A Novel Cofactor for p300 that Regulates the p53 Response Noriko Shikama, Chang-Woo Lee, Stephen France, Laurent Delavaine, Jonathan Lyon, Marija Krstic-Demonacos, Nicholas B La Thangue  Molecular Cell  Volume 4, Issue 3, Pages 365-376 (September 1999) DOI: 10.1016/S1097-2765(00)80338-X

Figure 1 Characterization of JMY (a) The primary amino acid sequence of JMY (983 amino acid residues). The S/T-P motifs in the N-terminal region (blue) are underlined, and the central p300-binding domain (from residue 1 to 119 and 469 to 558, respectively) is highlighted in yellow. The adenovirus E1A CR2-like motif, EVQFEILKCEE, is indicated in red (underlined residues being conserved in E1A CR2). The proline-rich C-terminal region is highlighted in green. (b) Diagrammatic summary of domains in JMY, showing the N-terminal S/T-P-rich region (blue), the central N-terminal and p300-binding domains (yellow), the adenovirus E1A CR2 homology region (red), and the proline-rich (green) region. (c) Expression of JMY determined by Northern blot analysis of RNA prepared from mouse tissues (Clontech). As a control, the level of β-actin RNA is shown. (d) Example of FISH mapping. (i) shows the FISH signal on human chromosomes, and (ii) shows the same metaphase spread stained with DAPI to identify chromosome 5. In (iii), a diagram of the FISH mapping results in shown. Each dot represents the double FISH signal detected on human chromosome 5. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)

Figure 2 JMY Interacts with p300 (a) Coimmunoprecipitation of JMY and p300 from U2OS cells transfected with pG4 (30 μg; lane 1) or pG4-p300611–2283 (30 μg; lane 2) together with pCMV-HA-JMY (30 μg; lanes 1 and 2). After extraction, immunoprecipitation was performed with anti-Gal4 monoclonal antibody followed by immunoblotting with anti-HA monoclonal antibody; the cell extract alone is shown in lane 3. In lanes 4 and 5, the cell extract was immunoblotted with an anti-peptide JMY antibody in the absence (lane 4) or presence (lane 5) of competing homologous peptide. The JMY 110 kDa polypeptide is indicated. (b) Immunoprecipitation of JMY with p300 was performed from untransfected HeLa cell extracts with the control anti-HA monoclonal antibody (lane 1) or the anti-p300 monoclonal antibody Ab-1 (lane 2) and thereafter immunoblotted with an anti-peptide JMY antiserum. Lane 3 indicates the endogenous JMY polypeptide in the HeLa cell nuclear extract. (c) Domains in JMY that bind to p300. A summary (left) of the data derived from the biochemical binding assay (right) where the indicated JMY derivatives (lanes 7–30) were assessed for ability to bind to baculovirus-expressed flag-p3001135–2414. As positive and negative controls, the binding of p53 and luciferase to p300 was assessed (lanes 1–6). In all the treatments, the input (IN) polypeptides were in vitro translated and binding assessed to p300 as described. The black dot (lane 22) shows the JMY469–558 polypeptide. The data for JMY1–119 are based on the two-hybrid results in (d). (d) Two-hybrid assay in mammalian cells: the indicated pG4-p300 expression vectors (0.5 μg), illustrated to the bottom of the figure, were introduced into SAOS2 cells either alone or together with pVP16-JMY469–558 or pVP16-JMY1–119 (0.5 μg) and the reporter pG5-luc. The data represent the relative activity of luciferase to β-galactosidase (derived from the internal control pCMV-β-gal) and are the average of several treatments. (e) Direct interaction between JMY and p300. Affinity-purified His-JMY (1 μg; lane 2 where JMY is indicated by the bracket) was incubated with either flag-p300 (1 μg; lane 3 shows the bound protein, with flag-p3001135–2414 indicated by a black dot) or control flag (lane 4) beads as described. The level of His-JMY bound to either the control flag or flag-p300 beads was assessed by immunoblotting with anti-JMY (lanes 6 and 7, respectively). The input His-JMY (lane 5; IN about 10%) is shown for comparison. Lanes 1–4 show a coomassie blue stained gel, lane 1 presenting the molecular weight standards. Lanes 5–7 show the immunoblot with anti-JMY. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)

