Fig. 3: The identified genes are upregulated by EWSR1::FLI1, but they do not form an interconnected co-regulatory circuitry in Ewing sarcoma cells.
From: The hegemonic EWSR1::ETS oncoprotein overrules core regulatory circuitry principles in Ewing sarcoma
A Heatmap of normalized ChIP-Seq intensity in EW-1 cells for the indicated TFs. 2065 peaks identified in super-enhancers for the three MTF candidates (POU3F2, SOX6, IKZF2) are displayed. The peaks are ranked by their intensity (order: FLI1, mean (POU3F2, SOX6, IKZF2)). Read density is displayed within a 5 kb window around the peak center, and color scale intensities are shown in normalized coverage. The black bar indicates peaks with mSat. Regions with MYC amplification are removed. B Barplot showing the distribution of co-bound TF within the top 100 SEs (EW-1 cell line): 1 (only POU3F2, SOX6 or IKZF2), 2 (POU3F2 & SOX6, POU3F2 & IKZF2 or SOX6 & IKZF2), 3 (POU3F2 & SOX6 & IKZF2). Knockdown of EWSR1::FLI1 leads to a decrease in CRC gene expression in EW-1 (C) and TC-71 (D) Ewing cells. Gene expression analysis by RT-qPCR was performed after transfection with siEF1 and compared to siRNA Control (siCT), and results were normalized using RPLP0 (48 h post-transfection for EW-1, n = 3 and 72 h post-transfection for TC-71, n = 6). E, F Heatmap of RT-qPCR analysis in EW-1 and TC-71, respectively, after transfection with siRNAs and comparing them to siRNA Control (siCT) and normalizing the results using RPLP0 (48 h post-transfection, n = 3). G, H show proliferation rate using Incucyte technology in EW-1 and TC-71, respectively, after transfecting cells with siRNAs (n = 3). I, J Cell count analysis after siRNAs transfection (3 days post-transfection for EW-1 and 2 days post-transfection for TC-71, n = 3). The data were shown as the mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001 : two-tailed Student’s t-test. ns non-significant.
