Locus Link ID 3714 6236 57801 9021 4804 2049 8913 28514 23345 284207 5155 4772 9123 10966 7107 10568 Gene Symbol JAG2 RRAD HES4 SOCS3 NGFR EPHB3 DLL1 SYNE1 METRNL PDGFB NFATC1 SLC16A3 RAB40B GPR137B Gene Name Jagged 2 RLoagti2osa 2, 2, 1.9, 1.9 Ras-related associated with diabetes 3, 2.8, 3.4, 2.8 Hairy and enhancer of split 4 Suppressor of cytokine signaling 3 1.2, 1.1, 1.2, 1.1 Nerve growth factor receptor EPH receptor B3 CACNA1G Calcium channel, alpha 1G Delta-like 1 Meteorin Spectrin repeat containing, nuclear 1.3, 1.2 envelope 1 Platelet-derived growth factor beta 4.1, 1.9, 3.4, 1.7 Nuclear factor of activated T-cells 1.1, 1, 1.2, 1.1 Solute carrier family 16, member 3 2.7, 2.6, 2.8, 2.5 RAB40B, member RAS family G-protein receptor 137B 2.4, 2.5 5.4, 5.4 1.2, 1.2 1.8, 2 5.3, 5.4 2, 2.2 1.1, 1 1.1, 1 4.1, 4 4.1, 4.1 Locus Link ID 387763 1852 5979 7090 9244 54855 10018 30812 4616 8535 131583 6299 2643 4487 4062 816 Gene Symbol DUSP9 RET TLE3 CRLF1 FAM46C BCL2L11 SOX8 GADD45B CBX4 FAM43A SALL1 GCH1 MSX1 LY6H CAMK2B Gene Name Log2 Ratios LOC387763 Hypothetical LOC387763 Dual specificity phosphatase 9 Ret proto-oncogene Transducin-like enhancer of split 3 Cytokine receptor-like factor 1 Family with sequence similarity 46, 3.3, 1.6, 3.3, member C 1.3 BCL2-like 11 SRY-box 8 Growth arrest and DNA damage-inducible 45 Chromobox homolog 4 Family with sequence similarity 43, member A Sal-like 1 GTP cyclohydrolase 1 Msh homeobox 1 Lymphocyte antigen 6 complex, l ocus H Calcium/calmodulin-dependent kinase 2 1, 1 1.3, 1.2 2.4, 2 1.3, 1.3 3, 2.9 1.2, 1.3 6.5, 6.3 1.3, 1.4 3.3, 3.3 1.2, 1.2 1.4, 1.3 1.4, 1.2 1.9, 1 4.3, 4.2 1.2, 1.1, 1, 1.1, 1.1, 1 Table 1. Putative polycomb target genes reactivated by SYT-SSX2. SYT-SSX2 Antagonizes Polycomb SLC34A2 Solute carrier famiy 34, member 2 164633 CABP7 Calcium binding protein 7 aNonbolded log2 ratios represent hits in microarray replicate 1 bolded log2 ratios represent replicate 2. doi:10.1371/journal.pone.0005060.t001 from a decrease in Bmi1 facilitation of Ring1B E3-ligase activity towards H2A, as the population of depleted Bmi1 corresponded largely to the pool that interacted with Ring1B. Whether the reduction in Bmi1 alters the positioning of Ring1B relative to H2A and/or affects the polyubiquitination status of Ring1B remains to be elucidated. To date, this is the only specific polycomb function that has been shown to be compromised by SYT-SSX2, suggesting that only a subset of polycomb target genes may be affected by the chimera. In addition, we presented evidence to suggest that the Bmi1 depletion and histone H2A hypoubiquitination correlated with the reactivation of polycomb-repressed genes. To demonstrate this derepression, we adopted a genomewide approach that relied on microarray analysis to determine which transcripts were induced by SYT-SSX2. The output from these analyses were compared with another study whereby polycomb target promoters were identified utilizing a combination of chromatin immunoprecipita- tion and microarray analysis. One important limitation of these comparison studies is that the genomewide analysis conducted by Lee et al. (2006), as well as others, were performed in the context of embryonic stem (ES) cells. However, studies performed on a prototypic polycomb target promoter –the NGFR gene- corrob- orated our central observation of Bmi1 attenuation by SYT-SSX2. Further chromatin studies in U2-OS and other mesenchymal cells are ongoing. Although the influence of indirect effects cannot be discounted and unlisted genes may be down-regulated to undetectable levels, the microarray results support the notion that SYT-SSX2 may predominantly activate rather than suppress genes. Notably, a large number (more than half) of the 33 derepressed polycomb targets were also upregulated in the microarrays of the SYT-SSX2-derived murine synovial sarcomas as well as human synovial sarcoma tissues [23,32,33,34,35]. The overlap includes either the same factors such as Jagged2 (JAG2), Delta-like 1 (DLL1), Sal-like 1 (SALL1), dual specificity phosphatase 9 (DUSP9), nerve growth factor receptor (NGFR), ephrin B3 receptor (EPHB3), suppressor of cytokine signaling 3 (SOC3), Msh-homeobox 1 (MSX1), chromobox homolog 4 (CBX4), BCL2-Like11 (BCL2L11), cytokine receptor-like factor 1 (CRLF1), member RAS family (RAB40), solute carrier family 16 (SLC16), nuclear factor of activated T-cells (NFATC1), or members of the same signaling families such as JAG1, SALL2, MSX2, transducin-like enhancer of split 3 (TLE3), G-protein receptors (GPR), ephrin receptors (EPHB1,2,4), NFATC4, CBX2,5, and platelet-derived growth factor (PDGF). Several of the polycomb-silenced transcripts upregulated by SYT-SSX2 corresponded to proteins that are implicated in tumor formation and progression. Several members of the Notch signaling pathway, including DLL1, JAG2 and HES4, displayed increased steady state mRNA in the presence of SYT-SSX2. Activating mutations of Notch signaling receptors or overexpression of the Notch ligands (DLL, JAG) have been observed in hematological, breast, cervical, and intestinal tumors [38]. Increased Notch signaling can promote cellular transformation, epithelial-to-mesenchymal transition and tumor vascularization in the appropriate context. Another gene, EphB3, corresponds to a receptor of the ephrin signaling pathway, a cascade whose deregulation contributes to tumor cell shape, adhesion, migration and angiogenesis [7]. Finally, PDGF-B corresponds to a ligand in the platelet derived growth factor pathway that can mediate tumor migration and capillary network formation. Reactivation of these genes by SYT-SSX2 could PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5060