1. Volume 18, Issue 4, Pages 447-459 (May 2005)
The Transcriptional Coactivator Yes-Associated Protein Drives p73 Gene-Target Specificity in Response to DNA Damage 
Sabrina Strano, Olimpia Monti, Natalia Pediconi, Alessia Baccarini, Giulia Fontemaggi, Eleonora Lapi, Fiamma Mantovani, Alexander Damalas, Gennaro Citro, Ada Sacchi, Giannino Del Sal, Massimo Levrero, Giovanni Blandino 
Molecular Cell 
Volume 18, Issue 4, Pages (May 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 YAP Is a Selective Transcriptional Coactivator of p73
(A–F) H1299 cells were transiently transfected with indicated combinations of plasmids encoding p73α (A–F), p73γ (A), or p53 (A and C) (25 ng/60-mm dish), and full-length human YAP (100 ng/dish) (A–F) together with BAX (A and B), p53AIP1 (C), p21waf1 (D–E), and TK (F) luciferase reporter plasmids (50 ng/dish). Bars indicate SD.
(B and E) Increasing amounts (100, 200, and 500 ng/dish) of YAP plasmid were transfected with the indicated luciferase reporter plasmids. Bars indicate SD.
(G) PML+/+ and (H and I) PML−/− MEFs were transiently transfected with indicated combinations of plasmids encoding p73α (25 ng/dish) (G–I), full-length human YAP (200–500 ng/dish) (G–I), wild-type PML (1 μg/dish) (I), and SUMO (−) mutant PML (1 μg/dish) (I) together with BAX (G–I) luciferase reporter plasmids (50 ng/dish). The total amount of transfected DNA in each dish was kept constant by the addition of empty vector wherever necessary. Transfection efficiency normalization was performed by cotransfecting a β-gal activity expression vector and assessing β-gal activity in cell lysates. Cell extracts were prepared 36 hr later and subjected to determination of luciferase activity. Results are represented as fold induction over the control. Histograms show the mean of three experiments, each performed in quadruplicate. Bars indicate SD.
(L) U2OS cells seeded on glass coverslips were transfected with pEGFP-YAP together with either pcDNA3-PMLIV (row b), pcDNA3-PMLIV SUMO (−) encoding a variant of PMLIV that cannot be sumoylated due to mutations in all the three known sumoylation sites (Fogal et al. 2000) (row c), or with empty pcDNA3 plasmid (row a). Cells were then fixed and sequentially incubated with polyclonal anti-PML antiserum (PG-M3, Santa Cruz) and TRITC-conjugated anti-rabbit secondary antibody (Sigma). Images were analyzed by confocal scanning laser microscopy with a Zeiss Axiovert 100 M microscope attached to a LSM 510 confocal unit.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
YAP Is Recruited In Vivo onto p73 Apoptotic Target Genes in Response to DNA Damage
(A) Subcellular localization of GFP-tagged YAP is modulated by p73 expression. H-p73α/GFP and H-p73α/YAP-GFP cells were fixed and stained with Hoechst either in absence or 24 hr after the addition of Pon A (2.5 μM) to the culture medium; aliquots containing 100 μg of total cell protein were subjected to immunoblotting with a monoclonal anti-p73 antibody and anti-GFP polyclonal serum (top); histograms in the lower panel represent the mean of three experiments in which the number of cytoplasmic and nuclear GFP-stained cells were counted. Cell clones expressing different levels of both GFP-YAP and GFP alone were employed. LY (Calbiochem) at the concentration of 25 μM was added for 6 hr to the H-p73α/YAP-GFP cells pretreated with the suboptimal concentration of 1.25 μM Pon A for 12 hr. Bars indicate SD.
(B) YAP potentiates p73α-mediated apoptosis. H-p73α/GFP and H-p73α/YAP-GFP cells were treated as indicated in the Experimental Procedures section. Floating and adherent cells were collected 48 hr after addition of cisplatin, fixed in cold methanol, washed in PBS, and resuspended in 1 ml PBS containing 50 μg/ml RNase and 50 mg/ml propidium iodide. The content of DNA was measured by an Epics XL analyzer. Percentages of sub-G1 fractions are shown. Histograms represent the mean of three experiments. Bars indicate SD.
