(E) Horizontal bar plot showing the ratio of transcription factor motif density between TNFα-gained and TNFα-lost SEs. Error bars represent 95% confidence intervals of the median determined by empirical resampling. The median p65 level was calculated in each bin. SEs were ranked by change in BRD4 and binned (50/bin). (D) Line plot showing the median levels of p65 binding (rpm/bp) at SEs in either TNFα (-) light blue or TNFα (+) dark blue conditions. Change in BRD4 levels at SEs are colored by intensity of change (green to red). X-axis shows the log 2 fold change in BRD4 signal. (C) All genomic regions containing a SE in TNFα(-) and TNFα(+) ECs are shown ranked by log 2 change in BRD4 signal (treated vs. (A,B) Gene tracks of ChIP-Seq signal (rpm/bp) for p65, BRD4, H3K27ac, H3K4me3, and RNA Pol II at the CCL2 gene (A) or SOX18 (B) locus in TNFα(-) (top) and TNFα(+) (bottom) ECs. The effect of co-treatment with vehicle (top), BAY (NF-kB inhibitor, middle) and JQ1 (bottom) are shown. (H) Line plots of kinetic ChIP-PCR showing enrichment (% input normalized to time 0) of p65 and BRD4 at an NF-κB binding site in the VCAM1 (left) SE and TEK TE (right) in ECs treated with TNFα (25 ng/mL 0, 5, 15, 30, 60 min). (F,G) Schematic of transcription factor motif binding sites at the VCAM1 SE (red box) (F) or TEK TE (grey box) (G) loci in ECs treated with TNFα. Significance of the difference between distributions determined using a two-tailed t test. (E) Boxplot of p65 binding signal (rpm) at all active gene promoters (TSS), TEs and SEs in TNFα treated ECs. (D) Boxplot of absolute change in BRD4 signal in response to TNFα measured at all enhancers in TNFα(-) and TNFα(+).
(C) Boxplots of median enhancer length (kb), signal (rpm) and density (rpm/bp) in TNFα-gained enhancers. (B) Pie charts displaying characteristics of TE and SE regions including number of loci, size and BRD4 signal. Genes associated with enhancers that are considered typical or super are colored gray and red respectively. The cutoff discriminating TEs from SEs is shown as a dashed line. Enhancers are defined as regions of BRD4 ChIP-Seq binding not contained in promoters. (A) Ranked plots of enhancers defined in resting (top) or TNFα(+) (bottom) ECs ranked by increasing BRD4 signal (units rpm). The x-axis depicts genomic position with TNFα gained typical enhancers (TE, gray) and SEs (SE, red) and promoter regions (white) marked. (G,H) Gene tracks of ChIP-Seq signal for p65, BRD4, and H3K27ac at the VCAM1 and TEK gene loci in untreated (top) or TNFα(+) (bottom) ECs. ChIP-Seq signal (rpm/bp) is depicted by color scaled intensities. Rows are ordered by max p65 in each region. Each row shows ± 5kb centered on p65 peak. (F) Heatmap of p65 (blue), BRD4 (red) and H3K27ac (yellow) levels in resting ECs and after TNFα (25 ng/mL, 1 hr). (E) Pie chart of p65 binding site distribution in EC genome in TNFα(+). Error bars are standard error of the mean (SEM). (D) Bar plots showing cell count normalized expression levels of SELE and VCAM1 in ECs±TNFα (25 ng/mL, 3 hrs).
(C) Images showing adhesion of calcein-labeled THP-1 monocytes to ECs ± TNFα (25 ng/mL, 3 hrs). (B) Western blot for p65, Ku-70, and Tubulin in cytosolic (left) and nuclear (right) protein fraction lysates in ECs±TNFα. (A) Images of ECs ± TNFα stained for p65 (red) or DAPI (blue) (25 ng/mL, 1 hr) cells. BET bromodomain inhibition abrogates super enhancer-mediated inflammatory transcription, atherogenic endothelial responses, and atherosclerosis in vivo. A chemical genetic approach reveals a requirement for BET bromodomains in communicating enhancer remodeling to RNA Polymerase II and orchestrating the transition to the inflammatory cell state, demonstrated in activated endothelium and macrophages. Mass action of enhancer factor redistribution causes momentous swings in transcriptional initiation and elongation. Inflammatory super enhancers formed by nuclear factor-kappa B accumulate at the expense of immediately decommissioned, basal endothelial super enhancers, despite persistent histone hyperacetylation. Stimulation with tumor necrosis factor alpha prompted a dramatic and rapid global redistribution of chromatin activators to massive de novo clustered enhancer domains. To explore the role of chromatin-dependent signal transduction in the atherogenic inflammatory response, we characterized the dynamics, structure, and function of regulatory elements in the activated endothelial cell epigenome. Proinflammatory stimuli elicit rapid transcriptional responses via transduced signals to master regulatory transcription factors.