Fig 2: ETS1 amplified VEGF downstream transcription. a, b UpSet plots45 showing the overlap of ETS1-bound peaks (a) or ETS1-associated genes (b) between the four time points (0, 1, 4, 12 h). The bar represents the number of peaks shared by the time points indicated by the colored dots and not by the time points indicated by the gray dots. Most ETS1 peaks were not shared between time points, but most ETS1-associated genes were shared. c VEGF stimulated ETS1 chromatin occupancy. Genome browser view of ETS1 occupancy at the KDR locus at the indicated times of VEGF stimulation. Pink highlights regions with greater VEGF occupancy over the time course. d Genome-wide gain of ETS1 signal at ETS1-bound regions during VEGF stimulation time course. ETS1 binding increased genome-wide at 4 and 12 h. Mann–Whitney U-test. Dark line and boxes represent the median and 25th and 75th percentiles. The whiskers represent median ± 1.5 times the interquartile range. e. VEGF-induced changes in gene expression correlated to changes in ETS1 promoter occupancy. Analysis was limited to expressed genes with ETS1 promoter occupancy. Expression and ETS1 signal at the indicated time point of VEGF treatment are expressed as fold change compared to hour 0. Each point was calculated by grouping genes with similar ETS1 occupancy change. Correlation and p-values were plotted using a linear regression model. f VEGF-induced gene activation requires ETS1–BRD4 interaction. HUVEC cells were transduced with lentivirus expressing the indicated proteins, transfected with siRNA, or treated with small molecule JQ1. Gene expression after the indicated number of hours of VEGF stimulation was measured by qRT-PCR and displayed as a heat map. Log10 fold-change compared to time 0 was row scaled. Linear minimum and maximum fold-change values for each row are listed on the right

Fig 3: RUNX2 is a target of BRD4 and of BRD4 inhibitor JQ1. (A) Western blot analysis of BRD4 expression in TPC1, BCPAP, MDA-MB231 and MCF7 cell lines. (B) ChIP analysis of RUNX2 P2 promoter and active ENH regions with anti-BRD4 antibodies in TPC1, BCPAP, MDA-MB231 and MCF7 cells. An unrelated DNA region upstream of the P2 promoter was used as a negative control. The bars represent the average enrichment of the indicated genomic regions in the immunoprecipitated DNA expressed as percentage of the input. (C) Proliferation curve of TPC1, BCPAP, MDA-MB231 and MCF7 cells treated with the indicated concentrations of JQ1 or mock (DMSO). D) qRT-PCR analysis of RUNX2 in TPC1, BCPAP, MDA-MB231 and MCF7 cells treated with the indicated concentrations of JQ1 or mock (DMSO). (E and F) qRT-PCR and Western blot analysis of BRD4 expression in the indicated cells lines treated with siRNA against BRD4 or control siRNA. G) qRT-PCR analysis of RUNX2 expression in the indicated cell lines treated with siRNA against BRD4 or control siRNA. All data are expressed as mean values ± SEM. * P < 0.05. N = 2.

Fig 4: MYC Co-binds and Co-regulates STAT5 and TLX1 Target Genes(A) In silico i-CisTarget analysis for enriched transcription factor motifs found within in regulatory regions of genes that are positively or negatively regulated by STAT5.(B) Read density heatmaps of ChIP-seq signals on TLX1 binding locations for different transcription factors or epigenetic marks ranked on TLX1 signal strength (top) or STAT5 signal strength (bottom) in ALL-SIL cells.(C) Venn diagram showing the total amount of TLX1 and STAT5 peaks that fall within the 41,229 H3K27ac peaks (left) and the total amount of STAT5 + TLX1 peaks that overlap with MYC peaks (right) in ALL-SIL cells.(D–G) ChIP-seq tracks (performed on ALL-SIL cells) of STAT5, TLX1, MYC, p300, BRD4, and H3K27ac at canonical STAT5 regulated genes OSM (D), PIM1 (E), BCL2 (F), and MYC (G) loci.(H) qRT-PCR in ALL-SIL cells treated with MYC siRNAs for 48 hr. Data are presented as mean ± SD. Statistical significance calculated using unpaired two-tailed t test with equal variance.(I) qRT-PCR of STAT5 target genes in ALL-SIL cells treated with 500 nM JQ1 for 6 hr. Data are presented as mean ± SD. Statistical significance calculated using unpaired two-tailed t test with equal variance.(J) ChIP-seq tracks (performed on ALL-SIL cells) of STAT5, TLX1, MYC, p300, BRD4, and H3K27ac at the MYC enhancer locus, 1.4 Mb downstream of the MYC gene.

Fig 5: A-485-sensitive promoter H3K18/27ac and high super-elongation complex association at 2XPI genes.(a) Average H3K18ac, H3K27ac, and H3K9ac near transcription start sites (TSSs) for all active genes, 2XPI genes, and 2Xin genes after DMSO (dimethyl sulfoxide) or A-485 treatment. (b) Metagene plots of AF9 and ENL ChIP-seq counts across all active genes and 2XPI genes after DMSO (dimethyl sulfoxide) or A-485 treatment. (c) Average BRD4 near TSSs for all active genes and 2XPI genes after DMSO (dimethyl sulfoxide) or A-485 treatment.