SYT-SSX2 Antagonizes Polycomb Figure 3. Mapping of the Polycomb association region on SYT-SSX2. (A) Schematic of wildtype SYT-SSX2 and the truncation mutants generated within the SSX2 region. (B) Immunofluorescent colocalization studies of the SYT-SSX2 truncation mutants (HA, red) and Bmi1 (green), with merging of the two channels displayed as well. Also indicated is the average percentage of SYT-SSX2 (or mutant) proteins aggregating with polycomb bodies (a minimum of 100 polycomb foci counted per replicate; n = 3). Image magnification was at 636. (C) Plot of Bmi1 fluorescence in U2OS cells expressing the SXdel mutants and the POZ control vector. Average fluorescence (20 cells per replicate; n = 3) of Bmi1 was compared between POZ- and SXdel-infectants. The Bmi1 fluorescence in the POZ nuclei was consistently equivalent to that of uninfected cells. Data analysis was performed using MetaMorph software. (D) Loss of Bmi1 immunoreactivity correlates with association of SYT-SSX2 to polycomb complexes. Western blotting of cells infected with indicated FLAG-tagged proteins using Bmi1, FLAG and alpha-tubulin-specific antibodies. doi:10.1371/journal.pone.0005060.g003 using both antibodies (Figure 4B). This argues against a Bmi1 post- translational modification, since an epitope-masking modification should not interfere with the ability of the polyoma tag antibody to recognize tagged Bmi1. In another experiment, lysates from either pOZ or SYT-SSX2-infected cells were blotted for endogenous Bmi1 using both a monoclonal and a polyclonal Bmi1-specific antibodies. Both antibodies revealed decreased Bmi1 levels in lysates from SYT-SSX2-transduced cells (Figure 4B), again negating the possibility of epitope-masking by a Bmi1 post- translational modification. Moreover, mass-spectrometry analysis of Bmi1 present in SYT-SSX2 cellular lysates failed to detect phosphorylated, acetylated or methylated residues (data not shown). In the final experiment, the half-life of 2PY-Bmi1 was measured using pulse-chase experiments in both pOZ and SYT- SSX2 infectants. Not surprisingly, Bmi1 in SYT-SSX2-expressing cells exhibited accelerated degradation when compared to pOZ control (Fig. 4C). We confirmed the correct identity of the immunoprecipitated 2PY-Bmi1 by peptide competition studies Figure 4. Loss of Bmi1 immunoreactivity in SYT-SSX2-infected cells results from depletion of the Bmi1 protein. (A) Real time RT-PCR of Bmi1 in U2OS lysates expressing the indicated proteins. (B) Left panel: overexpressed polyoma epitope-tagged Bmi1 (2PY-Bmi1) was infected with either SYT-SSX2 or pOZ backbone. Cell lysates were immunoblotted with Bmi1 antibody, then stripped and reprobed with an anti polyoma tag antibody. Immunoblotting for alpha-tubulin was used as a loading control. Right panel: cell lysates of pOZ and SYT-SSX2-infected cells were immunoblotted for either mouse monoclonal or rabbit polyclonal Bmi1 antibody. Immunoblotting for alpha-tubulin was used as a loading control. (C) Densitometry plotting of pulse-chase analysis of 2PY-Bmi1 in pOZ and SYT-SSX2-infected cells. 2PY-Bmi1-expressing cells infected with pOZ and SYT-SSX2 were labeled for 1 hr. with S35–labeled Methionine and Cysteine. Labeled cells were chased at the indicated timepoints and immunoprecipitated with a polyoma tag antibody. Immunoprecipitated 2PY-Bmi1 band intensities were quantitated by densitometry and plotted with pOZ and SYT-SSX2 experiments indicated. Pulse-chase experiment was successfully replicated (n = 2). (D) Immunoprecipitation studies demonstrating the specificity of the immunoprecipitated 2PY-Bmi1 band used in pulse-chase analysis. Negative controls using polyoma peptide blocking (lane 3), no antibody (lane 4) and na¨ıve U2OS cells (lane 1) are indicated. doi:10.1371/journal.pone.0005060.g004 PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5060