1. Volume 8, Issue 6, Pages 1243-1254 (December 2001)
Acetylation of p53 Activates Transcription through Recruitment of Coactivators/Histone Acetyltransferases 
Nickolai A. Barlev, Lin Liu, Nabil H. Chehab, Kyle Mansfield, Kimberly G. Harris, Thanos D. Halazonetis, Shelley L. Berger 
Molecular Cell 
Volume 8, Issue 6, Pages (December 2001)
DOI: /S (01)

2. Figure 1
In Vitro Acetylation and Biochemical Properties of Wild-Type p53 and the Acetylation Site Substitution Mutant p53
(A) Schematic diagram of p53 domain structure. Abbreviations are as follows: AD, activation domain; DBD, DNA binding domain; RD, repression domain; and OD, oligomerization domain. Positions of lysines mutated into arginines (K→R) are indicated.
(B) EMSA analysis of p53wt and p53mut (K320/373/381/382R). The indicated p53 was incubated with 32P-labeled DNA 30 bp probe derived from the p53 binding site within the p21 promoter. Monoclonal antibody 421 to the p53 C terminus and cold competitor DNA oligomer were added as indicated. Free probe was run out of the gel to enhance the separation of p53 and p53 supershifted DNA complexes.
(C) Gel filtration analysis of p53wt and p53mut (K320/373/381/382R). The oligomerization state was determined by size exclusion chromatography. Elution position from the column was monitored by UV absorbance at A280. The elution position of molecular weight standards is indicated.
(D) In vitro acetylation of purified recombinant wild-type p53 (p53wt) and p53 acetylation site substitution mutant K320/373/381/382R (p53mut). Purified full-length CBP or the HAT domain of PCAF was used to acetylate equal amounts of p53wt and p53mut in vitro. Coomassie-stained gel is shown above, and fluorogram is shown below. Percent acetylation is relative to theoretical maximum (see Experimental Procedures for details).
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
Transcriptional Activation by p53 or p53 Acetylation Site Substitution Mutants
(A) p53-dependent reporter assay in U2OS cells. Test HA-p53 was cotransfected with a dominant-negative p53 and an alkaline phosphatase reporter driven by a p53 binding site derived from the p21 promoter. The level of activity was determined relative to transfected wild-type p53 (set to 100%). An insert below shows anti-HA Western blot analysis of test HA-p53 protein levels for the wild-type and p53 acetylation substitution mutants, compared to the control transfected sample. The anti-tubulin Western immunoblotting was used as a loading control to normalize for total amount of protein in each sample.
(B) RT-PCR analysis of p21 mRNA in H1299 cells. Wild-type p53 or p53 bearing substitution mutations in acetylation sites (K320/373/381/382R) was transfected into H1299 cells. Following irradiation (15 Gy), total RNA was purified and cDNA was prepared. The GAPDH-specific PCR product was used as an internal control. The level of p21 RT-PCR signal was calculated as a percent of GAPDH signal (set at 100%). The protein levels of wild-type or mutant p53 in transfected cells were determined by Western blot analysis using the DO1 anti-p53 antibody.
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3
Cell Cycle Arrest by p53 and p53 Acetylation Site Substitution Mutants in Irradiated U2OS and H1299 Cells
(A) Left: U2OS cells were transfected with plasmids encoding GFP and wild-type p53 or acetylation site-defective mutants. Cells were transfected with a tumor-derived p53 dominant-negative mutant (p53W248) to eliminate endogenous p53. The cells were irradiated at 5 Gy, and 12 hr later the DNA was stained by propidium iodide. The GFP-positive population of cells was sorted, and the DNA content was determined by FACS analysis. Right: To verify that all the acetylation site substitution mutants of p53 were nuclear, U2OS cells were transfected with test HA-p53 constructs and were analyzed by indirect immunofluorescence using HA-antibody. Nuclear distribution of HA-p53 proteins (green) was verified by staining cells for DNA with Hoechst dye (dark blue). The average transfection efficiency of U2OS cells was 20%–30%.
(B) Tet-off-inducible H1299 cells expressing either vector alone (control), wild-type p53 (p53wt), or acetylation site-defective mutant of p53 (K320/373/381/382R) (p53mut) were irradiated as in (A), and the DNA was stained by propidium iodide. The DNA content was determined by FACS analysis. The protein levels of p53 in each cell line were determined by Western blot analysis using HA-antibody. Tubulin was used as a loading control. The indirect immunofluorescence was performed as in (A), in this case on transiently transfected H1299 cells. The transfection efficiency was 80%–90%.
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 ChIP Assay of p53 at the p21 Promoter in U2OS Cells
(A) Schematic representation of p21/WAF/CIP1 promoter (not to scale). Relative distance between p53 binding sites and the TATA box is indicated. Also shown are positions of primers and approximate lengths of PCR products obtained in ChIP and RT-PCR.
