Synovial sarcoma is an aggressive soft tissue cancer that typically afflicts young adults. This disease is characterized by a persistent t(X;18)(p11;q11) translocation event that juxtaposes the SYT (SYnovial sarcoma Translocated) gene on chromosome 18 with an SSX gene (either SSX1 or SSX2) on the X chromosome [22]. The resulting chimeric product, SYT-SSX, generates a fusion protein derived from both genes. The t(X;18)(p11;q11) rearrange- ment is detected in greater than 95% of synovial sarcoma tumors and is thought to play a crucial role in the genesis and progression of this cancer [22]. Previous studies have revealed that SYT-SSX1 possesses inherent transforming activity in both cell culture systems and nude mouse models [23]. The oncogenic capacity of SYT- SSX2 was also demonstrated in a transgenic mouse model whereby SYT-SSX2, expressed in Myf5 lineage myoblasts, generated synovial sarcoma-like tumors with 100% penetrance [24]. Recently, it was demonstrated that SYT-SSX2 disrupted cellular positioning by remodeling the cytoskeleton and altering both cytoarchitecture and microtubule stability. The former was caused by activation of the ephrin pathway [25]. Despite these recent advances in the understanding of the biological conse- quences of the SYT-SSX chimeras, the specific mechanisms through which they execute these functions are unclear. The SYT-SSX fusion product and its wildtype SYT and SSX counterparts are nuclear proteins believed to function as regulators of gene expression. Several studies have confirmed an interaction between the N-terminal domain of SYT (SNH; also present in SYT-SSX) with the SWI/SNF family of chromatin remodeling complexes [26–28] and the histone acetyltransferease p300 [29]. The specific contribution of SYT and SYT-SSX to chromatin modification and the biological consequences of these associations are currently unknown. Recently, colocalization of both wildtype SSX and oncogenic SYT-SSX with the nuclear aggregates of polycomb complexes was reported [30]. A functional correlation between polycomb and the oncogene was also described [31–37]. These associations suggest- ed that SYT-SSX likely modulates polycomb gene silencing function, an effect that could ultimately contribute to synovial sarcoma pathogenesis. However, a specific description of the functional consequence of SYT-SSX on polycomb repression is lacking. In this report, we demonstrate and further characterize the interaction between the SYT-SSX2 oncogene and the PRC1 polycomb complex. We also show that de novo expression of SYT- SSX2 induces a significant depletion of Bmi1 protein, an effect that is mediated at the post-translational level. Downregulation of Bmi1 was associated with attenuated monoubiquitination of histone H2A, a Bmi1-specific function. These events correlated with derepression of a variety of polycomb target genes involved in diverse cellular processes. These studies demonstrate for the first time polycomb antagonism as one possible mechanism by which SYT-SSX2 contributes to the epigenetic deregulation of normal transcription. They show negative regulation of polycomb function by a reputed oncogene and propose a novel paradigm regarding polycomb silencing and cancer-related proteins. Results SYT-SSX2 colocalizes and associates with polycomb components Previous reports have demonstrated colocalization between SYT-SSX and polycomb bodies, using Bmi1 and Ring1A as markers of polycomb complexes. These studies suggested that an interaction, whether direct or indirect, exists between these proteins. Similarly, using a retroviral system that allowed expression of SYT-SSX2 in U2OS cells (human osteosarcoma) SYT-SSX2 Antagonizes Polycomb at levels similar to those detected in primary synovial sarcoma cells (Syn1 cells- Figure 1A; in synovial sarcoma, SYT-SSX2 expression is driven by the robustly active promoter of SYT), we were able to detect substantial colocalization of SYT-SSX2 with the polycomb protein Bmi1 (Figure 1B). U2OS cells were chosen for their shared mesenchymal origin with synovial sarcoma tumors. Overlapping distribution was most readily visualized in the Bmi1-containing polycomb bodies, where approximately 86% of these subnuclear structures were co-occupied by SYT-SSX2 aggregates. Colocali- zation of SYT-SSX2 was also observed with the polycomb complex component Ring1B (Figure 1B), where co-aggregation was slightly higher (93%). the SYT portion of the chimera alone (SYTdel8) failed to display significant colocalization (Figure 1B), indicating that targeting to polycomb bodies required the presence of the SSX2 component in the SYT-SSX2 fusion protein. Further analysis confirmed that when expressed alone, the SSX2 component of the chimera is capable of localizing to polycomb bodies. SSX2 was detected in 78% of Bmi1- and 75% of Ring1B- containing foci (Figure 1B). These indirect immunofluorescence experiments, in accordance with others, suggested that SYT-SSX2 might physically interact with polycomb complexes. We next wanted to determine whether a direct interaction between SYT- SSX2 and Bmi1 or Ring1B was responsible for targeting SYT- SSX2 to polycomb foci. Using in vitro GST pull-down assays, we detected specific binding between SYT-SSX2 and Ring1B (Figure 1D) but failed to demonstrate a similar interaction with the Bmi1 protein (Figure S1). These results suggest that SYT- SSX2 colocalization with the Bmi1/Ring1B complex in polycomb bodies is likely mediated by its direct binding to Ring1B. Further characterization of the SYT-SSX2/Ring1B association is under- way. Loss of Bmi1 immunoreactivity in SYT-SSX2-infectants During the Bmi1/SYT-SSX2 colocalization experiments, we observed a substantial reduction in Bmi1, but not Ring1B, fluoresecent intensity in SYT-SSX2-expressing cells (Figure 1 B and C). This reduction in Bmi1 fluorescence was quantified by MetaMorph software as approximately 50% of wildtype levels (Figure 1C). There was an apparent loss of not only the Bmi1 pool distributed diffusely throughout the nucleoplasm, but in the Bmi1 pool present in polycomb bodies as well. No change in the Bmi1 nuclear pool was observed in pOZ vector control and wildtype SYT-infectants. We also noted that localization of SSX2 in the Bmi1- and Ring1B-bearing foci was not accompanied by an attenuation of the Bmi1 signal (Figure 1B). Interestingly, the specific loss of Bmi1 immunofluorescence associated with SYT- SSX2 expression was consistently accompanied by an apparent decrease of Bmi1 protein levels on immunoblots of SYT-SSX2- transduced cellular extracts, using the same Bmi1 monoclonal antibody (Figure 2A). Conversely, the levels of other polycomb complex subunits, such as Ring1B and YY1 in SYT-SSX2 infectants were unchanged (Figure 2B), highlighting the specificity of this depletion effect for Bmi1. Consistent with the Bmi1 fluorescence signal, the singular expression of the SYT or the SSX2 fragment present in the chimera (SYTdel8, SSX2) did not affect Bmi1 protein levels (Figure 2C). Altogether, these results suggest that the SSX2 domain targets SYT-SSX2 to polycomb foci. However, its fusion to SYT is required to cause diminution of Bmi1 cellular levels. This effect is therefore specific to the SYT- SSX2 oncogene. To gain a better insight into the decreased immunoreactivity of Bmi1 in SYT-SSX2-infectants, we generated a series of truncation mutants of SYT-SSX2 and expressed them in U2OS cells. The first set of deletions, named SXdel1-del4 (Figure 3A), contained PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5060