therefore ultimately facilitate tumorigenesis and/or tumor pro- gression in synovial sarcoma. The finding that a reputed oncogene can suppress certain aspects of polycomb silencing was somewhat unexpected. The majority of work examining the role of polycomb silencing in cancer has established that oncogenicity is often associated with a polycomb gain of function. However, several lines of evidence, including the observed reactivation of the polycomb target Hox genes [19,21] and downregulation of some polycomb proteins in limited cancers [16], have suggested that polycomb derepression can equate to tumorigenesis in the appropriate context. In addition, the transforming activity attained by a loss-of-function mutant of the polycomb subunit PC2 provides more direct evidence that polycomb silencing can also be impaired in cancer. Recently, a landmark study also demonstrated that a consistent decrease in global ubiquityl-histone H2A occurs in prostate cancer, suggesting that H2A hypoubiquitination may serve as a tumor marker in this disease [21]. The significance of the work presented in the current study lies in the novel demonstration of disrupted polycomb silencing caused by an oncogene. A crucial and precarious balance in Polycomb function is needed for maintaining the homeostasis of the cell. This balance in polycomb gene silencing is mediated by processes including histone H2A ubiquitination, chromatin compaction, RNA poly- merase II inhibition, histone hypoacetylation and DNA methyl- ation. When any of these functions are perturbed through a gain of polycomb silencing, such as when polycomb components are overexpressed, tumor-promoting phenotypes are induced through the repression of tumor suppressors. However, a shift towards impairment of certain polycomb silencing functions can also promote cancer through the reactivation of oncogenic genes. In this regard, it is important to emphasize that many polycomb gene targets are overexpressed in cancer, including members of the Wnt, Notch and ephrin signaling pathways [9–11]. Although it has yet to be determined, a possible mechanism explaining aberrant signaling through these pathways in cancer may reside in the suppression of polycomb silencing. Based on the available evidence, we believe that SYT-SSX2 protein is recruited to a subset of polycomb repressive complexes within the nucleus via direct and/or indirect interactions with polycomb components or other co-residing chromatin modifiers. This SYT-SSX2 recruitment promotes the displacement and/or degradation of Bmi1 protein, which alters normal Bmi1-mediated functions, such as histone H2A monoubiquitination. In addition, the SYT-SSX2 chimera, through physical interactions [29,30], may recruit both the SWI/SNF chromatin remodeling complex and p300 acetyltransferase to this polycomb-repressed chromatin. The aberrant targeting of these proteins may then promote changes that culminate in the transcription of genes that were once silenced by polycomb complexes. The protein products of these upregulated genes could then ultimately contribute to some aspect of synovial sarcoma pathogenesis. Taken together, these studies provide an interesting insight into the molecular function of SYT-SSX2. When carried further, these analyses of chromatin deregulation by