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Table S1. PAX5 fusions reported in B-cell ALL Fusion PAX5-ETV6 Exona 4 4 4 4 4 4 4 7 7 5 6 5 6 7 6 8 5 8 8 PAX5 aa Exona 1-158 3 1-158 3 1-158 3 1-158 3 1-158 3 1-158 2 1-158 3 1-303 2 1-303 2 1-260 2 1-260 7 1-201 12 1-260 7 1-303 4 1-260 4 1-337 3 1-201 8 1-337 8 1-303 3 Fusion aa 55-452 55-452 55-452 55-452 55-452 12-452 55-452 27-1001 27-1001 44-end 60-705 291-705 60-7-5 74-1311 208-1258 70-436 358-436 358-436 70-436 No.b D.N.c 1 n.d. 2 n.d. 2 Yes 2 Yes 7 n.d 1 n.d. 3 n.d. 2 Yes 1 n.d. 2 n.d. 1 Yes 1 Yes 2 n.d. 1 Yes 1 n.d. 2 Yes 1 Yes 1 n.d. 1 n.d. Pred. Oligod Yes-SAM Yes-SAM Yes-SAM Yes-SAM Yes-SAM Yes-SAM Yes-SAM Yes Yes Yes-CC Yes-CC unknowne Yes-CC weak-CCf unknown Yes-CC Yes-CC Yes-CC Yes-CC Reference(s) (Cazzaniga et al., 2001) (Strehl et al., 2003) (Mullighan et al., 2007) (Kawamata et al., 2008) (An et al., 2008) (An et al., 2008) (Coyaud et al., 2010) PAX5-ELN (Bousquet et al., 2007) (Coyaud et al., 2010) PAX5-PML (Nebral et al., 2007) PAX5-FOXP1 (Mullighan et al., 2007) (Kawamata et al., 2008) (Coyaud et al., 2010) PAX5-ZNF521 (Mullighan et al., 2007) PAX5-AUTS2 (Kawamata et al., 2008) PAX5-C20orf112 (Kawamata et al., 2008) (Kawamata et al., 2008) (Nebral et al., 2009) (An et al., 2008) (An et al., 2009) PAX5-JAK2 5 1-201 19 812-1132 2 n.d. Yes-CC (Nebral et al., 2009) 5 1-201 19 812-1132 1 n.d. Yes-CC (Coyaud et al., 2010) PAX5-BRD1 5 1-201 1 1-1058 (+4)g 1 n.d. Yes-CC (Nebral et al., 2009) PAX5-POM121 5 1-201 5 1-999 (+88)g 1 n.d. unknown (Nebral et al., 2009) g 5 1-201 4 1-999 (+72) 1 n.d. unknown (Coyaud et al., 2010) PAX5-HIPK1 5 1-201 9 662-1210 1 n.d. unknown (Nebral et al., 2009) PAX5-DACH1 5 1-201 5 376-706 1 n.d. Yes-CC (Nebral et al., 2009) PAX5-DACH2 5 1-201 3 213-599 1 n.d. Yes-CC (Coyaud et al., 2010) PAX5-GOLGA6 6 1-260 3 96-693 1 n.d. Yes-partial CC (Coyaud et al., 2010) PAX5-LOC392027 4 1-158 1 n.a.h 1 n.d. unknown (An et al., 2008) PAX5-NCoR1 5 1-201 43 2226-2440 1 Yes unknown (Coyaud et al., 2010) PAX5-SLCO1B3 4 1-158 1 n.a. h 1 n.d. unknown (An et al., 2008) PAX5-TAOK1 5 1-201 19 17 aai 1 n.d. unknown (Coyaud et al., 2010) PAX5-ASXL1 4 1-158 4 125-1541 1 n.d. unknown (An et al., 2008) (An et al., 2009) j 3’reg 1-391 1 n.d. unknown (An et al., 2009) PAX5-KIF3B 7 1-303 6 656-747 1 n.d. Yes CC (An et al., 2008) (An et al., 2009) PAX5-truncated 5 1-201 intergen. 55 add. aa 1 Yes unknown (Coyaud et al., 2010) a Based on RT-PCR, bNumber Identified, c Possess dominant negative activity, dPredicted motif for oligomerization-CC refers to coiled-coil, but these are amphipathic helices that may mediate tetramer formation as in the case of PML (Antolini et al., 2003), enot in-frame, results in 34aa fused to C-terminus of PAX5, f does not contain NuRD binding domain, has zinc finger regions that probably mediate homo-oligomerization, g Includes additional amino acids not found in exons, hn.a. - not available, ireverse orientation to its open reading which results in an additional 17 amino acid tail. jrefers to 3’UTR after last PAX5 exon. Supplemental Figures Figure S1. Alignment of C20S homologs * indicates identity. : indicates similar amino acids Figure S2. Luciferase reporter assays demonstrating YFP-PAX5-wt and YFPPAX5-C20S fusion proteins have similar activities to PAX5-wt and PAX5-C20S, respectively. (A) An empty, wt PAX5, or YFP-PAX5-wt expression vector was co-transfected with a reporter gene containing three repeats of PAX5 recognition sites derived from the murine CD19 promoter and the luciferase gene. Luciferase activity was measured 24-48 hours after the transfection into 293T cells. The results show the averages of three experiments. (B) PAX5-C20S suppressed the transcriptional activity