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Simon et al. Supplementary information: Table of contents p.1 Supplementary material and methods p.2-4 • PoIy(I)-poly(C) Treatment • Flow Cytometry and Immunohistochemistry • Western Blotting • Quantitative RT-PCR • Fluorescence In Situ Hybridization • RNA-Seq • Exome capture • Sequencing Supplementary Figures and Tables Suppl. items Description pages Figure 1 Inactivation of Ezh2 affects normal thymocyte development 5 Figure 2 Ezh2 mouse leukemias express cell surface T cell receptor 6 Figure 3 Expression of EZH2 and Hox genes in T-ALL 7 Figure 4 Additional mutation et deletion of chromatin modifiers in T-ALL 8 Figure 5 PRC2 expression and activity in human lymphoproliferative disease 9 Figure 6 PRC2 regulatory network (String analysis) 10 Table 1 Primers and probes for detection of PRC2 genes 11 Table 2 Patient and T-ALL characteristics 12 Table 3 Statistics of RNA and DNA sequencing 13 Table 4 Mutations found in human T-ALLs (see Fig. 3D and Suppl. Fig. 4) 14 Table 5 SNP populations in analyzed human T-ALL samples 15 Table 6 List of altered genes in T-ALL for DAVID analysis 20 Table 7 List of David functional clusters 31 Table 8 List of acquired SNP tested in normal non leukemic DNA 32 1 Simon et al. Supplementary Material and Methods PoIy(I)-poly(C) Treatment. pIpC (GE Healthcare Lifesciences) was dissolved in endotoxin-free D-PBS (Gibco) at a concentration of 2 mg/ml. Mice received four consecutive injections of 150 μg pIpC every other day. The day of the last pIpC injection was designated as day 0 of experiment. Flow Cytometry and Immunohistochemistry. Flow cytometric analyses were performed using BD Canto and BD FACSDiva 4.1 software (BD Pharmingen). Data were analysed using FlowJo V7. Antibodies used were: phycoerythrin (PE)-conjugated anti- CD4, allophycocyanin (APC)-conjugated anti-CD8, fluorescein isothiocyanate FITC)- conjugated anti-CD45 R (B220), FITC-conjugated anti-Ly6-G (GR1), PE-Cy5-conjugated anti-TCRβ, biotin-conjugated anti-TCR γδ (BD Biosciences); FITC-conjugated anti-Ly76 (Ter119), FITC-conjugated anti-CD11b, APC-Alexa Fluor 750-conjugated anti-CD3, and streptavidin-PE-Cy7 (eBiosciences). 5 μm tissue sections were prepared using standard histological techniques. Immunohistochemical reactions were carried out using primary rat anti-CD3 (AbD Serotec) or rabbit anti-Ki-67 (Biocare Medical) antibodies, secondary biotin-conjugated anti-rat or anti-rabbit antibodies (Vector). Location of bound antibodies was revealed using streptavidin-conjugated horseradish peroxidise and 3,3- diaminobenzidine according to the manufacturer’s instructions (Ventana Medical Systems). Sections were counterstained with hematoxylin. Images of tissue sections and the Wright stain–dyed cytospin preparations of bone marrow and spleen at 40-fold magnification were acquired using a Zeiss Axio-Imager Z1 microscope with an 40x / 1.3 Plan-Apochromat, oil, DIC objective and Canon MK2 camera fitted with LmScope adapter (Micro-Tech- Lab) and Canon EOS utility software. The resulting JPEG files were transformed into TIFF files using Adobe Photoshop version 11. Western Blotting. Total cellular lysates were prepared by boiling cells in 1X Laemli buffer. Primary antibodies used for western blotting were anti-EZH2 (Cell Signaling Technology, 3147) and anti-α-tubulin (Cell Signaling Technology, 2144), anti-EZH1 (Abcam, 13665), anti-SUZ12 (Cell Signaling Technology, 3737); anti-Histone H3 (Abcam, AB1220), anti-Histone H3 (tri methyl K27) (Millipore, 07-449) and anti-Histone H3 (di methyl K27) (Abcam, ab24684), anti-Histone H3 (mono methyl K27) (Millipore 07- 448). Secondary anti–rabbit and anti–mouse HRP-conjugated antibodies were from SantaCruz Biotechnology. 2 Simon et al. Quantitative RT-PCR. Total RNA from the bone marrow, spleen, and thymus of wild- type and sick Ezh2F/Δ mice was isolated using Trizol™ according to the manufacturer’s protocol (Invitrogen, CA) and treated with DNase I (Invitrogen, Carlsbad, CA) before cDNA synthesis. Reverse transcription of total RNA was performed using Superscript II- reverse Transcriptase and random hexamers according to the manufacturer’s protocol (Invitrogen). Gene expression assays were designed using Roche Universal ProbeLibrary Assay Design software and were tested for maximum efficiency by standard curve analysis. Primer sequences and Universal ProbeLibrary numbers listed in supplementary Table 1. Reference endogenous control Taqman® assay for Gapdh (glyceraldehyde-3-phosphate dehydrogenase) was purchased from Applied Biosystems (Carlsbad, CA). Gene expression analysis was performed on the ABI 7900HT Fast Real- Time PCR System (Applied Biosystems) or the Light Cycler 480 Real-Time PCR system (Roche). Reactions were performed in 384-well plates with an initial step of 3 minutes at 95˚C, followed by 40 cycles of: 5 sec at 95˚C and 30 sec at 60˚C. To determine the level of target gene expression, threshold cycle (Ct) values of target genes were normalized to endogenous control gene (Gapdh) (Ct = Ct target – Ct Gapdh). All reactions were done in triplicate and the average values