Figure 1.
Pax3 negatively regulates p53 protein, but not mRNA levels in ESC just as in mouse embryos.
(A) Real time RT-PCR of p53, Pax3, and Nestin mRNA in stage 1 (open bars) and stage 3, days 2–8 (solid bars) ESC. Nestin mRNA is expressed in neuroepithelium and in ESC-derived neuronal precursors [29], [30] and served as a control for a marker of neuroepithelial neuronal precursors. Each mRNA was normalized to rRNA. In (A), (B), and (D) values represent the mean ± SEM (n = 3 culture dishes). *p<0.01 vs. stage 1. (B) Quantification (band intensity in arbitrary units) of immunoblots of p53, Pax3, and Nestin normalized to β-actin in stage 1 and stage 3 ESC harvested on days as indicated in (A). (C) Indirect immunofluorescence of p53 (green) and Pax3 (red) in stage 1 and stage 3 ESC. Cells were counterstained with DAPI (blue) to visualize nuclei. Cells incubated with secondary antibodies alone generated no detectable signals (not shown). (D) Percent Pax3 or p53 positive cells in stage 1 and stage 3 ESC. Values represent the mean ± SEM (n = 10 fields). (E) Real time RT-PCR of Pax3 and p53 mRNA in stage 1 ESC transfected with empty vector (Control), or vector encoding w.t. Pax3. Each mRNA was normalized to rRNA. ***p<0.0001 vs. control cells. (F) Immunoblot of Pax3 or p53 in stage 1 ESC stably transfected with empty pCMV vector or pCMV-Pax3. (G) Immunoblot of Pax3 or p53 in stage 1 or stage 3 ESC. Stage 3 ESC were untransfected, or transfected with empty shRNA vector (pSingle), pSingle expressing a scrambled shRNA sequence (scrambled), and 3 different Pax3 shRNA sequences. Stage 3 cultures were treated or not with doxycycline during days 4–6.
Figure 2.
Pax3 stimulates p53 degradation and ubiquitination and physically associates with p53 and Mdm2.
(A) Pulse labeling with 35S-met to determine the rate of p53 synthesis in stage1 and stage 3 ESC. Quantitation of 35S-p53 is described in the supporting online material. (B) Pulse-chase labeling to determine the t12 of p53 in stage 1 and stage 3 ESC. (C) Quantitation of ubiquitinated p53/total p53 in stage 1 and stage 3 ESC following immunoprecipitation of p53 and immunoblotting using anti-ubiquitin or anti-p53 antibodies. *p<0.05 vs. stage 1. (D) Whole cell extracts of stage 1 or stage 3 ESC were immunoprecipitated using antibodies against p53, Pax3 or Mdm2, and then immunoblotted using antibodies against p53, Pax3, and Mdm2. (E) Schematic diagram of full-length w.t. Pax3, Pax3 structural domains, and a Splotch Pax3 (Sp) protein product that were expressed as GST and FLAG fusion proteins. N-term, amino-terminus of Pax3 through the homeodomain; C-term, carboxy-terminus distal to the homeodomain (including the trans activation domain); DBD, DNA-binding domain (PD through HD); ID, the trans-activation inhibitory domain (amino-terminal to the PD); PD, paired domain; OCT, octapeptide (carboxy-terminal of the PD to amino-terminal of the HD); and HD, homeodomain. The Splotch cDNA deletes exon 4 and lacks coding sequence for part of the PD and the OCT but retains the HD. Numbers refer to amino acid positions of w.t. Pax3. (F) Immunoblot using antibodies against p53 (upper panel), Mdm2 (middle panel), or GST following incubation of whole cell lysates from stage 1 ESC with GST-Pax3 fusion proteins linked to glutathione-sepharose beads. (G) Immunoblot using antibodies against p53 (upper panel), Mdm2 (middle panel), or FLAG following incubation of whole cell lysates from stage 1 ESC that had been transiently transfected with plasmids encoding FLAG-tagged Pax3 fusion proteins with antibody against FLAG linked to M2 beads.
Figure 3.
Pax3 stimulates Mdm2-mediated ubiquitination of p53 in vitro.
(A) In vitro ubiquitination reactions of GST-p53 with GST-Mdm2 (150 ng), and 0–1600 ng GST-Pax3. Ubiquitination was assayed by immunoblot using 53 antibodies. The position of GST-p53 is indicated by a heavy arrow, and the positions of ubiquitinated p53 are indicated by narrow arrows. (B) In vitro ubiquitination reactions as in (A) except that GST without any Pax3 coding sequences was used. (C) In vitro ubiquitination reactions performed with only GST-Mdm2 or 400 ng GST-Pax3. (D) In vitro ubiquitination reactions of GST-p53 with GST-Mdm2 and 400 ng GST fusion proteins of w.t. Pax3, Pax3 structural domains, or Splotch Pax3. (The GST-p53 and GST-Mdm2 vectors used here encoded murine p53 and Mdm2, although similar results were obtained using human p53 and Mdm2 fusion proteins (data not shown).)
Figure 4.
The Pax3 domains that associate with p53 and Mdm2 stimulate p53 ubiquitination and down regulation in vivo.
(A) Quantitation of in vivo ubiquitination of p53 in stage 1 ESC that were untransfected, or transiently transfected with 4 µg FLAG-tagged vectors encoding w.t. Pax3, Pax3 structural domains, or Splotch Pax3. *p<0.05, **p<0.01. (B) Immunoblot analyses of whole cell extracts from Stage 1 ESC that were untransfected, or transiently transfected with 1–4 µg FLAG-tagged vectors as in (A) using antibodies against FLAG, p53, or β-actin. (C) Quantitation of FLAG or p53 relative to β-actin from three replicate transfected culture dishes assayed as in (B).