Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Tornado plots of p300, CBP and H3K27ac ChIP-seq in A673 cells at EWS::FLI1 enhancer regions. ( B ) Tornado of MLL3/4 complex members and H3K4me1/2 ChIP-seq in A673 cells at EWS::FLI1 enhancer regions. ( C ) ChIP-seq tracks at NKX2-2 enhancer and CCND1 enhancer. Enhancer regions highlighted in orange. ( D ) MACS2 peak overlaps of p300, CBP, MLL4, and UTX with EWS::FLI1 enhancer regions. ( E ) Correlation of peak intensity of EWS::FLI1 with p300, MLL4, UTX, and WDR5. R2 value for each correlation shown on each graph.

Article Snippet: MLL3 , CST , 53641S.

Techniques: ChIP-sequencing

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( 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. .

Article Snippet: MLL3 , CST , 53641S.

Techniques: Western Blot, Extraction, Control, MANN-WHITNEY, ChIP-sequencing, Modification

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) p300 ChIP-qPCR in DMSO control and dCBP-1 conditions in SKNMC cells from two biological replicates. ( B ) p300 and CBP western blot from nuclear extracted SKNMC cells treated with A-485 for 6 h and 24 h. ( C ) H3K18ac ChIP-qPCR in SKNMC and A673 following 6 h dCBP-1 treatment from two biological replicates. ( D , E ) H2BK20ac and H3K27ac ChIP-qPCR at NKX2-2 enhancer in SKNMC and A673 following 6 h dCBP-1 treatment. Graphs shown for two biological replicates. ( F ) H3K27ac ChIP-qPCR at NKX2-2 enhancer following dCBP-1 (6 h) treatment from at least three biological replicates. ( G , H ) H3K18ac and H3K27ac ChIP-qPCR at NKX2-2 and CCND1 enhancer in SKNMC cells following 6 h A-485 treatment from at least two biological replicates. Error bars where present depict standard error of the mean from three biological replicates. ( I ) H3K18ac normalized rpk at EWS::FLI1 enhancers in DMSO and A-485 (6 h) condition in EWS-502 cells. ChIP-seq from one biological replicate. ( J ) H3K27ac normalized rpk at EWS::FLI1 enhancers in DMSO and A-485 (6 h) condition in EWS-502 cells. ChIP-seq from one biological replicate. ( K ) Histone modifications at EWS::FLI1 enhancers in sgGFP control and sgMLL3/4 DKO conditions in A673 cells. ( L ) H3K4me1 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC and A673 cells from two biological replicates. ( M ) H3K4me2 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC cells from two biological replicates. ( N ) H3K27ac ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in SKNMC and A673 cells from two biological replicates. ( O ) H3K27ac ChIP-seq fold change at all EWS::FLI1 enhancers in MLL3/4 DKO and A-485 (6 h) treatment compared to DMSO control in SKNMC cells. Kruskal–Wallis 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.

Article Snippet: MLL3 , CST , 53641S.

Techniques: ChIP-qPCR, Control, Western Blot, ChIP-sequencing

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A , B ) eRNA fold change at all EWS::FLI1 enhancers and EWS::FLI1-sensitive enhancers in dCBP-1 (6 h) and A-485 (6 h) treatment compared to DMSO control. Kruskal–Wallis test, (dCBP-1; **** P < 0.0001, A-485; *** P = 0.0608, P = 0.0006 ns, P = 0.99). 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. eRNA signal calculated from three biological replicates, ( C ) RT-qPCR for eRNA of NKX2-2, CCND1 and PPP1R1A enhancers following 6 h treatment of A-485, CCS1477, and dCBP-1 treatment in SKNMC cells. Error bars represent standard error of the mean from at least three biological replicates. Kruskal–Wallis test, exact P values indicated on figure. ( D ) Example tracks at NKX2-2 and CCND1 enhancers. ( E ) RNA polymerase II ChIP-qPCR at NKX2-2 enhancer in SKNMC cells following 6 h dCBP-1 treatment from two biological replicates. ( F ) Fold change in eRNA RNA-seq reads at all EWS::FLI1 enhancers and at sensitive enhancers from both degrader settings following MLL3/4 DKO. Mann–Whitney U test, ns P > 0.01. 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. eRNA signal calculated from three biological replicates. ( G ) Fold change in eRNA RNA-seq reads at all EWS::FLI1 enhancers and at sensitive enhancers from both degrader settings following MLL3/4 DKO compared to A-485 treatment. 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. eRNA signal calculated from three biological replicates. ( H ) EWS::FLI1 normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in sgGFP control and sgMLL3/4 DKO. Mann–Whitney U test, *** P = 0002, **** 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. ( I ) Example tracks at NKX2-2 and CCND1 enhancers. ( J ) p300 normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in sgGFP control and sgMLL3/4 DKO. Mann–Whitney U test, ns P > 0.01. 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. ( K ) EWS::FLI1 normalized rpk at all EWS::FLI1 enhancers compared to sensitive enhancers in DMSO and. A-485 conditions in SKNMC cells. Mann–Whitney U test, ** P = 0.021, **** 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. ( L ) FLI1 ChIP-qPCR in DMSO control and dCBP-1 condition in SKNMC cells from two biological replicates. ( M ) Histograms showing EWS::FLI1 (Black—Control and Red—DKO/A-485) ChIP-seq reads per 10 million ( Y axis) at NKX2-2 enhancer. ( N ) Fold change in EWS::FLI1 ChIP-seq signal at all EWS::FLI1 enhancers following MLL3/4 DKO and A-485 treatment. Mann–Whitney U test, ns P > 0.01. 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. .

