Fig 2: EWS-WT1 DNA occupancy profiles are distinct from wild type WT1, and depend on its EWS prion-like domains.a Schematics of the constructs encoding for EWS, WT1, EWS-WT1 and the EWS(YS37)-WT1 mutant proteins. Tyrosines (Y) mutated into serines (S) are depicted as black bars within the EWS N-terminal prion-like domain of the EWS(YS37)-WT1 mutant. b Barplot depicts the number of genome-wide ChIP-seq binding sites observed for each protein as in Fig. 5a (+and -KTS isoforms for WT1, EWS-WT1 and EWS(YS37)-WT1) after their individual expression in MeT-5A cells. c Pie charts show the genomic distribution for the binding sites of the + and -KTS isoforms of WT1 and EWS(YS37)-WT1 upon their individual expression in MeT-5A cells. d Heatmaps depict V5 (WT1 + KTS or -KTS) and H3K27ac ChIP-seq signal densities in MeT-5A cells expressing either WT1 isoform, at the subset of consensus EWS-WT1 binding sites bound by either EWS-WT1 -KTS or +KTS as in Fig. 4d. 10-kb windows in each panel are centered on EWS-WT1 binding sites as defined in JN-DSRCT1 cells. Chromatin profiling was performed twice in WT1 transduced MeT-5A cell line. e Heatmaps showing V5 (EWS(YS37)-WT1 + KTS or -KTS) and H3K27ac ChIP-seq signal densities in MeT-5A cells expressing either EWS(YS37)-WT1 -KTS or +KTS mutant isoforms, at the subset of consensus EWS-WT1 binding sites bound by either EWS-WT1 -KTS or +KTS as in Fig. 4d. 10-kb windows in each panel are centered on EWS-WT1 binding sites as defined in JN-DSRCT1 cells. Chromatin profiling was performed twice in EWS(YS37)-WT1 transduced MeT-5A cell line. f EWS-WT1 + KTS and -KTS isoforms precipitate in a dose-dependent manner upon b-isox treatment in lysates from HEK293T-transfected cells. The experiments were independently repeated twice with similar results. Source data are provided as a Source Data file. g, h Wild-type WT1 and mutant EWS(YS37)-WT1 isoforms show weak precipitation properties when b-isox is added to lysates from HEK293T-transfected cells. * non-specific binding of WT1 proteins is detected in absence of b-isox. The experiment was independently repeated twice with similar results. Source data are provided as a Source Data file.

Fig 3: EWS-WT1 depletion leads to strong changes in chromatin activity and connectivity.a Left: Composite plot shows EWS-WT1 ChIP-seq signals in JN-DSRCT1 cells lentivirally induced with a shRNA targeting WT1. 10 kb window centered on EWS-WT1 binding sites (n = 6823). Right: Immunoblotting shows EWS-WT1 protein levels in the same conditions. The experiment was independently repeated twice with similar results. Source data are provided as a Source Data file. b Histogram depicts changes in H3K27ac ChIP-seq signals at EWS-WT1 binding sites upon the fusion protein knockdown in JN-DSRCT1 cells as in Fig. 3a. 4711 EWS-WT1 binding sites show H3K27ac signal decrease of at least twofold in both experiments. Twofold changes are highlighted in red (decreases) and blue (increases). c Left: Representative ChIP-seq tracks showing chromatin changes at the FGFR4 locus upon EWS-WT1 knockdown in the JN-DSRCT1 cell line. Right: Corresponding changes in FGFR4 expression levels as detected by RNA-seq. d Histograms show changes in H3K27ac Hi-ChIP looping signals in JN-DSRCT1 cells upon EWS-WT1 knockdown at sites initially bound by EWS-WT1 on one side (n = 19606), both sides (n = 4929) or none (n = 27426). H3K27ac Hi-ChIP in shRNA transduced JN-DSRCT1 was performed once. e Left: Example shows marked changes in H3K27ac Hi-ChIP signal at the VEGFA locus upon EWS-WT1 depletion in JN-DSRCT1 cells. Right: Corresponding changes in VEGFA expression levels as detected by RNA-seq. f Left: Volcano plot depicting gene expression changes in JN-DSRCT1 cells upon EWS-WT1 knock-down. y axis is capped at a maximum adjusted p value of −100. Genes connected to EWS-WT1 with twofold changes and adjusted p value of 0.05 are shown in red. Right: Boxplots show gene expression levels in control (red) and EWS-WT1-depleted cells (blue) for genes initially connected to CTCCC and/or TCC repeats bound by EWS-WT1 (n = 2176), other EWS-WT1 sites (n = 2584) or sites not associated with EWS-WT1 (n = 7150). n represents the number of genes in each category. p value in CTCCC and/or TCC repeats = 2.16e–73, in other EWS-WT1 = 0.08, in not connected to EWS-WT1 = 0.24. Median value is shown as a line within the boxplot, which spans from the 25th to 75th percentiles. Whiskers indicate a 1.5× inter-quartile range. p values were calculated using two-sided t tests. H3K27ac Hi-ChIP in shRNA transduced JN-DSRCT1 was performed once. Expression profiling was performed four times in shRNA transduced JN-DSRCT1.