(C and D) Crosslinked chromatin derived from H-p73α/YAP-GFP cells treated with CDDP (C) or Eto (D) was immunoprecipated with antibodies to p73 or YAP or in the absence of antibody and analyzed by PCR with specific primers for the indicated regulatory regions. Input: nonimmunoprecipitated crosslinked chromatin.
(E) Crosslinked chromatin derived from H-p73α/YAP-GFP treated as in (C) was immunoprecipated with antiacetylated histone H4 or p300 antibodies or in the absence of antibody and analyzed by PCR with specific primers for the indicated regulatory regions. Input: nonimmunoprecipitated crosslinked chromatin.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Endogenous p73α, YAP, and p300 Proteins Are Concomitantly Recruited onto the Regulatory Region of p53AIP1 in Response to CCDP and Doxorubicin
(A) Coimmunoprecipitation between endogenous YAP and p73 proteins in CDDP-treated HCT116(3) cells. Cell lysates were immunoprecipitated with anti-YAP or anti-IgG polyclonal sera, blotted onto nitrocellulose, and probed with anti-p73 (top row) and anti-YAP (top row) antibodies. Positions of protein molecular size markers are indicated on the left.
(B) Floating and adherent HCT116(3) cells treated as indicated were collected and counted with a Thoma’s hemocytometer. Cell viability was determined by the ability to exclude trypan blue. Histograms represent the mean of three experiments. PARP cleavage in CDDP-treated HCT116(3) cells is analyzed by immunoblotting (top). Bars indicate SD.
(C) CDDP treatment induces nuclear translocation of YAP. HCT116(3) cells treated with CDDP as in (A) and (B) were stained with anti-YAP policlonal serum as described in the Experimental Procedures. Histograms represent the mean of three experiments in which the number of cytoplasmic and nuclear YAP-stained cells was counted. Bars indicate SD.
(D) Crosslinked chromatin derived from HCT116(3) cells treated as in (B) was immunoprecipitated with anti-p73, anti-YAP, anti-p300, anti-acetylated histone H4, or in absence of antibody and analyzed by PCR for the regulatory regions of p53AIP1, p21waf1, and TK.
(E) RNA preparations derived from HCT116(3) cells treated as in (B) were subjected to RT-PCR analysis. Specific primers for the detection of p53AIP1 (top) and p21waf1 (bottom) transcripts were used. Amplification of aldolase A (Ald-A) was used to normalize equal loading of each RNA preparation.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Silencing of YAP Decreases p300 Recruitment onto p53 AIP1 Target Gene and Reduces p73-Mediated Apoptosis
(A) Specific anti-YAP, anti-p73, and control anti-GFP siRNAs were transfected into HCT116(3) cells exposed to apoptotic dosages of either CDDP or Doxo. Cell viability was assessed by trypan bleu exclusion. The efficiency of siRNA-mediated reduction of YAP and p73 expression was assessed by immunoblotting (top). Bars indicate SD.
(B) Time course of PARP cleavage (top) and cell death (bottom) in HCT116(3) cells exposed to 7.5 μg/ml of CDDP. Bars indicate SD.
(C) RNA preparations derived from HCT116(3) cells treated as in (A) were subjected to RT-PCR analysis. Specific primers for the detection of p53AIP1 (top), BAX (middle), and p21waf1 (bottom) transcripts were used. Amplification of Ald-A was used to normalize equal loading of each RNA preparation. Bars indicate SD.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
YAP Potentiates p73-p300 Functional Interactions in Response to DNA Damage
(A and B) Recruitment of p300 (A) and histone H4 acetylation (B) onto the p53AIP1 target gene after CDDP treatment is decreased in HCT116(3) cells transfected with YAP-specific siRNA. In (B), the amount of p53AIP1 or TK DNAs present in immunoprecipitations from HCT116(3) cells transfected with siRNAs directed against YAP or GFP (control) and treated or not with CDDP was calculated relative to the input (as described in the Experimental Procedures). Bars indicate SD.
(C) AKT controls YAP recruitment onto p73 apoptotic target genes. HCT116(3) cells were transfected with a plasmid encoding the T308D/S473D double mutant of Akt that is constitutively active (CA-AKT) (Bellacosa et al., 1998). Constitutive phosphorylation of YAP by CA-AKT impairs its in vivo recruitment onto the p53AP1 promoter in cells exposed to CDDP.
