(Figure 4D). Regression analysis on the pulse chase curves revealed a half-life of Bmi1 at 13.1 hours in pOZ vector-infected cells and 4.9 hours in SYT-SSX2 expressants. These studies suggest that decrease in Bmi1 protein stability is the contributing factor to Bmi1 signal depletion in SYT-SSX2-infected cells. The decision to use the 2PY-Bmi1 in the pulse-chase experiments was based on the fact that expressed 2PY-Bmi1 exhibited similar behavior to that of endogenous Bmi1 in SYT-SSX2 expressants and on the high immunoprecipitating efficiency of the 2PY antibody (Figure 4B). Expression of the SYT-SSX2 compromises the integrity of Bmi1/Ring1B complex and impairs its function in histone H2A ubiquitination From our aforementioned colocalization experiments, it was evident that at least a subpopulation of Bmi1 depleted in the presence of SYT-SSX2 corresponded to locations that also contained Ring1B (polycomb bodies). We thus wanted to confirm whether the pool of Bmi1 apparently depleted in SYT-SSX2- infectants corresponded to the functional population of Bmi1 normally complexed with Ring1B. The integrity of the Bmi1/ Ring1B interaction was therefore assessed by coimmunoprecipi- tating (co/IP) Bmi1 from SYT-SSX2 and pOZ vector control infectants using anti-Ring1B antibodies. Western blotting of these co/IPs revealed that although equivalent amounts of Ring1B were immunoprecipitated from vector and SYT-SSX2 expressing cells, the quantity of Ring1B-interacting Bmi1 was substantially reduced in SYT-SSX2 infectants (Figure 5A, lower panel). The pronounced depletion of Bmi1 from the Bmi1/Ring1B complex by SYT-SSX2 raised the possibility that some Bmi1- mediated function related to polycomb silencing may be compro- mised in the presence of SYT-SSX2. Bmi1 can contribute to polycomb silencing through the facilitation of Ring1B-dependent histone H2A ubiquitination [8]. The loss of Bmi1-Ring1B association in the presence of SYT-SSX2 suggested that a corresponding alteration in histone H2A monoubiquitination might occur as a consequence. To ascertain whether the diminished Bmi1/ Ring1B association translated into a loss of H2A K119 ubiquitina- tion, we performed Western blotting analysis to determine the global levels of ubiquityl-H2A in SYT-SSX2-infectants versus controls. Immunoblotting demonstrated that while the levels of total histone H2A and tubulin were unchanged, those of ubiquitinated H2A were dramatically reduced following ectopic expression of SYT-SSX2 (Figure 5B). Taken together, these results demonstrate a general loss of Bmi1-specific histone-modifying function caused by SYT-SSX2- mediated depletion of Bmi1. Genomewide analysis of SYT-SSX2-infectants demonstrates reactivation of polycomb target genes The depletion of Bmi1 and the associated diminution of ubiquityl-H2A provided evidence to support the notion that SYT- SSX2 antagonizes polycomb silencing. Nonetheless, the definitve means to demonstrate a reversion of polycomb-mediated tran- scription inhibition is to examine whether expression of SYT- SSX2 induces reactivation of polycomb-repressed genes. We performed genomewide analysis of SYT-SSX2-infectants using microarray technology to elucidate the gene expression patterns altered by this oncogene. Hybridization of total RNAs derived from SYT-SSX2 and pOZ vector-infectant transcripts to Affyme- trix microarray platforms was performed and transcript changes were expressed as Log2 ratios of SYT-SSX2 signal intensity to pOZ control [where the Log2 ratio (x) represents 2x fold increases (or decreases) in transcript level]. Only differences of two-fold or greater that recurred in both replicates were designated for further SYT-SSX2 Antagonizes Polycomb analysis. Of the differentially expressed genes, 123 displayed transcriptional upregulation whereas 33 demonstrated transcription- al downregulation (Microarray of upregulated and downregulated target genes by SYT-SSX2 is available upon request). Gene profiles in SYT-SSX2-expressing cells repeatedly displayed changes in the expression patterns of key proteins involved in cell cycle control, signal transduction, cytoskeletal organization and transcriptional regulation. Many corresponded to previously described SYT-SSX targets (e.g. IGF2) or genes known to be differentially expressed in synovial sarcoma [e.g. EPHB3, TLE3; 25]. In order to determine which of these genes correlated with polycomb targets, the Affymetrix microarray data was compared to another genomewide analysis performed by Lee et al., 2006, [11] in human embryonic cells. These studies, which combined chromatin immunoprecipitation of polycomb-bound promoters with microarray analysis, were designed to determine polycomb- binding regions across the genome. They identified approximately 8% of the human embryonic genome as binding to polycomb complexes and these genes have been designated as putative polycomb targets. In order to compare our SYT-SSX2-differen- tially expressed genes with this genomewide study, we utilized the Vanderbilt University run, web-based program called WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/). This program was used to perform bollean operations to determine what percentage of SYT-SSX2-regulated transcripts corresponded to putative polycomb targets. Of the 123 genes upregulated by SYT-SSX2, 33 of them (26%) represent polycomb- repressed genes. These genes are listed in Table 1. This percentage of polycomb reactivated genes represented a greater than 3-fold enrichment over the percentage of genes (8%) that could be theoretically detected by chance alone [11]. These data point towards a trend in polycomb target gene transcript upregulation following ectopic expression of SYT-SSX2. Antagonism of polycomb by SYT-SSX2 To confirm the overlap between the SYT-SSX2 microarray expression profile and the reported polycomb gene targets [11], upregulated transcription of a representative group of shared targets was validated by relative RT-PCR (Figure 5C). The effect of SYT-SSX2 on the Ring1B/Bmi1 complex was further characterized on a representative promoter- that of the NGFR gene- by chromatin immuno-precipitation (CHIP). Our choice of an appropriate target for such studies was based on the persistent upregulation of NGFR in all the microarrays performed on SYT- SSX2-expressing mesenchymal cells (Figure 5C and data not shown). Our CHIP analyses confirmed that the NGFR promoter is indeed a direct target of SYT-SSX2 (Figure 5D, upper panel). They also established that while Ring 1B is present at the promoter in vector control- and SYT-SSX2 expressants (Figure 5D, middle panel), Bmi1 was no longer detected on the NGFR region in SYT-SSX2 cells (Figure 5D, lower panel). These results are consistent with the Ring 1B- and Bmi1- SYT-SSX2 colocalization data (Figure 1B) and Bmi1 diminution upon SYT- SSX2 expression. In summary, the SYT-SSX2 oncogene appears to influence gene-expression programs through its association with and subsequent antagonism of polycomb components in mesenchymal cells. This function likely constitutes part of its transforming activity in synovial sarcoma. Discussion In this study, we demonstrate a consistent and significant depletion of the polycomb complex protein Bmi1 following de novo PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5060