of wt PAX5 in a dominant negative fashion. Expresssion vectors for wt PAX5 and PAX5-C20S were cotransfected at ratios of 1:1 and 1:0.2 as indicated by the ramps. Luciferase activity was measured 24-48 h after the transfection into 293T cells. The results show the averages of three experiments. (C) YFP-PAX5-C20S was co-transfected with wt PAX5 and the luciferase reporter. Expresssion vectors for wt PAX5 and YFP-PAX5-C20S were co-transfected at ratios of 1:1 and 1:0.2 as indicated by the ramps. Luciferase activity was measured 24-48 h after the transfection into 293T cells. The results show the averages of three experiments. Figure S3. Immuno-blotting of lysates from luciferase assays with anti-YFP antibody to show relative protein levels. Lysates from reporter assays in Figures 2A, 4A, 5C, and 6B and C were resolved on SDS-PAGE gels, transferred to nitrocellulose membranes, and blotted with rabbit antiFP Living colors antibody from Clontech. YFP-PAX5-wt is the upper band marked by arrows. A and B above the blots refer to lysates from duplicate assays performed in parallel. Note YFP-wtPAX5 expressed at higher levels in every assay except the assay with YFP-PAX5-L344P. Figure S4. Luciferase assay with YFP-PAX5-ΔDBD-C20S, and sub-cellular localization of YFP-labeled constructs. (A) YFP-PAX5-ΔDBD-C20S does not inhibit the transcriptional activity of PAX-wt. YFPPAX5-C20S or YFP-PAX5-ΔDBD-C20S were co-transfected with YFP-PAX5-wt and the reporter gene. Luciferase activity was measured 24 hours after transfection into H1299 cells. (B) YFP-PAX5-wt, YFP-PAX5-C20S, and YFP-C20orf112 localize to the nucleus. Fluorescence signals of YFP (without a NLS), YFP-PAX5-wt, YFP-PAX5-C20S, or YFPC20orf112 were detected by confocal microscopy. Nuclei were stained with DAPI. Supplement: Figure S5 The potential Groucho binding sequence does not contribute to PAX5-20S DN-activity. SAM domain of PAX5-ETV6 mediates its DNactivity. (A) FRAP assays in H1299 cells using YFP-PAX5-C20S with a point mutation in the octapeptide sequence (Y179E) of PAX5. YFP-PAX5-C20S (black circles, N=5, t1/2 200 s) and YFP-PAX5-(Y179E)-C20S (blue diamonds, N=6, t1/2 200s). (B) Luciferase reporter and YFP-PAX5-wt were co-transfected with YFP=PAX5C20S, YFP-PAX5(Y179E)-C20S, YFP-PAX5-wtp53tetra, or YFP-PAX5-(Y179E)wtp53tetra. Luciferase activity was measured 24 h post-transfection into H1299 cells. (C) FRAP assays of YFP-PAX5-ETV6 (t1/2 200 s), YFP-PAX5-SAM (t1/2 200 s), and YFP-PAX5-C20S (t1/2 200 s). (D) PAX5-ETV6 or PAX5-SAM were transiently expressed with wt PAX5 using 1:1 and 1:0.2 ratios of the expression vectors for wt PAX5 and the PAX5-fusions (indicated by the ramps) and activation of the PAX5 luciferase reporter assayed in 293T cells. Additional supplementary Information is available at the Oncogene website Supplemental Experimental Procedures FRAP procedure and analysis The settings for FRAP experiments were as follows: 800 Hz, 8× zoom, and an image format 512×256. Fluorescence emission from EYFP was detected with the range of photomultiplier tube (PMT) 2 set to 525–55 nm (with a maximal gain of 680). Bleaching of a circle with a 2 μm radius was performed with laser line 514 nm set to maximal power. Individual FRAP experiments were performed for each construct and then averaged to generate a single FRAP curve. Average background values were determined outside of cells (ROI2) and subtracted from all raw data points of bleach region (ROI1) as well as inside the nucleus outside of the bleach area (ROI3) before further analysis as follows (Phair et al., 2004): Bn = (ROI1 – ROI2)/(ROI3 – ROI2). Pb = average of pre-bleach signal calculated in Bn. Normalized Fluorescence Intensity FIN = Bn/Pb. These data were then normalized