1. An Acetylation Switch in p53 Mediates Holo-TFIID Recruitment
Andrew G. Li, Landon G. Piluso, Xin Cai, Brian J. Gadd, Andreas G. Ladurner, Xuan Liu 
Molecular Cell 
Volume 28, Issue 3, Pages (November 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Direct Interaction between Acetylated p53 and TAF1 Double Bromodomains
(A) U2OS cells were treated with either control (−) or TSA (+). The p53-TAF1 interaction was analyzed by IP with anti-p53 antibody followed by IB with anti-TAF1 antibody. Five percent of input was loaded on the gel.
(B) Wild-type p53 and HA-tagged TAF1 and bromodomain mutants (DD, N1481/1604D) were overexpressed in H1299 cells. The p53-TAF1 interaction was analyzed by IP with anti-TAF1 antibody followed by IB with anti-p53 antibody. Ten percent of input was loaded on the gel.
(C) The GST-NTK, HAT, CTK, and DBrD were tested for binding with either highly acetylated or control p53. Ten percent of input was loaded on the gel.
(D) Schematic representation of the TAF1 protein. NTK, N-terminal kinase domain (1–414); HAT, histone acetyltransferase (516–986); CTK, C-terminal kinase domain (1404–1893); and DBrD, double-bromodomain (1380–1646).
(E) GST-DBrD was tested for binding with either acetylated p53 (p53Ac) or acetylation mutant (5KR).
(F) Wild-type TAF1 and the DD mutant were tested for binding with acetylated p53.
(G) Acetylation status of highly acetylated or control p53.
(H) Locations of N1481D and N1604D in each BC loop of the TAF1 DBrD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 TAF1 Recognizes a Diacetyl-K373/K382 Mark in Activated p53
(A) A series of 26-mer peptides corresponding to the C-terminal domain of p53 (residues 364–389) in various acetylation states were synthesized.
(B) 100- and 1000-fold molar excess of peptides to full-length acetylated p53 were used to compete the DBrD-acetylated p53 interaction.
(C–E) Excess of acetylated and unacetylated peptides was used to block the acetylated p53/TAF1 interaction (C and D) and the acetylated p53/TFIID interaction (E). Ten percent of input was loaded on the gel.
(F) Saos2 cells were transfected with wild-type or p53 acetylation mutants. The p53-TAF1 interaction was analyzed by IP with anti-TAF1 antibody followed by IB with anti-p53 antibody. Ten percent of input was loaded on the gel.
(G) The acetylation levels of K373 and K382 on overexpressed p53 proteins were determined.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Dynamics of p53-Mediated TAF1 Recruitment and Assembly of TFIID Complex on the p21 Promoter
(A) (Upper panel) Schematic representation of p53 binding sites (5′ and 3′) and core promoter (pp) of human p21 promoter and illustration of primer pairs used for PCR. (Lower panel) ChIP analysis of human p21 promoter in response to UV-induced DNA damage. U2OS cells were either untreated (M) or subjected to UV irradiation and assayed at the time points indicated on the top. Binding sites that were amplified by PCR are indicated on the top of each panel. Antibodies used in ChIP are listed on the right. PCR products were resolved by agarose-gel electrophoresis and visualized by ethidium bromide staining.
(B) A model for recruitment of TFIID to the p21 promoter.
(C) RT-PCR analysis reveals the p21 RNA levels at the time points indicated on the top.
(D and E) PCR amplification using the forward primer for the 3′ site and the reverse primer for the pp region (3′ pp) on sheared and unsheared DNA and using a pair of control primers (cc) before and after DNA damage.
(F) Sequential-ChIP assays were performed before (−) or 8 hr after (+) DNA damage. T, anti-TAF1 antibody (rabbit); P, anti-p53 antibody (mouse); M and R, mouse or rabbit control IgG.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Acetylated p53 and TAF1 Directly Interact on an Immobilized p21 Promoter
(A) From top to bottom: schematic representation of Dynabead-immobilized p21 promoter. Acetylated p53 binds to the immobilized p21 promoter. Immunoblots of p53 and TAF1 bound to the immobilized promoter in the presence or absence of 100- and 1000-fold molar excess acetylated (6Ac or 2Ac) or unacetylated (unAc) p53 peptides. Immunoblots of p53 and TAF1 bound to the immobilized promoter in the presence or absence of excess K8/K16 acetylated (2Ac) or unacetylated (unAc) H4 peptides. Twenty percent of input was loaded on the gel.
(B) (Upper) Schematic representation of the mutant p21 promoter. (Lower) Immunoblots of p53 and TAF1 bound to the immobilized wild-type or mutant p21 promoter.
(C) (Upper) Schematic representation of Dynabead-immobilized minimal p53-responsible promoter that only contains p53 binding sites and adenovirus E4 TATA box. (Middle) Acetylated p53 binds to the immobilized promoter. (Lower) Immunoblots of p53 and TAF1 bound to the immobilized promoter in the presence or absence of 100- and 1000-fold molar excess acetylated (Ac) or unacetylated (unA) p53 peptides.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Promoter Recruitment of TAF1 Depends on the Diacetyl-p53/Bromodomain Interaction
(A) p53 acetylation is required for TAF1 recruitment.
(B) The TAF1 DBrD is required for the recruitment.
(C) Histone H4 acetylation on p21 promoter. In (A)–(C), H1299 cells were transfected with the plasmid combination listed on the top. The 3′ and 5′ p53-binding sites and the core (pp) promoter amplified by PCR are indicated on the left. Antibodies used in ChIP are listed on the right.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
p53 Acetylation and Bromodomain Function Are Required for p53-Mediated p21 Activation
(A) H1299 cells were transfected with a p21-Luc reporter construct combined with a p53-expression vector (wild-type or acetylation mutants K373R, K382R, and 2KR) and increasing amounts of TAF1-expressing vector. The p53 protein levels were monitored using specific antibody.
(B) H1299 cells were transfected with a p21-Luc reporter construct combined with p53 and increasing amounts of wild-type TAF1 or bromodomain mutants. The TAF1 protein levels were monitored using specific antibody.
(C) H1299 cells were transfected with a p53E4TATA-Luc reporter construct combined with a p53-expression vector and increasing amounts of wild-type TAF1 or the DD mutant.
(D) H1299 cells were transfected with a Gal4E4TATA-Luc reporter construct combined with a Gal4VP16-expression vector and increasing amounts of wild-type TAF1 or the DD mutant.
(E) H1299 cells were transfected with cyclin D1- or c-fos-Luc reporter combined with increasing amounts of wild-type TAF1 or the DD mutant. In (A)–(E), error bars show average ± SD from three to four independent experiments. The average fold of activation relative to no TAF1 cotransfection is shown on the top of each bar.
(F) The DD mutant interacts with other subunits of TFIID. HA-tagged wild-type TAF1 and DD mutants were overexpressed in H1299 cells. Cell lysates were analyzed by IP with anti-HA antibody and IB with anti-TAF1, TAF4, TAF5, TAF9, and TBP antibodies. Ten percent of input was loaded on the gel.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7 The Dynamic Interaction between p53 and TAF1
U2OS cells were either untreated (M) or subjected to UV irradiation and fractionated to chromatin-bound and free fractions at the time points indicated on the top. The TAF1-p53 interaction was assayed by anti-TAF1 IP followed by IB with anti-p53, anti-TAF4, and anti-TBP antibodies. Two percent of input was loaded on the gel.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions