EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma

Abstract

The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.

Figure 1. EWS-FLI1 binds enhancer elements in Ewing sarcoma cell lines and primary tumors

(A) Heatmaps depict EWS-FLI1, H3K4me1 and H3K27ac signal intensities for 1604 EWS-FLI1-bound distal regulatory elements. Rows: 10 kb regions, centered on EWS-FLI1 peaks, ranked by overall signal intensities of H3K4me1 and H3K27ac. (B) Heatmaps depict EWS-FLI1 and H3K4me3 signals for 181 EWS-FLI1-peaks overlapping with transcriptional start sites (TSS). Rows: 10 kb regions, centered on EWS-FL1 peaks, ranked by overall signal intensities of H3K4me3. EWS-FLI1-binds to enhancers with variable levels of activity as demonstrated by the presence of the H3K4me1 mark and different levels of the H3K27ac activation mark. In contrast EWS-FLI1 is primarily found at active promoters. (C) Composite plots showing average levels of H3K27me3 signals at EWS-FLI1 binding sites (left), compared to genome-wide signals at H3K27me3 peaks (right). (D) Examples of active distal regulatory elements near known EWS-FLI1 target genes in Ewing sarcoma cell lines (A673 and SKMNC) and a primary tumor. Tracks show EWS-FLI1, H3K27ac and H3K4me1 signals. EWS-FLI1 binding is highlighted in gray. See also Figure S1 and Table S1.

Figure 2. EWS-FLI1 activates or represses enhancers depending on the underlying DNA sequence and differential recruitment of p300

(A) Heatmaps (left) and composite plots (middle) depicting H3K27ac and p300 signal intensity changes across EWS-FLI1 peaks after EWS-FLI1 knock-down in SKNMC cells at indicated time points. Binding sites are classified as repressed if EWS-FLI1 depletion results in increased H3K27ac and p300 signals (top, 330 sites, 1.5 fold increase in H3K27ac), or activated if depletion results in decreases in H3K27ac and p300 (bottom, 1011 sites, 1.5 fold decrease in H3K27ac). Right: de novo motif analysis of repressed peaks shows strong enrichment for the canonical ETS factor family motifs (p = 1e⁻¹²⁹, top); activated peaks show enrichment for consecutive GGAA repeat elements (p = 1e⁻⁸⁷⁸, bottom). (B) Signal tracks for representative repressed binding sites (ENC1 and RAB3GAP2) in SKNMC cells. EWS-FLI1, H3K27ac and p300 signals for shGFP or shFLI1 infected cells are shown. The genomic sequence for the EWS-FLI1 binding site near ENC1 is provided (single GGAA). (C) Signal tracks for representative activated binding sites (NKX2-2 and NPY1R) as in (B). The genomic sequence for the EWS-FLI1 binding site near NKX2-2 is provided (GGAA repeats). Areas of EWS-FLI1 binding are highlighted in gray. These data suggest that repression and activation of EWS-FLI1 bound sites rely on two distinct chromatin remodeling mechanisms, dictated by the underlying genomic sequence and the differential recruitment of p300. See also Figure S2.

Figure 3. EWS-FLI1 binding leads to opening of chromatin and recruitment of chromatin remodeling complexes to induce de novo active enhancers at DNA repeats lacking regulatory functions in other contexts

(A) Boxplots for DNAseI signals at activated (top) or repressed (bottom) EWS-FLI1 binding sites across 112 cell lines profiled by ENCODE. SKNMC cells are shown in red. (B) Conservation scores (PhastCons) in 100 vertebrate species for 2 kb intervals centered on activated or repressed EWS-FLI1 binding sites. (C) Left: Comparison of H3K27ac changes at EWS-FLI1 binding sites after introduction of EWS-FLI1 in MSCs or after EWS-FLI1 depletion in SKNMC cells. Activated EWS-FLI1 binding sites are boxed with a dashed line. Right: Boxplots of H3K27ac (top) and H3K4me1 (bottom) signal intensities at 1011 EWS-FLI1 activated sites after introduction of EWS-FLI1 in MSCs (blue) compared to an empty vector control (black). Signals for both enhancer marks are significantly induced following EWS-FLI1 expression. (D) Composite plots of WDR5 and H3K4me1 signals at activated EWS-FLI1 binding sites in MSCs expressing EWS-FLI1 or infected with an empty vector. Signals in SKMNC cells are shown on the right panel for comparison. (E) Heatmaps depict signals for ATAC-seq, WDR5, H3K4me1 and H3K27ac at activated binding sites as in C, either from empty vector infected or EWS-FLI1-expressing MSCs. (rows: ATAC-seq 2 kb region, WDR5-H3K4me1-H3K27ac 10 kb regions, centered on EWS-FLI1 peaks). (F) Signal tracks for FLI1, H3K4me1, H3K27ac, WDR5 and ATAC-seq at the NKX2-2 locus in SKNMC cells, and MSCs expressing EWS-FLI1 (E-F) or empty vector control (Co). EWS-FLI1 expression in MSCs leads to nucleosomal rearrangement, WDR5 recruitment, and de novo deposition of both enhancer marks H3K4me1 and H3K27ac, recapitulating the open active chromatin architecture of SKNMC cells. (G-H) 3C-qPCR analysis of long-distance interactions between the NKX2-2 promoter and the corresponding EWS-FLI1-bound distal regulatory element in SKNMC (G) and primary mesenchymal stem cells (H). A strong interaction is present between the distal regulatory element and the promoter of NKX2-2 in SKNMC cells. No significant interaction is observed in MSCs until introduction of EWS-FLI1 leads to DNA looping (H) to produce a conformation similar to SKNMC cells. The human NKX2-2 locus is depicted above each graph. The x-axes represent distances (kb) from the NKX2-2 promoter. A red arrow denotes the HindIII fragment serving as anchor, black and blue arrows denote the analyzed HindIII fragments. P: promoter; E: enhancer. Error bars represent standard deviations. See also Figure S3.

Figure 4. EWS-FLI1 represses conserved distal regulatory elements by displacing endogenous wild type ETS factors

(A) Boxplots of H3K27ac signal intensities at distal elements corresponding to EWS-FLI1 peaks (repressed sites = blue, activated sites = black) in H1 embryonic stem cells, H1-derived NPCs (neural progenitor cells), H1-derived MSCs, bone marrow-derived MSCs, osteoblasts, skeletal muscle myoblasts (HSMM) and dermal fibroblasts (NHDF). Repressed EWS-FLI1 bound distal elements in Ewing sarcoma are active in normal mesenchymal cell types but not in H1 ES cells or H1-derived NPCs. (B) Composite plots show EWS-FLI1 (left) and ELF1 (right) signal intensities for 330 repressed EWS-FLI1 binding sites upon EWS-FLI1 depletion in SKNMC cells. (C) Heatmaps depict signals for EWS-FLI1, ELF1 and p300 at the same repressed sites (rows: 2 kb regions centered on EWS-FL1 peaks). ELF1 binding is observed at many of these sites upon EWS-FLI1 depletion. (D) Signal tracks for EWS-FLI1, H3K27ac, p300 and ELF1 at the ENC1 locus in SKMNC cells. After EWS-FLI1 depletion ELF1 binding leads to p300 recruitment and enhancer activation. Areas of EWS-FLI1 binding are highlighted in gray. See also Figure S4s.

Figure 5. Combined analysis of epigenetic states, transcriptional changes and chromatin conformation identifies the tyrosine kinase VRK1 as a direct EWS-FLI1 target gene in Ewing sarcoma

(A) Box-plots show Z-scores for gene expression changes vs Z-scores for H3K27ac chromatin changes after EWS-FLI1 knock-down in SKNMC and A673 cells (48 hr). Z-scores provide a measure of effect size and consistency between cell lines. The nearest expressed genes in SKNMC and A673 cells were assigned to each binding site. (B) Heatmaps depict fold changes in H3K27ac and gene expression in A673 and SKNMC Ewing cells after EWS-FLI1 knock-down (48 hr). Genes were ranked by the combined significance of H3K27ac and gene expression changes (average z-score). The top 100 activated or repressed enhancer binding sites and genes are shown in the heatmap and the top 10 annotated genes are listed on the right. (C) Track signals for FLI1, H3K27ac and RNA-seq in SKNMC after EWS-FLI1 depletion (96 hr) identify an active regulatory element distal to VRK1 (top). The same enhancer element is present in primary Ewing tumors (middle), and is generated de novo by EWS-FLI1 expression in MSCs (bottom). P: promoter; E: enhancer. (D-E) 3C-qPCR analysis of long-distance interactions between the VRK1 promoter and the corresponding EWS-FLI1-bound distal regulatory element in SKNMC (D) and A673 cells (E). The human VRK1 locus is depicted below each graph. The x-axes represent distances (kb) from the VRK1 promoter. Red arrow denotes the HindIII fragment serving as anchor, black and blue arrows denote the analyzed HindIII fragments. P: promoter; E: enhancer. Error bars represent standard deviations. See also Figure S5, Table S3 and S4.

Figure 6. VRK1 is highly expressed in primary Ewing sarcoma and its depletion strongly reduces tumor proliferation and survival in vitro and in vivo

(A) VRK1 is expressed in the majority of Ewing sarcoma cells, as assessed by immunohistochemistry of primary tumors (magnification: 400x, scale bar: 50 uM). (B) Left: VRK1 mRNA expression in A673 and SKNMC cells after EWS-FLI1 depletion (shFLI1) compared to control (shGFP). Right: VRK1 mRNA expression in MSCs after introduction of EWS-FLI1 (pLIV EWS-FLI1) compared to control cells (pLIV empty). Error bars represent standard deviations. (C) Proliferation rates and relative apoptosis (D) of a panel of tumor cell lines after VRK1 knock-down compared to control (shGFP). Error bars represent the standard deviation of three replicates. Ewing sarcoma cells display selective high sensitivity toward VRK1 depletion, compared to Saos-2 (osteosarcoma) and HeLa cells. (E-F) VRK1 depletion markedly reduces tumor growth in immunocompromised mice, as assessed by tumor weight (E) and volume (F) 3 weeks after subcutaneous injection of control or VRK1-depleted SKNMC cells. Error bars represent standard deviations.

Figure 7. Mechanisms of enhancer remodeling driven by EWS-FLI1

Schematic illustrating the two distinct chromatin remodeling mechanisms underlying EWS-FLI1 divergent transcriptional activity: enhancer induction and activation (upper panel) with recruitment of WDR5 and p300 at GGAA repeats, and enhancer repression (lower panel) with displacement of endogenous ETS transcription factors and p300 at single GGAA canonical ETS motifs.