based on the bleach depth, generating curves set to zero (based on the last bleach point) to pre-bleach intensity, calculated as follows: FIN – FIinitial point post bleach The time starting at the last bleach point was set to time zero. The half-times of recovery (t1/2) were calculated based on the time at half-maximal recovery. For curves that didn’t reach maximal recovery under the time measured for the experiment, then t1/2 was estimated by extrapolating the curves to maximum recovery. X-axis: time, Y-axis: normalized fluorescence signal, error bars represent SEM. Gel filtration Nuclear extracts (Dignam et al., 1983) from transfected H1299 or 293T cells were fractionated by gel filtration through a Superose 6 10/300 L column (GE Healthcare), equilibrated with D300 buffer (20 mM Hepes, pH 7.4, 300 mM KCl, 1 mM DTT, 10 mM -mercaptoethanol, 0.1% modified NP-40). 3-5 mg was fractionated at a flow rate of 0.2 ml/min at 4C using the AKTA FPLCTM controller. 0.25 ml fractions were collected, followed by SDS-page and Western blot analysis. Dextran Blue (2 MDa) was used to determine the void volume (7.5 ml, fraction 30). Protein standards were apoferritin (440 kDa), aldolase (158 kDa), ovalbumin (44 kDa), and aprotinin (6.5 kDa). Immunoblotting and immunoprecipitation For immunoprecipitations, anti-GFP(FL) cat# sc-8334 from Santa Cruz Biotechnology was used. Immunoblotting used PAX5-N-terminal specific antibody from Millipore cat# AB4227, which detected both wild-type and PAX5-fusions and anti-GFP antibody. 293T cells were used for transfection following the protocol from Qiagen using Effectene transfection reagent. Electrophoretic Mobility Shift Assays Recombinant PAX5-Exon5 and PAX5-C20 proteins were expressed in E. coli, and purified by Ni-affinity followed by Superose 6 gel filtration chromatography. DNA binding reactions contained 2 nM 32P-labeled 45 bp probe (Czerny et al., 1993) in 60 mM KCl, 1 mM MgCl2,10 mM HEPES (pH 7.9), 0.5 mM EDTA, 1 mM DTT, 0.05 g/ml bovine serum albumin, 0.1% NP-40, 1 ng/ml poly (dA-dT), and 3 % glycerol. For competition experiments, unlabeled oligonucleotide (45 bp DNA used as probe without 32P-labeling) or unlabeled plasmid DNA with the 45 bp probe sequence inserted was incubated with components of the binding buffer and labeled oligonulceotide. PAX5-Exon5 or PAX5C20S from the peak fraction of the Superose column was added for 15 min at room temperature and samples (without tracking dye) loaded for electrophoresis at 75V for 90 min on a 6% native polyacrylamide gel containing 1xTBE and 0.1% NP-40. Quantitative analysis of the radioactive signals was performed with a PhosphorImgaer. Data were analyzed using Imagequant 5.2 software, and graphed using Kaleidagraph software. The data were fit by the following model: y = Bottom + ((Top-Bottom)/(1+(IC50/x)Hill coefficient)), where y is the fraction bound, x is the concentration of the competitor, Top is the maximum signal of probe bound without competitor, Bottom is the signal of probe with all competitor bound, IC50 is the concentration of protein halfway between the baseline and the maximum, and the Hill coefficient describes the ‘steepness’ of the curve (Fauth et al., 2010). Supplemental References An, Q., Wright, S. L., Konn, Z. J., Matheson, E., Minto, L., Moorman, A. V., Parker, H., Griffiths, M., Ross, F. M., Davies, T., et al. (2008). Variable breakpoints target PAX5 in patients with dicentric chromosomes: a model for the basis of unbalanced translocations in cancer. Proc Natl Acad Sci U S A 105, 17050-17054. An, Q., Wright, S. L., Moorman, A. V., Parker, H., Griffiths, M., Ross, F. M., Davies, T., Harrison, C. J., and Strefford, J. C. (2009). 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