A. Ratio of the number of GGAA repeats in EWS-FLI1-bound regions to the number of repeat in 50000 randomly picked regions. B. Ability of EWS-FLI1 to modulate transcription of a reporter gene depending upon the number of GGAA repeats. Firefly relative to Renilla luciferase activity is shown. Control experiments with the empty pGL3-promoter vector were set to 1.

A. Enrichment of GGAA motifs in EWS-FLI1-bound sequences. Frequencies of each of 4096 possible 6mer nucleotides found for the 246 identified EWS-FLI1 specific regions (black circle) and for regions identified in the control experiment (white circle) are represented along the Y axis whereas frequency of the same 6mers in the genome is represented on the X axis. B. GGAA repeat enrichment is a common feature of Ewing cell lines. Number of sequences found in A673 (grey circle) and SK-N-MC (white circle) for each type of binding site. C. Consensus motif assessed with MEME algorithm (E-value = 4.1×10⁻⁴⁶) in regions other than GGAA microsatellites.

A. Proportion of EWS-FLI1-bound regions, as compared to the proportion of random regions, around EWS-FLI1 regulated genes. The proportion of EWS-FLI1-bound regions as a function of the distance to the transcription start sites of EWS-FLI1-up or -down regulated genes (solid lines) is shown. As a control, a similar function is indicated for 1500 randomly chosen regions (dashed line). B. Gene Set Enrichment Analysis (GSEA) of genes flanking EWS-FLI1-bound microsatellites. The 94 genes flanking the 80 microsatellites>9R regions (upper panel) as well as the 144 genes flanking the non-microsatellites regions (lower panel) were used as gene set. The expression dataset resulted from previously described EWS-FLI1 inhibition experiments of A673 and SK-N-MC Ewing cell lines [37], [40], ranked using the signal-to-noise algorithm. A strong enrichment of genes flanking EWS-FLI1 bound GGAA microsatellites among EWS-FLI1 up-regulated genes is observed (upper panel). C–F. Regions upstream of EWS-FLI1 up-regulated genes are enriched in GGAA-microsatellites. The number of microsatellites with either 3 to 9 GGAA repeats (grey line) or more than 9 repeats (black line) was calculated for each 1 Kb window from 1 Kb to 1 Mb upstream of the transcription start sites. The numbers of GGAA repeats along DNA are shown for (C) 17000 known genes (control distribution), (D) 582 EWS-FLI1-up-regulated genes, (E) 558 EWS-FLI1-down-regulated genes and (F) 561 genes that are expressed in A673 and SK-N-MC cell lines but not regulated by EWS-FLI1. The control distribution shown in C is also indicated on part D, E and F.