(D) Expression of YAP favors the binding of the acetyltransferase p300 to p73α and its acetylation in response to DNA damage. Cell lysates from untreated and CDDP-treated HCT116(3) cells transfected with the indicated combinations of expression vectors were immunoprecipitated with an anti-HA monoclonal antibody and probed with antiacetylated lysines, anti-p300, and anti-p73-specific antibodies, respectively. Bottom: aliquots containing 100 μg of total cell proteins were subjected to immunoblotting with anti-p300, anti-GFP, and anti-actin antibodies, respectively.
(E) YAP efficiently binds nonacetylatable p73. H1299 cells were transiently transfected with either wild-type p73α or mutant p73α(KKK) (Costanzo et al., 2002) expression vectors. Cell extracts were assayed by GST pull down with either purified GST-WWYAP or GST alone.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 YAP Contributes to p73 Accumulation in Response to DNA Damage
(A) Silencing of YAP by specific siRNAs impairs p73 accumulation in cells exposed to CDDP. HCT116(3) cells were transiently transfected with p300 (6 μg) and p73αHA (3 μg) expression vectors and either the specific YAP(b) siRNA or with the control GFP siRNA. Cell lysates were analyzed by immunoblotting with the indicated antibodies.
(B) The p73 Y487P mutant, which is unable to bind YAP and cannot be transcriptionally coactivated (Strano et al., 2001), does not accumulate upon overexpression of YAP. H1299 cells were transiently transfected with the pcDNA-YAP (full-length human YAP) (4 μg) and either the pcDNA-73αHA (2 μg) or the pcDNA-73αHA Y487P (2 μg) plasmids. Cell lysates were analyzed by immunoblotting with the indicated antibodies.
(C) The p73 Y487P mutant displays a reduced half-life when compared to wild-type p73α. H1299 cells were transiently transfected with either the pcDNA-p73αHA (1 μg) or the pcDNA-p73αHA Y487P (1 μg) plasmids. After 24 hr, the cells were treated with 20 mg/ml of cyclohexamide (CHX) for 2 hr to block de novo protein synthesis. Cell lysates, prepared at the indicated times, were processed by immunoblotting with anti-HA antibodies and quantified by scanning densitometry. The results of three independent experiments (mean ± SEM [Figure 5C] are shown).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
YAP Requires p73 to Relocalize into the Nucleus and to Promote Cisplatin-Induced Cell Death
(A) Subcellular localization of endogenous YAP in untreated and CDDP-treated p73−/− fibroblasts and in their p73β-reconstituted derivatives. Histograms represent the mean of three experiments in which the number of cytoplasmic and nuclear YAP-stained cells was counted (see the Experimental Procedures).
(B) Crosslinked chromatin derived from p73−/− and p73β reconstituted fibroblasts treated with CDDP as in (A) was immunoprecipitated with anti-YAP polyclonal serum and analyzed by PCR for the regulatory regions of KILLER/DR5, p21waf1, and TK genes.
(C) p73−/− and p73β reconstituted fibroblasts were transiently transfected with the indicated plasmids and treated with 7.5 μg/ml of CDDP for 24 hr. Cells were stained and analyzed for the subG1 fraction as described in the Experimental Procedures. A representative experiment out of three performed is shown. 50 μg of total cell lysates derived from the indicated cells were resolved by SDS-10% PAGE and transferred to nitrocellulose membranes. The blot was probed for p73 (top) and for actin (bottom).
(D) Proposed model for p73 gene target specificity modulation by YAP in response to DNA damage. DNA damage causes p73 accumulation (Gong et al., 1999; Agami et al., 1999; Costanzo et al., 2002), release of YAP from cytoplasmic multiprotein complexes containing and AKT, and YAP relocalization into the nucleus (Basu et al., 2003). PML is required to localize YAP into the NBs to coactivate p73. The interaction with YAP promotes p73 stabilization, binding to p300, and its acetylation. Under apoptotic conditions, the transcriptionally active complex that contains acetylated p73α, YAP, and p300 assembles onto the regulatory regions of the p53-p73 complex proapoptotic target genes p53AIP1 and BAX.
Molecular Cell  , DOI: ( /j.molcel )
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