Figure 2. Loss of Bmi1 immunoreactivity following de novo expression of SYT-SSX2. (A) Extracts derived from pOZ vector, SYT and SYT-SSX2 cells were analyzed by Western blotting for levels of Bmi1 and alpha-tubulin (loading control). (B) Loss of immunoreactivity is specific to Bmi1. pOZ and SYT-SSX2-infectants were lysed and SYT-SSX2 Antagonizes Polycomb immunoblotted for Bmi1, Ring1B, YY1, FLAG (to detect ectopically expressed proteins) and alpha-tubulin. (C) The decrease in Bmi1 signal is a specific function of SYT-SSX2 chimera. Extracts from uninfected cells, pOZ, SYT, SYT-SSX2, SYTdel8 and SSX2 were immunoblotted for Bmi1, FLAG-tagged ectopically expressed proteins and alpha-tubulin. The FLAG-tagged protein visualized in pOZ-infected cells is due to the generation of an irrelevant FLAG/HA-tagged peptide by the vector. The molecular weight of the SSX2 domain is ,10 Kd. For its detection we ran the SSX2 lysates on a separate 18% SDS-PAGE system. The remaining lysates were resolved on a 10% SDS-PAGE. U: uninfected cells. doi:10.1371/journal.pone.0005060.g002 truncations between 15–23 amino acids in length. Mutants SXdel1 and SXdel2, corresponding to deletion of amino acids 1–35 of the translocated SSX2 domain (Figure 3A), retained their ability to colocalize with polycomb complexes (Figure 3B). Conversely, mutants SXdel3 and SXdel4, which lack amino acids 35–78, lost their association with polycomb bodies. This suggested that the C- terminal 44 amino acids contain the region essential for localization of SYT-SSX2 to polycomb complexes. We further refined the mapping of SYT-SSX2- polycomb association by creating smaller truncations within the C-terminal 44 amino acids of SSX2; these mutants were designated SXdel5-del9 (Figure 3A). Of these mutants, only SXdel5 retained its polycomb colocaliza- tion ability (Figure 3B), suggesting that the C-terminal 34 amino acids of SYT-SSX2 contain the polycomb association motif. This C-terminal stretch lacks homology to any known protein-protein interaction domains and represents a novel interface for association with polycomb components or their interacting proteins. Like its SXdel4 counterpart, mutant SXdel8 with truncated amino acids 55–65 expressed poorly (Figure 3D), suggesting that region maintains proper stability of SYT-SSX2. As anticipated, expression of the SYT-SSX2 mutants that co- localized with polycomb complexes (SXdel1, del2, and del5) induced depletion of endogenous Bmi1 protein (Fig. 3B and D). These same three mutants also exhibited a decreased Bmi1 fluorescence in their nuclei (Fig. 3C). The remaining SYT-SSX2 mutants that failed to be targeted to polycomb bodies had no effect on Bmi1 cellular levels. Taken together, these experiments suggest that SYT-SSX2 requires close association with polycomb complexes in order to induce a decrease in Bmi1 immunoreac- tivity. Loss of Bmi1 immunoreactivity is due to a decrease in Bmi1 protein stability Several explanations can account for the decrease in Bmi1 signal intensity in SYT-SSX2 infectants observed on cellular extracts immunoblots and by indirect immunofluorescence; these include a decrease in the transcription of Bmi1, a decreased half- life of Bmi1 protein and a post-translational modification that impairs epitope recognition by the Bmi1 monoclonal antibody used in our studies. We did not detect changes in Bmi1 mRNA levels following transduction of SYT-SSX2 by both relative RT- PCR (Figure S2) and real time RT-PCR (Figure 4A), arguing against transcriptional inhibition as the mechanism for Bmi1 depleted signal. We then carried out three different studies to ascertain whether the lack of Bmi1 immunoreactivity resulted from decreased Bmi1 protein stability or a de novo Bmi1 modification. In the first experiment, U2OS cells were transduced with Bmi1 cDNA that was fused to two copies of the polyoma epitope tag (2PY-Bmi1). These cells were re-infected with either pOZ retroviral backbone or SYT-SSX2 cDNAs and lysed 48 hours later. Immunoblotting for both the polyoma tag (2PY) and Bmi1 revealed signal depletion in the SYT-SSX2-infectants PLoS ONE | www.plosone.org 4 April 2009 | Volume 4 | Issue 4 | e5060