Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas

Clear cell sarcoma (CCS) is a rare soft tissue sarcoma caused by the EWS/ATF1 fusion gene. Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice develop secondary sarcomas immediately after EWS/ATF1 induction, but only in soft tissue. EWS/ATF1 expression induces oncogene-induced senescence in most cell types in sarcoma-iPSC mice but prevents it in sarcoma cells. We identify Tppp3-expressing cells in peripheral nerves as a cell-of-origin for these sarcomas. We show cell type-specific recruitment of EWS/ATF1 to enhancer regions in CCS cells. Finally, epigenetic silencing at these enhancers induces senescence and inhibits CCS cell growth through altered EWS/ATF1 binding. Together, we propose that distinct responses to premature senescence are the basis for the cell type-specificity of cancer development.


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Sample size Sample size is described in experimental materials and methods section, figures or figure legends. RT-qPCR experiments were performed in triplicate (Figures 3c, 3f, 5b, S4b, S4f, S5g, S8a, S8d) and duplicate ( Figure S4e). To confirm difference of Oct3/4 and Nanog expression level between established iPSC clones, the results from 6 ( Figure S1b) and 3 ( Figure S2b, S2c) independent clones were listed. RT-PCR experiments were performed in 7 (Figure 4d) or 6 (Figure S1f) or 5 ( Figure S2e) independent clones. More than 6 (Figures 1h, 1i, 4i, 6b, 6e) and independent mice were collected to ensure statistical power of detection. Microarray was performed in 1 or 2 sample in each condition (Figure 4e, 7a, S5f). To confirm knockdown efficiency, RT-qPCR was performed in 1 sample in which was used for microarray ( Figure S5e). To confirm difference of global gene expression between established iPSC clones, microarray was performed in 2 independent iPSCs derived from same sarcoma cell line (G1297) ( Figure S1C). ChIPseq data were obtained from 2 sample in each condition (Figures 7b, 7c, 7d, 7f, S7b, S7c, S7d, S7e, S7f). Representative heat map results from one of two ChIP seq data listed in Figure S7g. Bisulfite sequencing was performed at least 7 replicate clones in each cell types. With respect to Sarcoma-iPSC, more than 2 independent clones derived from different sarcoma cell line (G1297 and K11) were used (Figure 1e-1i). Cell growth assay was performed in 3 (Figure 3a, 7e, 7h, 7i, S8b, S8c, S8e, S8f) or 2 (Figure 3d) samples in each condition. Array CGH analysis was performed in 1 sample in each conditions (Figure 1b, S1g, S4a). Exome analysis and direct sequencing were performed 1 sample in each cell types (Figure 1c, S1h, S4a). SA-β-gal positive cell ratio was calculated from more than 3 are from identical cell culture condition (Figure 3b and 3e, 7g). For MP-CCS-SY xenograft studies, the size of more than 12 clumps were calculated in each condition (Figure 7j).
Data exclusions Since we used chimeric mice to evaluate pathological data, we excluded mice with no chimeric contribution. We also excluded a few mice which unexpectedly died during Dox treatment. We didn't excluded any other data from the analysis to avoid the arbitrary selection.

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