were used for quantification. Total RNA from human leukemia specimens was isolated using TrizolTM according to the manufacturer’s protocol (Invitrogen). After DNase I treatment, the total RNA was reverse transcribed using random primers and MMLV-reverse transcriptase as recommended by manufacturer (Invitrogen). Taqman-based quantitative RT-PCR assays were performed in triplicate as described above. Primer and probe sequences for human qRT-PCR assays are listed in Supplementary Table 1. Fluorescence In Situ Hybridization. Preparation of interphase nuclei from bone marrow or peripheral blood cells and fluorescence in situ hybridizations (FISH) experiments were performed using standard procedures. The following commercial probes were used to characterize the leukemic samples: Vysis LSI CDKN2A SpectrumOrange/CEP9 SpectrumGreen Probes; Vysis LSI ETV6 Dual Color Break Apart Rearrangement probe; Vysis LSI BCR/ABL ES Dual Color Translocation Probe; Vysis LSI MYC Dual Color Break Apart Rearrangement probe (Abbott Molecular), and LPH049 TLX1 and LPH050 TLX3 Breakapart probes (Cytocell, Cambridge, UK). Bacterial artificial chromosomes (BACs), RP11-1140K8, covering the EED gene at chromosomal band 11q14.2, RP11-28C14 covering the EZH2 gene at band 7q36.2, and RP11-640N20 covering the SUZ12 gene at band 17q11.2, were selected from the UCSC 3 Simon et al. genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway) and obtained from BACPAC Resources Center (Children's Hospital Oakland Research Institute, Oakland, CA; http://bacpac.chori.org/). BAC probes were labeled with Spectrum Orange-dUTP by nick translation according to manufacturer’s instructions (Abbott Molecular), denatured and hybridized on pretreated slides. Slides were incubated at 37°C for 16 hours in a humidified chamber, washed 10 seconds in 0.4X SSC/0.3% NP-40 and in 2X SSC/0.1% NP-40 solutions at 55°C and counterstained with DAPI II (Abbott Molecular). FISH signals were captured using CytoVision® software version 3.6 (Leica Microsystems). BACs were validated on normal metaphases and a 10% cut-off level for deletion (mean ± 3 SD) was established by analyzing 1500 nuclei from 5 different normal blood samples. Centromeric probes labeled with Spectrum Green were co-hybridized with the BAC located on the same chromosome (CEP 11, CEP7 or CEP17, Abbott Molecular). A minimum of 200 interphase nuclei was analyzed by two experienced observers for each leukemic sample. RNA-Seq. Total RNA from bone marrow or blood samples (~5 million cells) was isolated using TrizolTM as recommended by the manufacturer (Invitrogen), and then further purifed using RNeasy columns (Qiagen). Sample quality was assessed using Bioanalyzer RNA Nano chips (Agilent). Transcriptome libraries were generated from 4μg total RNA using the TruSeq RNA Sample Prep Kit (v2) (Illumina) following the manufacturer’s protocols. Exome capture. Genomic DNA was extracted from bone marrow or blood samples (~5 million cells) using the DNeasy Blood and Tissue Kit as recommended by the manufacturer (Qiagen). Genomic libraries were constructed from 1μg of genomic DNA using the TruSeq DNA Sample Prep Kit (v2) (Illumina) following the manufacturer’s protocols. The DNA was sheared using a Covaris S2 instrument using the “TruSeq enrichment” settings as specified in the Illumina protocol. Target capture was done using the TruSeq Exome Enrichment Kit (Illumina) as recommended by the manufacturer. Four samples were pooled per enrichment reaction. Sequencing. Paired-end (2 x 100bp) sequencing was performed using the Illumina HiSeq2000 machine running TruSeq v3 chemistry. Cluster density was targeted at around 600-800k clusters/mm2. Two transcriptomes or a pool of four exomes were sequenced per lane. 4 Simon et al. A B 50 6 WT Δ/Δ 40 2% 86 +- 2% 25 +- 3% 4.2+- 4% 30 20 Δ/Δ WT Ezh2 + + 10 5 +- 2% 7 - 1% 51 - 2% 26 +- 2% Cells/thymus ( x 10 ) 0 CD8 CD4 Supplementary Figure 1. Inactivation of Ezh2 affects normal thymocyte development. A) Total Δ/Δ thymocyte numbers in WT and Ezh2 mice at 12 days after pIpC treatment. Mean +- SD, n=4. B) Distribution of thymocyte subsets in WT and Ezh2Δ/Δ mice at 12 days after pIpC treatment. Note that Ezh2 inactivation leads to accumulation of immature CD4-CD8-, and disappearance of CD4+CD8+ cell populations. Numbers shown represent average proportions (mean +- SD, n=2) of CD8+, CD8+CD4+, CD4+ and CD8-CD4- thymocytes. 5 Simon et al. A BM Liver Kidney Kidney WT 10 µm 10 µm 20 µm 10 µm ∆/∆ 10 µm 10 µm 10 µm 10 µm CD3 Ki67 Supplementary Figure 2. A) Left panels: infiltration of lymphoid cells in the bone marrow (BM), liver and kidney of sick Ezh2∆/∆ mouse visualized by immunohistochemical detection of CD3, 40X ∆/∆ magnification. Righ panels: accumulation of actively proliferating cells in the kidney of sick Ezh2 mouse visualized by immunohistochemical detection of the proliferating cell antigen Ki67, 40X magnification. B Lin- CD3+ cells 98 1.7 WT ∆ ∆ 0.6 97 Supplementary Figure 2. B) Ezh2 / leukemias express cell surface T cell receptor (TCR) γδ.TCR expression was examined on cells isolated ∆/∆ #7 from enlarged lymph nodes of sick Ezh2 mice.