzed by western blot using anti-Tip60 antibody (CLHF) and anti-p53 antibody (DO-1).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Tip60 Is Required for the Acetylation of the Endogenous p53 Protein at K120 and for the p53-Dependent Induction of p21 and Puma, but Not Mdm2, in Response to DNA Damage
(A) The colorectal cancer cell line LS174TR1 was treated with the proteasome inhibitor MG132 (6 hr) or 10 nM actinomycin D (0, 2, 4, 8, and 12 hr). Cell extracts were analyzed by western blot using the antibodies against p53 (FL), p21, and actin.
(B) Twenty-four hours after transfection with the Tip60-specific siRNA, U2OS-Flag/HA-Tip60 cells were treated with or without 10 nM actinomycin D (8 hr). Western blot analysis of the cell extracts and the immunoprecipitates was performed as in (A).
(C) Tip60 and p53 were ablated by siRNA in as in (B). The cell extracts from the U2OS-Flag/HA-Tip60 cells treated without or with 10 nM actinomycin D (8 hr) or 20 μM etoposide (6 hr) were analyzed by western blot.
(D) Tip60 was ablated in U2OS cells by using a different Tip60-specific siRNA as described in the Experimental Procedures section. The cell extracts from U2OS cells treated without or with 20 μM etoposide (6 hr) were analyzed by Western blot using the antibodies against Tip60 (CLHF), p53 (FL), puma, p21, Mdm2, and actin.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Tumor-Associated Mutation K120R of p53 Abrogates Induction of Puma, but Not Mdm2
(A) H1299 cells were infected at ∼70% confluence with the adenoviruses expressing the wild-type p53 or the mutant p53-K120R proteins, or the control adenoviruses for 0, 8, 12, 18, and 24 hr. Western blot analysis of the cell extracts was carried out with antibodies against p53 (FL), p21, puma, Mdm2, and actin.
(B) ChIP assay of the Mdm2, p21, and Puma promoters. H1299 cells were infected at ∼70% confluence with the adenoviruses, as indicated, for 18 hr, treated with 1% formaldehyde for 10 min, and processed for ChIP. The crosslinked DNA-protein complexes were immunoprecipitated with the control IgG, the antibodies against p53 (FL), Tip60 (N17 + K17), and Ac-H4. The presence of the assorted promoter DNA was detected by PCR.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Loss of Acetylation of p53 at K120 Renders It Defective in the Induction of Apoptosis, but Not of Cell-Growth Arrest
(A) Normal H1299 cells and H1299 cells infected with the control adenoviruses or those expressing p53 or p53-K120R for 40 hr were harvested, fixed in cold 95% methanol, and analyzed for DNA content by flow cytometry. The apoptotic cells, identified by sub-G1 DNA content, were represented in the M1 population, whereas the cells in the M2 population are those with sub-G1 and G1 DNA content.
(B) H1299 cells were infected for 24, 36, and 40 hr with the adenoviruses and assayed for apoptosis as in (A). Quantitated apoptotic cells were plotted against the infection time and represented by the colored bars.
(C) Twenty-four hours after infection with the adenoviruses as in (A), H1299 cells were treated with 10 μM BrdU for 1 hr and immunostained with the anti-BrdU antibody. The stained nuclei are shown in red.
(D) H1299 cells were infected for 12, 18, and 24 hr with the adenoviruses as in (A), and incorporation of BrdU was assayed as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Model of Three Differential Levels of Activation of p53 Targets by Tip60
See text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions