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. Author manuscript; available in PMC: 2023 Apr 14.
Published in final edited form as: Clin Cancer Res. 2022 Oct 14;28(20):4466–4478. doi: 10.1158/1078-0432.CCR-22-0384

Figure 2. EWS::FLI1 creates and activates a de novo HOXD13 enhancer in the developmentally conserved TAD.

Figure 2.

A) Top- Schematic of the murine HOXD TAD regulatory regions with the associated enhancers for each region (colored circles). Middle- The murine HOXD C-DOM region (mm9) with the highlighted five enhancer (I-V) regions necessary for Hoxd13 expression in limb development. Bottom- The corresponding human syntenic region (hg19) with annotation of human-specific GGAA microsatellite site (posterior HOX enhancer: PHE). ChIP-seq tracks of EWS::FLI1, H3K27ac, and H3K4me1 binding at the PHE region in Ewing sarcoma cells (2). B) ChIP-seq tracks of H3K27ac and H3K4me1 at the PHE of primary Ewing sarcoma (2) and osteosarcoma (38) tumor samples. C) ChIP-seq tracks of EWS::FLI1 and H3K27ac at the PHE region in Ewing cells following EWS::FLI1 knockdown (2). D) ChIP-qPCR for EWS::FLI1, E) H3K27ac, and H3K4me1 in Ewing sarcoma cells. F) ChIP-qPCR for EWS::FLI1, H3K27ac, and H3K4me1 in U2OS cells. ChIP-qPCR for G) EWS::FLI1, H) H3K27ac after EWS::FLI1 knockdown. Negative control: an inert intergenic region in chr2. VRK1 enhancer: positive control GGAA enhancer site (2). C-DOM: centromeric domain; T-DOM: telomeric domain. Error bars representative of SEM from three independent replicates. * p<0.05; ** p<0.01; Two-way ANOVA; Sidak’s multiple comparison test; Two-tailed t-test.