GAAC-39, RET-F: 59-GGATTTCGGCTTGTCCCGAG-39, RET-R: 59-CCATGTGGAAGGGAGG-GCTC-39, PDGF-B F: 59-GATCCGCTCCTTTGATGATC-39, PDGF-B R: 59- GTCTCA-CACTTGCATGCCAG-39, HES4-F: 59-CACCG- CAAGTCCTCCAAG-39, HES4-R: 59-TCACCTCCGCCAGA- CACT-39. IGF2-F: 59-GAAGTCGATGCTGGTGCTTCT-39, IGF2-R: 59-TGAACGCCTCGAGCTCCTTG-39, DLL1-F: 59- GTTCGAACTGAAGCT-GCAGGA-39, DLL1-R: 59- AGAATCTGTGTGGAGAGCTTC-39, JAG2-F: 59-AAGAC- CTGAACTACTGTGGCA, JAG2-R: 59-GCATGGCTTCCCT- TCACACT-39, EPHB3-F: 59-GGTGACGTCTGAGCTGG- CATG-39, EPHB3-R: 59-TCATCTGGCGCAATGGTG-TCC- 39. GAPDH loading control was amplified using the following primers, GAPDH-F: 59-TGATGACATCAAGAAGGTGGT- GAAG-39 and GAPDH-R: 59-TCCTTGGAGG-CCATGTGG- GCCAT-39. Reaction conditions for PCR amplification are as follows: 1 cycle of 4 min. at 94uC; 32 cycles of 1 min. at 94uC, 1 min. at 56uC and 1 min. at 72uC; 1 cycle of 10 min. at 72uC. In vitro Binding Assays The generation of GST, GST-p300-CH3, GST-Bmi1 and GST-SYT-SSX2 lysates was performed as previously described [28]. In vitro translation of SYT-SSX2 and Ring1B was carried out using the coupled TnT rabbit reticulocyte in vitro transcription/ translation system (Promega Co; Madison, WI). Transcription/ translation was carried out in the presence of SP6 RNA polymerase and 20 mCi S35-methionine. Binding reactions of in vitro translated proteins and GST-fused proteins were performed in the presence of binding buffer (20 mM Tris pH 8.0, 100 mM NaCl, 0.1% NP-40, 1 mM DTT) for 1 hr. at 4uC. Beads were washed in ice-cold binding buffer 3 times, then boiled in Laemmli sample buffer. Reactions were electrophoresed on a 10% SDS- polyacrylamide gel, dried and exposed to x-ray film for autoradiography. Indirect Immunofluorescence For indirect immunofluorescence studies, coverslips were fixed in 3% paraformaldehyde/ 2% sucrose, washed with PBS and permeabilized in 0.2% Triton X-100. Cells were blocked in 3% goat serum and incubated with anti-Bmi1 (1:100), anti-Ring1B (1:100) and anti-HA (1:200) antibodies for 2 hrs, then with Alexa- conjugated secondary antibodies (Molecular Probes; Eugene, OR) for 30 min. Cells were visualized using a Zeiss (Axioplan 2) fluorescence microscope. Microarray Analysis Total RNA was extracted from infected NIH3T3 cells using the RNAeasy Miniprep Kit (Qiagen; Valencia, CA) according to the manufacturer’s instructions. For microarray experiments, RNA was submitted to the Vanderbilt Microarray Shared Resource Facility for hybridization with Affymetrix Human Genome U133 GeneChip arrays. Gene expression intensity 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]. Analysis of microarray data was carried out by the WebGestalt Program, maintained by Vanderbilt University and available online at http://bioinfo.vanderbilt.edu/ webgestalt/. Lysis and Immunoprecipitations To generate extracts for Western blotting analysis of ubiquiti- nated H2A, infected cells were lysed and boiled directly in Laemmli sample buffer. Lysate preparation for immunoprecipitation and SYT-SSX2 Antagonizes Polycomb other Western blotting procedures were performed in IP buffer (50 mM Tris pH 8.0, 250 mM NaCl, 0.5% NP-40 and protease inhibitor cocktail). Lysates used for immunoprecipitation were incubated with primary antibodies (all at 1 mg/mL final concentration) for 4 hrs at 4uC. Samples were then incubated with Protein G-sepharose (Amersham Biosciences; Uppsala, Sweden) for 30 min at 4uC. Immunoprecipitates were then washed in IP buffer and boiled in Laemmli sample buffer for Western blot analysis. Pulse Chase Experiments, Peptide Blocking and Regression Analysis U2OS cells that stably expressed 2PY-Bmi1 were infected with pOZ and SYT-SSX2 vectors as described earlier. At 48 hrs post- infection, cells (26105 cells per timepoint for each experimental condition) were starved in Methionine-minus DMEM medium (containing 5% dialyzed fetal bovine serum) for 1 hr at 37uC. Cells were then labeled with 500 mCi of S35 labeled Methionine/ Cystine diluted in Methionine-minus medium (containing 5% dialyzed fetal bovine serum) for 1 hr at 37uC. Cells were then chased at indicated time-points with Methionine-positive DMEM supplemented with 10% fetal bovine serum. Cells from each timepoint were processed and immunoprecipitated with anti- polyoma tag antibody (1 mg/mL) as described before. Immuno- precipitates were run on 10% polyacrylamide gels, which were subsequently dried and exposed to x-ray film for autoradiography. Immunoprecipitated 2PY-Bmi1 was quantitated using densitom- etry computer software (FluorChem 9800; Alpha Innotech, San Leandro, CA). To confirm the correct identity of the immuno- precipitated 2PY-Bmi1 species, 16106 cells were labeled overnight with S35 labeled Methionine/Cystine. Cells were immunoprecip- itated with anti-polyoma antibodies as before, except that the following conditions were included: na¨ıve U2OS cells, no antibody control, preclearing of lysate with Sepharose beads (30 min. at 4uC) and overnight preincubation of polyoma antibody with polyoma epitope tag peptide (20 mg/mL; Covance, Emeryville, CA). For the regression analysis, densitometry values for pOZ and SYT-SSX2 were plotted on GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) for linear regression analysis and half- life calculations. Half-lives were assessed by determining the unpaired half-life time-point values from a relative intensity value of 0.5. Chromatin Immuno-Precipitation CHIP was performed on pOZ- and SYT-SSX2-derived nuclear lysates according to the protocol described by Boyer et al. [40]. 26107 cells were used for each IP reaction. After DNA precipitation, PCR of the NGFR promoter region was performed with the following primers: NGFR-forward: 59-GCAGTTAGG- GAGCAAGGCTCC-39 and NGFR-reverse: 59-GGTGGGA- AGCAGAGGCAAAGG-39. SYT-SSX2 was IP’ed with the SSX2-specific antibody described in Pretto et al. [39]. For Ring1B and Bmi1 CHIP, the MBL and Upstate antibodies described above were used, respectively. Supporting Information Figure S1 In vitro binding assays Found at: doi:10.1371/journal.pone.0005060.s001 (0.54 DOC) Figure S2 SYT-SSX2 does not alter Bmi1 mRNA levels Found at: doi:10.1371/journal.pone.0005060.s002 (0.10 DOC) MB MB PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5060