the fusion protein will be beneficial for targeting synovial sarcoma. Moreover, they will enhance our understanding regarding the regulation of polycomb components. Materials and Methods Cells and Reagents The U2OS osteosarcoma cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal SYT-SSX2 Antagonizes Polycomb bovine serum (FBS). Antibodies for Western blotting and immunofluoresence included anti-Bmi1 (Upstate; Lake Placid, NY), anti-Ring1B (MBL; Woburn, MA), anti-YY1 (Santa Cruz Biotechnology; Santa Cruz, CA), anti-tubulin (Sigma; St. Louis, MO), anti-ubiquitinated H2A (Upstate; Lake Placid, NY), anti- H2A (Upstate; Lake Placid, NY) anti-HA (Sigma; St. Louis, MO) and anti-FLAG (Sigma; St. Louis, MO). The SSX2-specific polyclonal antibody and the SV11 monoclonal antibody were described in Pretto et al, 2006 [39]. Plasmids The SYT, SYTdel8, SYT-SSX2 cDNAs were inserted into the pOZ retroviral construct (a gift from P. Nakatani) as described previously [39]. Construction of pGST-SYT-SSX2 was previously described [29]. The LZRS-Bmi12PY-IRES-GFP and pGST-Bmi1 vectors were kind gifts from M. Lohuizen. The pCS2+-Ring1B vector for in vitro translation of Ring1B was a gift from A. Ciechanover. For construction of SYT-SSX2 deletion mutants, designated SYT-SSX2del1-del9, site directed mutagenesis was performed using pOZ-SYT-SSX2 as a template. Primers utilized in the mutagenesis reactions were as follows: SYTSSXDel1F-59- CCTTATGGATATGACCAGG-TGCCAGAAGCATCTGGC- 39; SYTSSXDel1R-59-GCCAGATGCTTCTGGCACCT-GGT- CATATCCATAAGG-39; SYTSSXDel2F-59-GGAAATGATTC- GGAGGAAAC-TACCTCTGAGAAGATT-39; SYTSSXDel2R- 59-AATCTTCTCAGAGGTAGTTTCC-TCCGAATCATTTC- C-39; SYTSSXDel3F-59-TGCCCCCCGGGAAAACCACACA- GA-CTGCGTGAGAGA-39; SYTSSXDel3R-59-TCTCTCAC- GCAGTCTGTGTGGTTTTC-CCGGGGGGCA-39; SYTSSX- Del4F-59-GAACATGCCTGGACCGGCCGCTGGAGG-AGA- CTA-39; SYTSSXDel4R-59-TAGTCTCCTCCAGCGGCCGG- TCCAGGCATGTT-C-39; SYT-SSXDel5F-59-TGCCCCCCG- GGAAAACCAGGACCCAAAAGGGGGGA-AC-39; SYT-SSX- Del5R-59-GTTCCCCCCTTTTGGGTCCTGGTTTTCCCG- GGGGG-CA-39; SYT-SSXDel6F-59-TCTCTCACGCAGTC- TGTGAGATCTCTCGTGAATCTT-39; SYTSSX-Del6R-59- TCTCTCACGCAGTCTGTGAGATCTCTCGTGAATCTT- 39; SYTSSXDel-7F-59-TCTGGACCCAAAAGGGGGAGAAA- ACAGCTGGTGATT-39; SYTSSXDel7R-59-AATCACCAGC- TGTTTTCTCCCCCTTTTGGTCCAGA-39; SYTSSXDel8F- 59-GGGAACATGCCTGGACCATTTATGAAGAGATCAGC- 39; SYTSSXDel8R-59-GCTGATCTCTTCATAAATGGTCCA- GGCATGTTCCC-39; SYTSSXDel9F-59-GAGAGAAAACAG- CTGGTGGCGGCCGCTGGAGGAGAC-39; SYTSSXDel9R- 59-GTCTCCTCC AGCGGCCGCCACCAGCTGTTTTCTC- TC-39. Retroviral Transduction of U2OS Cells The SYT, SYTdel8, SYT-SSX2 cDNAs were inserted into the pOZ retroviral vector and the infections were conducted as described previously [39]. RT-PCR For RT-PCR analysis, total RNA was first extracted from retrovirally-infected cells using the RNAeasy Miniprep Kit (Qiagen). 1 mg of total RNA was subjected to reverse transcription reaction using the Superscript II Reverse Transcription System (Invitrogen). A 1 ml volume of the cDNA reaction was amplified using PCR with the following specific primers, BMI1-F: 59- GGTACTTCATTGATGCCACAAC-39, BMI1-R: 59-CTGC- TGGGCATCGTAAGTATC-39, NGFR-F: 59-GGCACCTCCA- GAACAA-GACCTC-39, NGFR-R: 59-ACAGGGATGAGGT- TGTCGGTG-39, RRAD-F: 59-TTTA-CAAGGTGCTGCT- GCTGGG-39, RRAD-R: 59-TGCCGCTGATGTCTCAAT- PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5060