Figure 6. Fusion of Fragments of the EWSR1 Prion-like Domain to the FLI1 C Terminus Is Sufficient to Recapitulate EWS-FLI1 Activity.
(A) Schematic representation of EWS-FLI1 prion-like domain mutants used in experiments. SYGQ1 or SYGQ2 fragments are fused to the FLI1 C terminus.
(B) Dose-dependent precipitation assay in presence of b-isox after lentiviral expression of fusion proteins in U2OS cells.
(C)Heatmap shows changes in expression detected by RT-Qpcr for selected EWS-FLI1 target genes associated with GGAA repeats after infection of MSCs with either control vector, EWS-FLI1, SYGQ1-FLI1, or SYGQ2-FLI1 fusion proteins.
(D) In vitro sedimentation assay from bacterially expressed and purified SYGQ1-FLI1. (Top) Representative examples of invitro sedimentation assays are shown. The GST tag is cleaved as part of the assay and is used as a soluble internal control. (Bottom) Quantification of two independent experiments is shown.
(E) ATAC-seq signal intensity indicative of chromatin accessibility at GGAA repeat microsatellites in MSCs infected with either control, EWS-FLI1, or the SYGQ2-FLI1 fusion protein.
(F) Heatmaps of FLI1, BAF155, and H3K27ac ChIP-seq signal densities in MSCs treated with either control vector, EWS-FLI1, or the SYGQ2-FLI1 fusion protein. 10-kb windows in each panel are centered on EWS-FLI1-bound GGAA repeat enhancer sites (n = 812).
(G) Example ChIP-seq tracks of FLI1 (EWS-FLI1), H3K27Ac, and ATAC-seq signals over the NKX2–2 locus in MSCs expressing either control, EWS-FLI1, or the SYGQ2-FLI1 fusion protein.
(H) Principal-component analysis (PCA) plot showing PC1 for the 207 target genes associated with EWS-FLI1 GGAA repeats sites (x axis) and PC1 for the 158 remaining differentially expressed genes in EWS-FLI1-expressing cells (y axis). RNA-seq datasets are from MSCs infected with either control vector, EWS-FLI1, EWS(YS37)-FLI1, or the SYGQ2-FLI1 fusion protein.
See also Figure S7.