Article Snippet: MLL3 , CST , 53641S.

Techniques: Control, Quantitative RT-PCR, ChIP-qPCR, RNA Sequencing, MANN-WHITNEY, ChIP-sequencing

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Western blot analysis in dCBP-1, SAHA and combined SAHA and dCBP-1 treatment. ( B ) FLI1 ChIP-qPCR in DMSO, SAHA and combined SAHA and dCBP-1 treatment. Graphs depict two biological replicates. ( C ) H3K27ac ChIP-seq normalized rpk at EWS::FLI1 enhancers in dCBP-1, SAHA and combined SAHA and dCBP-1 treatment. 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 ) RT-qPCR at EWS::FLI1 enhancers in SKNMC cells following 24 h treatment of either 100 nM dCBP-1, 1 μM SAHA or dual treatment from two biological replicates. ( E ) RNA polymerase II ChIP-qPCR at NKX2-2 enhancer in SKNMC cells following 6 h A-485 and 6 h dCBP-1 treatment from two biological replicates. ( F ) Fold change in eRNA RNA-seq reads at EWS::FLI1-sensitive enhancers following MLL3/4 DKO compared to A-485 treatment. 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. eRNA expression calculated from three biological replicates. ( G ) p300 ChIP-qPCR in sgGFP control and sgMLL3/4 DKO conditions in A673 cells. Error bars represent standard error of the mean from at least three biological replicates. ( H ) Western blot analysis of EWS::FLI1 from nuclear extracts of SKNMC cells treated with dCBP-1, A-485 or CCS1477. ‘L’ indicates protein ladder lane. ( I ) EWS::FLI1 ChIP-qPCR at NKX2-2 and CCND1 enhancers in SKNMC cells following 6 h A-485 treatment from two biological replicates.

Article Snippet: MLL3 , CST , 53641S.

Techniques: Western Blot, ChIP-qPCR, ChIP-sequencing, Quantitative RT-PCR, RNA Sequencing, MANN-WHITNEY, Expressing, Control

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) RT-qPCR for mRNA of NKX2-2, CCND1 and PPP1R1A genes following 6 h treatment of A-485, CCS1477 and dCBP-1 treatment in SKNMC cells. Error bars represent standard error of the mean from at least three biological replicates. Kruskal–Wallis test, exact P values indicated on figure. ( B , C ) Volcano plot depicting differential gene expression following 6 h A-485 and dCBP-1 treatment. Red dots and dashed lines represent genes with a log2FC of >−/+1. Gene expression data calculated from three biological replicates. Significant values measured using a negative binomial wald test. ( D ) Gene set enrichment scores showing top 20 negatively enriched gene sets following 6 h A-485 and 6 h dCBP-1 treatment, in order of enrichment in SKNMC cells. Color represents P .adjust values from a Benjamini-Hochberg (BH) procedure test. Gene ratio represents total differentially expressed genes in given gene set. ( E ) Z-scores of A673-sensitive EWS::FLI1 genes in DMSO, dCBP-1 and A-485 treatment conditions. ( F ) Overlap between downregulated A673-dHALO and EWS-502 dTAG-sensitive genes and genes downregulated following A-485 treatment (6 h). ( G ) Overlap between downregulated A673-dHALO and EWS-502 dTAG-sensitive genes and genes downregulated following dCBP-1 treatment (6 h). ( H ) Volcano plot depicting differential gene expression following 7 days of MLL3/4 DKO in A673 cells. Red dots and dashed lines represent genes with a log2FC of <−/+1, FDR < 0.05. Significant values measured using a negative binomial wald test Gene expression data calculated from three biological replicates. ( I ) Overlap between all, and sensitive, downregulated EWS::FLI1-sensitive genes and genes downregulated following MLL3/4 DKO (6 h). .

Article Snippet: MLL3 , CST , 53641S.

Techniques: Quantitative RT-PCR, Gene Expression

Journal: EMBO Reports

Article Title: p300/CBP is an essential driver of pathogenic enhancer activity and gene expression in Ewing sarcoma

doi: 10.1038/s44319-025-00552-z

Figure Lengend Snippet: ( A ) Overlap between downregulated genes in A-485 and dCBP-1 conditions at two different log2FC cut off thresholds. ( B ) GSEA negatively enriched EWS::FLI1 signatures in both conditions. ( C ) Gene set enrichment scores showing top 20 negatively enriched gene sets following 6 h A-485 and 6 h dCBP-1 treatment, in order of enrichment in EWS-502 cells. Color represents P .adjust values from a Benjamini-Hochberg (BH) procedure test. Gene Ratio represents total differentially expressed genes in given gene set. ( D ) Z-scores of EWS::FLI1-sensitive enhancers and genes in DMSO, dCBP-1 and A-485 treatment conditions. ( E ) RT-qPCR at EWS::FLI1 genes in SKNMC cells following 24 h treatment of either 100 nM dCBP-1, 1 μM SAHA or dual treatment from two biological replicates. ( F ) Overlap between MLL3/4 DKO downregulated genes in SKNMC cells with genes downregulated in A673-dHALO setting and EWS::FLI1-sensitive genes. ( G ) Overlap between MLL3/4 DKO downregulated genes in SKNMC cells with genes downregulated in EWS::FLI1 KO setting in A673 cells. ( H ) Overlap between A-485 downregulated genes (either log2FC < -1 or log2FC < −0.5), MLL3/4 DKO downregulated genes (either log2FC < −1 or log2FC < -0.5) and EWS::FLI1 KO sensitive genes.

Article Snippet: MLL3 , CST , 53641S.

Techniques: Quantitative RT-PCR