(B) RT-PCR analysis of p21 RNA. Cells were mock irradiated or treated with ionizing radiation at 15 Gy and harvested 5 hr later. RT-PCR of GAPDH RNA was used a loading control. The signals were quantitated by Kodak 1D Digital Image Analysis Software (Eastman Kodak). Numbers below the lanes represent the ratio of p21 signal to GAPDH signal, which was set as 100%. Total p53 levels in U2OS cells were determined by Western blotting using DO-1 antibody.
(C) ChIP assay of p21 promoter. Cells were either mock irradiated or treated with ionizing radiation at 15 Gy and harvested 1.5 hr later. Antibodies to p53 and acetylated-p53 were used. PCR analysis (using primers to p21 or GAPDH promoter) is shown, using input DNA (1/100 of ChIP) or DNA after ChIP. The input PCR signal was set at 100%, and the numerical value of ChIP signal represents the percentage of input.
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5 ChIP Assay of p53 at the p21 Promoter in H1299 Cells
(A) Comparison of expression levels for endogenous p53 in U2OS cells and wild-type p53 (p53wt), or acetylation site-defective p53 (K320/373/381/382R) (p53mut) in transfected H1299 cells. Cells were irradiated at 15 Gy or mock irradiated. Protein amounts were normalized either by total protein or anti-tubulin Western blotting. p53 or HA-p53 was detected by DO-1 antibody.
(B) ChIP assay of H1299 cells mock transfected (control), transfected with wild-type p53 (p53wt), or transfected with mutant p53 (K320/373/381/382R) (p53mut). Cells were mock irradiated or irradiated at 15 Gy and were harvested 1.5 hr later. FL393 polyclonal antibodies to p53 were used. PCR and quantitation were done as in Figure 4.
(C) Reporter activation assay by Gal4DBD fusions of wild-type p53 (Gal4DBD-p53wt) and p53 bearing substitution mutations in acetylation sites (K320/373/381/382R) (Gal4DBD-p53mut) in H1299 cells. H1299 cells were transfected with reporter plasmid bearing E1B promoter with five Gal4 binding sites upstream of luciferase gene and with each of the following test constructs: empty vector, Gal4DBD, Gal4DBD-p53wt, or Gal4DBD-p53mut. Cells were irradiated at 15 Gy at 36 hr posttransfection and were grown for additional 12 hr. Activity represents the fold activation over background luciferase activity in the sample containing only the reporter. Total protein amounts were normalized, and Gal4DBD-p53 protein levels were determined by Western blotting using DO-1 antibody (shown below).
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6
ChIP of Coactivators and Histones at the p21 Promoter in U2OS and H1299 Cells
(A) U2OS cells and H1299 cells mock transfected (control), transfected with wild-type p53 (p53wt), or transfected with acetylation site-defective p53 (K320/373/381/382R) (p53mut) were irradiated at 15 Gy. Samples were tested by Western blot analysis for expression levels of endogenous CBP, TRRAP, and tubulin control.
(B) U2OS cells were irradiated or mock irradiated. ChIP assay of p21 promoter using antibodies to CBP, TRRAP, PBP, and acetylated histones H3 and H4 is shown below.
(C) H1299 cells were mock transfected (control), transfected with wild-type p53 (p53wt), or transfected with acetylation site-defective p53 (K320/373/381/382R) (p53mut), irradiated at 15 Gy, and harvested after 1.5 hr. ChIP assay as in (B).
(D) Tet-off-inducible H1299 cell lines expressing none (control), p53 wild-type (p53wt), or p53 bearing substitution mutations in acetylation sites (K320/373/381/382R) (p53mut) were transfected with Flag-CBP expression plasmid. Cells were treated as described in (B) 48 hr later. The protein levels of ectopically expressed Flag-CBP were monitored by immunoblotting using anti-Flag antibody (top). Whole-cell extract prepared from mock-transfected H1299 cells (w.c.e.) was used as a control for specificity of the Flag antibody. Tubulin served as a loading control. ChIP assay of p21 promoter using CBP antibody was performed on transfected samples. Quantitation of PCR signals was carried out as described earlier.
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 Binding of TRRAP and hGcn5 to Acetylated p53 In Vitro
(A) p53 was acetylated or mock acetylated and incubated with a HeLa extract P11 column fraction containing TRRAP. p53 was immunoprecipitated, and the amount of TRRAP and hGcn5 bound to the beads was determined by Western analysis. The lower panels are Westerns to p53 and to acetylated p53.
(B) GST alone, GST-p53C(WT), or substitutions (as indicated) were acetylated, bound to glutathione-sepharose beads, and incubated with HeLa nuclear extract. Bead bound material was washed and analyzed by immunoblot, using various coactivator antibodies. The lower panels are Westerns to GST or to acetylated p53.
Molecular Cell 2001 8, DOI: ( /S (01) )