SYT-SSX2 Antagonizes Polycomb Figure 5. Functional antagonism of Bmi1 in SYT-SSX2-expressing cells. (A) Lower panel: the Ring1B-interacting Bmi1 population is depleted in SYT-SSX2-expressing cells. pOZ and SYT-SSX2 infected cell extracts were immunoprecipitated (IP) with a Ring1B antibody. Immunoprecipitates were immunoblotted with the antibodies indicated to the left. FLAG antibody was used to detect ectopically expressed SYT-SSX2 (upper panel). (B) Extracts from pOZ and SYT-SSX2 infectants were immunoblotted with anti-ubiquityl H2A, total H2A and FLAG antibodies. Immunoblotting for alpha- tubulin was used as a loading control. (C) Increased transcription of SYT-SSX2-derepressed polycomb targets. Relative RT-PCR of a subset of SYT- SSX2-upregulated genes also shown to be polycomb-silenced targets [11]. GAPDH served as loading control. D) SYT-SSX2 (upper image), Ring 1B (middle image) and Bmi1 (lower image) occupancy on the NGFR promoter by CHIP-PCR in pOZ vector- and SYT-SSX2-expressing U2-OS cells. doi:10.1371/journal.pone.0005060.g005 expression of the SYT-SSX2 oncogene. This attenuation of Bmi1 appears to be mediated at the level of protein stability, although the specific mechanism by which SYT-SSX2 promotes Bmi1 degradation awaits further investigation. The expected outcome of Bmi1 degradation in the presence of SYT-SSX2 is the alteration of at least certain aspects of polycomb silencing function. Indeed, evidence exists to corroborate the expected impairment of polycomb gene regulation. First, we demonstrated the predicted loss of global histone H2A monoubiquitination resulting from SYT-SSX2-mediated Bmi1 depletion. This effect most likely stems PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5060