Fig 4: The transcriptional programs controlled by each EWS-WT1 isoform are required to induce in vivo tumor formation from mesothelial cell precursors.a Immunoblots of whole cell extracts from MeT-5A cells lentivirally transduced with either control vector, V5-EWS-WT1 + KTS, HA-EWS-WT1 -KTS or both EWS-WT1 isoforms concomitantly before subcutaneous injection into immunocompromised mice. GAPDH confirms equal protein loading. Source data are provided as a Source Data file. b MeT-5A cells co-expressing EWS-WT1 -KTS and +KTS isoforms generate tumors in immunocompromised mice. Boxplots show tumor weight 9 months after subcutaneous injection of 6 week-old female mice. 7 tumors were assayed per condition. (**) two-sided p value Wilcox rank-sum test = 0.001058. Median value is shown as a line within the boxplot, which spans from the 25th to 75th percentiles. Whiskers indicate minimum to maximum value range. The displayed results were obtained from a single in vivo experiment. Source data are provided as a Source Data file. c Micrographs show H&E staining of a primary DSRCT tumor (Top) and a tumor derived from MeT-5A cells co-expressing EWS-WT1 -KTS and +KTS isoforms implanted in mice as in (b) (Bottom). Scale bar: 300 μm. d UMAP plot based on RNA-seq showing clustering of MeT-5A-derived tumor xenografts with the JN-DSRCT1 cell line and primary DSRCT tumor samples. Comparison with dedifferentiated Liposarcoma (DDLPS), Myxofibrosarcoma (MFS), Malignant Peripheral Nerve Sheath Tumor (MPNST), SARC (other Sarcoma), Synovial Sarcoma (SS), Leiomyosarcoma arising in gynecologic location (uterine ULMS) and soft tissue (STLMS) sites, and Undifferentiated Pleomorphic Sarcoma (UPS) described in ref. 31.

Fig 5: DSRCT 3D tumor connectivity is dominated by EWS-WT1-centered chromatin interactions.a Circos plot shows genome-wide loops identified in JN-DSRCT1 cells by H3K27ac Hi-ChIP. Loops associated (red) or not (blue) with EWS-WT1 binding sites were determined based on EWS-WT1 ChIP-seq occupancy profiles. Inset: Barplot shows an enrichment of EWS-WT1 bound sites in Hi-ChIP loops genome-wide. b Distribution of loop interactions based on the presence or absence of EWS-WT1 binding in the JN-DSRCT1 cell line. c Boxplots show the number of loops (top) and interactions (bottom) between anchor sites in H3K27ac Hi-ChIP in absence or presence of EWS-WT1 binding in JN-DSRCT1 cells. Both the number of loops and interactions are increased at CTCCC clusters and TCC repeat sites bound by EWS-WT1 when compared to all EWS-WT1 binding sites, which includes other EWS-WT1 binding sites, and sites not bound by the fusion protein. Median value is shown as a line within the boxplot, which spans from the 25th to 75th percentiles. Whiskers indicate a 1.5× inter-quartile range. p values were calculated using two-sided t tests. n represents the number of chromatin loops in each category. d Plots showing the distribution of H3K27ac Hi-ChIP loops distances in presence or absence of EWS-WT1 binding at each anchor. The categories are combinations of: anchors not bound by the fusion protein (n = 15205), CTCCC clusters and/or TCC repeat sites bound by EWS-WT1 (both sides n = 645; one side only n = 4985), other EWS-WT1 binding sites (one side only n = 11940; both sides n = 2587), CTCCC clusters and/or TCC repeat sites bound by EWS-WT1 on one side and other EWS-WT1 binding site on the other side (n = 2077). The number of sites (n) for each category is indicated. Median distances for each category are shown as red dotted vertical lines. e Example shows representative image of the increases in H3K27ac Hi-ChIP looping distance for sites associated with EWS-WT1 binding (red, top) when compared to other H3K27ac sites (blue, bottom) on chromosome 8. See also Supplementary Fig. S3A. f Example shows H3K27ac Hi-ChIP looping maps and EWS-WT1/H3K27ac ChIP-seq signals at the VEGFA locus in JN-DSRCT1 cells. H3K27ac Hi-ChIP in JN-DSRCT1 cell line was performed once.