expressed in MM. Enrichment profile generated after a gene-set enrichment (GSE) analysis of the five PRC2 target genes (CIITA, GATA2, CDH6, CXCL12 and ICSBP/IRF8) when compared to Mayo clinic dataset (Chng, Kumar et al. 2007) (GSE6477) using software GSEA v 2.05 with FDR q-value 0.0181. Found at: doi:10.1371/journal.pone.0011483.s003 (0.68 MB TIF) Figure S4 DZNep and LBH589 deplete EZH2 protein expression in a concentration-dependent manner. (A): The RPMI 8226 cells and (B): The U-266-1984 cells were treated for 48 hours with 0.5, 2.5 and 10 mM DZNep; (C): the RPMI 8226 cells were treated for 72 hours with 4, 10 and 20 nM LBH589 and (D): the U-266-1984 cells were treated for 24 hours with 4, 20 and 100 nM LBH589 followed by western blot analysis using anti-EZH2 antibody. Actin was used to control for equal loading. Found at: doi:10.1371/journal.pone.0011483.s004 (0.14 MB TIF) Figure S5 DZNep and LBH589 reduce growth of the RPMI 8226 and U-266-1982 cells. (A) and (B): RPMI 8226 and U-266- 1984 were treated with the indicated concentrations of DZNep (48 hours) and LBH589 (24 hours) followed by AlamarBlue assay. At least 2 experiments were performed in triplicates; data are presented as mean percentage of control 6SD. Found at: doi:10.1371/journal.pone.0011483.s005 (0.08 MB TIF) Figure S6 DZNep and LBH589 have mild effect on growth of the normal SK 1064 cells and PBMC. (A) and (B): SK 1064 and PBMC were treated with the indicated concentrations of DZNep (48 hours) and LBH589 (48 hours) followed by resazurin assay. Experiment was performed in triplicates; data are presented as mean percentage of control 6SD. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque separation of buffy coats from healthy donors. References 1. Hallek M, Bergsagel PL, Anderson KC (1998) Multiple myeloma: increasing evidence for a multistep transformation process. Blood 91: 3–21. 2. Trudel S, Li ZH, Wei E, Wiesmann M, Chang H, et al. (2005) CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t(4;14) multiple myeloma. Blood 105: 2941–2948. 3. Bild AH, Yao G, Chang JT, Wang Q, Potti A, et al. (2006) Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 439: 353–357. 4. Mattioli M, Agnelli L, Fabris S, Baldini L, Morabito F, et al. (2005) Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma. Oncogene 24: 2461–2473. 5. Agnelli L, Fabris S, Bicciato S, Basso D, Baldini L, et al. (2007) Upregulation of translational machinery and distinct genetic subgroups characterise hyperdip- loidy in multiple myeloma. Br J Haematol 136: 565–573. 6. Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, et al. (2007) Frequent engagement of the classical and alternative NF-kappaB pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell 12: 115–130. 7. Zhan F, Hardin J, Kordsmeier B, Bumm K, Zheng M, et al. (2002) Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells. Blood 99: 1745–1757. 8. Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, et al. (2007) Promiscuous mutations activate the noncanonical NF-kappaB pathway in multiple myeloma. Cancer Cell 12: 131–144. 9. Shaughnessy JD, Jr., Zhan F, Burington BE, Huang Y, Colla S, et al. (2007) A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1. Blood 109: 2276–2284. 10. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, et al. (2008) An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40: 499–507. 11. Yu J, Rhodes DR, Tomlins SA, Cao X, Chen G, et al. (2007) A polycomb repression signature in metastatic prostate cancer predicts cancer outcome. Cancer Res 67: 10657–10663. 12. Simon JA, Kingston RE (2009) Mechanisms of polycomb gene silencing: knowns and unknowns. Nat Rev Mol Cell Biol 10: 697–708. Polycomb Silencing in MM Found at: doi:10.1371/journal.pone.0011483.s006 (0.09 MB TIF) Table S1 H3K27me3 targets among the 10% most under expressed genes in MM, MGUS and MM stage III, corresponding to figure 1a, 1b and 1c. Complete gene list of H3K27me3 targets (Bracken, Dietrich et al. 2006) among the 10% most under expressed genes in MM (Zhan, Hardin et al. 2002), MGUS (Zhan, Barlogie et al. 2007) and MM stage III (Agnelli, Fabris et al. 2007), corresponding to figure 1a, 1b and 1c. Found at: doi:10.1371/journal.pone.0011483.s007 (0.33 MB XLS) Table S2 Primers used in real-time qPCR for chromatin immunoprecipitation (ChIP). Primers used in real-time qPCR for chromatin immunoprecipitation (ChIP). Found at: doi:10.1371/journal.pone.0011483.s008 (0.03 MB DOC) Acknowledgments We are grateful to Charlotta Sandberg and Pernilla Martinsson for the excellent technical assistance and to Nicola Cahill and Fiona Murray for comments on the manuscript. Author Contributions Conceived and designed the experiments: AK MF ML CF KV F O¨ HJW. Performed the experiments: AK MF ML CF PA ME SD. Analyzed the data: AK MF ML CF PA. Contributed reagents/materials/analysis tools: PA A O¨. Wrote the paper: AK. Supervised the project, interpreted data and edited the manuscript: HJW KV F O¨. Developed the concept, performed the integrative genomics analysis and interpreted data: MF. Provided clinical expertise: A O¨. Edited the manuscript: KN. 13. Hansen KH, Bracken AP, Pasini D, Dietrich N, Gehani SS, et al. (2008) A model for transmission of the H3K27me3 epigenetic mark. Nat Cell Biol 10: 1291–1300. 14. Ohm JE, McGarvey KM, Yu X, Cheng L, Schuebel KE, et al. (2007) A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing. Nat Genet 39: 237–242. 15. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, et al. (2007) Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet 39: 232–236. 16. Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, et al. (2007) Epigenetic stem cell signature in cancer. Nat Genet 39: 157–158. 17. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, et al. (2006) The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439: 871–874. 18. Kondo Y, Shen L, Cheng AS, Ahmed S, Boumber Y, et al. (2008) Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation. Nat Genet 40: 741–750. 19. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K (2006) Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 20: 1123–1136. 20. Zhan F, Barlogie B, Arzoumanian V, Huang Y, Williams DR, et al. (2007) Gene-expression signature of benign monoclonal gammopathy evident in multiple myeloma is linked to good prognosis. Blood 109: 1692–1700. 21. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, et al. (2004) ONCOMINE: a cancer microarray database and integrated data-mining platform. Neoplasia 6: 1–6. 22. Chng WJ, Kumar S, Vanwier S, Ahmann G, Price-Troska T, et al. (2007) Molecular dissection of hyperdiploid multiple myeloma by gene expression profiling. Cancer Res 67: 2982–2989. 23. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, et al. (2007) The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev 21: 525–530. 24. Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, et al. (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298: 1039–1043. 25. Fiskus W, Pranpat M, Balasis M, Herger B, Rao R, et al. (2006) Histone deacetylase inhibitors deplete enhancer of zeste 2 and associated polycomb repressive complex 2 proteins in human acute leukemia cells. Mol Cancer Ther 5: 3096–3104. PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11483