Figure 3 JMY Augments p53-Dependent Transcription (a) The p53 reporter pBax-luc (2 μg) together with expression vectors for wild-type p53 (0.25 μg), JMY (4 or 8 μg), either alone or together with p300 (3 μg), were transfected into SAOS2 cells as indicated. (b) The p53 reporter pBax-luc (1 μg) together with expression vectors for p53 (0.5 μg) or p5322/23 (0.5 μg), together with JMY (5 μg) or p300 (2.5 μg), were transfected into SAOS2 cells as indicated. The values shown in (a) and (b) are the average of different readings and represent the relative level of luciferase to the β-galactosidase activity from the internal control. (c) (i) To assess the induction of endogenous Bax protein, expression vectors for wild-type p53 (10 μg) with or without JMY (40 μg) and p300 (40 μg) were transfected as indicated into SAOS2 cells. Extracts from transfected cells were prepared and immunoblotted for Bax and p53 as described. The Bax (top) and p53 (bottom) polypeptides are indicated. An untransfected cell extract is shown in lane 1. (ii) To assess the induction of endogenous p21 protein, expression vectors for wild-type p53 (5 μg) with or without JMY (15 μg) and p300 (15 μg) were transfected as indicted into SAOS2 cells. Extracts from transfected cells were prepared and immunoblotted for p21 as described. A cell extract transfected with empty vector is shown in lane 1. (d) To assess the level of p53, expression vectors for wild-type p53 (0.3 μg), JMY (1, 3, or 6 μg), or p300 (1, 6, or 12 μg) were transfected either alone or together as indicated into SAOS2 cells. Extracts from control or transfected cells were prepared and immunoblotted for p53 as described. The p53 polypeptide is indicated. (e) To assess the levels of p300 and JMY, expression vectors encoding p53 (0.1 μg), JMY (1, 2, and 5 μg), or p300 (where + = 2 μg and +++ = 6 μg) were transfected as indicated into SAOS2 cells. The level of protein expression was assessed by immunoblotting with an anti-HA antibody. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)

Figure 4 A Functional Role for JMY in Activating p53 Target Genes (a) The levels of endogenous p53, p300, and JMY in extracts prepared from untreated and actinomycin D (1 nM)–treated U2OS cells were assessed by immunoblotting with anti-p53, anti-p300, or anti-JMY antisera as described. (b) Immunoprecipitation of endogenous p53 from the U2OS cell extracts shown in (a) followed by immunoblotting with either anti-p300 (top) or anti-JMY (bottom) antisera was performed as described. About 10% of the endogenous p300 and JMY were coimmunoprecipitated with p53. (c) The p53 reporter pBax-luc (1 μg) together with expression vectors for wild-type p53 (0.1 μg), JMY (6 μg), or p3001572–1903 (2 or 6 μg) were transfected into SAOS2 cells as indicated. (d) The p53 reporter pBax-luc (0.5 μg) together with expression vectors for wild-type p53 (0.1 μg) or JMY1–403 (1 and 4 μg) were transfected into U2OS cells as indicated. The values shown in (c) and (d) represent the average of different readings and represent the relative level of luciferase to the β-galactosidase activity from the internal control. (e) Expression vectors for p53 either alone (i and ii) or together with JMY (iii and iv) were introduced into SAOS2 cells as described. Cells were fixed and treated with the anti-p53 monoclonal antibody 421 (i and iii) or assayed for the level of apoptosis by TUNEL (ii and iv); (i and ii) and (iii and iv) show, respectively, the same field of cells assayed for p53 and by TUNEL. (f) Quantitative comparison of the effect on apoptosis in SAOS2 cells caused by JMY, p300, or both in the presence or absence of p53. On the left side, the percentage of p53-positive cells that were TUNEL-positive was derived and compared to values obtained in the presence of JMY, p300, or both together. In the data, both the absolute percentage level of apoptosis together with the percentage stimulation in apoptosis relative to p53 alone is presented. The level of apoptosing cells represent the average of three independent readings. The TUNEL-positive population compared to the number of DAPI-positive cells in the absence of p53 was used to assess the level of apoptosis in the presence of JMY and p300, and the values shown were obtained from two separate assays. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)

Figure 5 Isoforms of JMY (a) Schematic diagram of wild-type JMY and JMYΔP isoform. (b) Protein sequence absent from the JMYΔP isoform. (c) Immunoblotting for wild-type JMY and JMYΔP (top) and p53 (bottom) in SAOS2 cell extracts transfected with expression vectors for wild-type JMY or JMYΔP (4 μg) and p53 (0.1 μg), as indicated. Immunoblotting with either anti-JMY or anti-p53 was performed as described. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)

Figure 6 Isoform-Specific Regulation of the p53 Response (a) The p53 reporters pBax-luc, pMDM2-luc, or pWWP-luc (1 μg) together with expression vectors for wild-type p53 (0.05 μg), wild-type JMY (5 μg), or JMYΔP (5 μg) were transfected into SAOS2 cells as indicated. The values shown represent the average of three readings and are the relative level of luciferase to β-galactosidase derived from the internal control. (b) Quantitative comparison of the effect on apoptosis in SAOS2 cells caused by wild-type JMY and JMYΔP in either the presence or absence of p53 determined by TUNEL assay, performed as described in Figure 4E and Figure 4F. The data show the absolute level of apoptosis caused by the different treatments together with the percentage stimulation relative to p53 alone. (c) SAOS2 cells were transfected with expression vectors for p53, wild-type JMY or JMYΔP in the presence or absence of p53, together with pCMV CD20, as indicated. At 36 hr after transfection, cells were identified by staining with anti-CD20 antibody, and DNA was stained with propidium iodide, and the proportion of G1, S, and G2/M phase cells determined as described. Molecular Cell 1999 4, 365-376DOI: (10.1016/S1097-2765(00)80338-X)