Fig 2: Loss of p300/CBP and MLL3/4 protein function alter the EWS::FLI1 enhancer histone modification landscape.(A) p300, CBP, H3K27ac and H3K18ac western blot following nuclear extraction, and histone extraction, of SKNMC cells in DMSO control and 6 h 100 nM dCBP-1 treatment at different concentrations. (B) H3K27ac and H3K18ac western blot following histone extraction in 6 h 1 μM A-485 treated SKNMC cells. (C) H3K18ac normalized rpk at all EWS::FLI1 enhancers compared to EWS::FLI1-sensitive enhancers in DMSO and dCBP-1 (6 h) condition in SKNMC cells. Mann–Whitney U test, ****P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. (D–F) Histone modification ChIP-seq normalized rpk at all EWS::FLI1 enhancers and sensitive enhancers in DMSO control and A-485 (6 h) condition in SKNMC cells. Mann–Whitney U test, **P = 0.089, ***P = 0.004, ****P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. (G) Tornado plots showing H3K18ac ChIP-seq signal at EWS::FLI1 enhancers in DMSO, A-485 and dCBP-1 conditions in SKNMC cells. (H) Example ChIP-seq tracks at EGR2 and CCND1 enhancers for H3K18ac in DMSO, A-485 and dCBP-1 conditions in SKNMC cells. (I) H3K4me1 normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in DMSO and A-485 condition in SKNMC cells. Mann–Whitney U test, ****P < 0.0001, *P = 0.0113. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. (J) MLL3, MLL4 and H3K4me1 western blot following nuclear extract and histone extraction in sgGFP control and sgMLL3/4 DKO A673 cells. (K) Tornado plots showing H3K4me1, H3K4me2 and H3K27ac ChIP-seq signal at EWS::FLI1 enhancers in sgGFP control and sgMLL3/4 DKO setting in SKNMC cells (L–N) H3K4me1, H3K4me2 and H3K27ac normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in sgGFP control and sgMLL3/4 DKO setting. Mann–Whitney U test, *P = 0.0421, ****P < 0.0001. Boxes depict the range between the first and third quartile. Central line within box shows the median value and whiskers highlight the maximum to minimum data points. ChIP-seq from one biological replicate. Source data are available online for this figure.