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. 2023 Aug 17;13(12):4765–4784. doi: 10.1016/j.apsb.2023.08.015

Figure 5.

Figure 5

Oxidized LDL regulates the open chromatin state of inflammatory genes in ECs in a BRD4-dependent super-enhancer associated with condensation. (A) ChIP-seq peaks (p65, BRD4, H3K27ac, H3K4me3 and RNA Pol II levels) and putative super-enhancer location around ICAM-1. The putative super-enhancer region is marked as the horizontal line in black. The promoter is marked as the purple line. (B) Quantitative analysis of chromosome conformation capture assays-qPCR (3C-qPCR) analysis of long-distance interactions between ICAM-1 promoter and seven enhancer loci in ox-LDL-treated HACECs combined with STING, IRF3, p65 or BRD4 siRNA (x-axis means seven enhancer loci of ICAM-1). (C, D) The ChIP-qPCR (C) and Re-ChIP (D) analysis of the binding of H3K27ac and BRD4, IRF3, or p65, separately, on the +1 kb region of ICAM1 in ox-LDL-treated HCAECs combined with STING, p65, IRF3 or BRD4 siRNA. The Re-ChIP was performed with 1st round pull-down of antibodies, including IRF3, p65, and BRD4 as well as 2nd round pull-down of H3K27ac antibody. (E) The color-coded schematic representation of the aligned amino acid sequence and corresponding prion-like domain disorder propensity plots for BRD4 using the PLAAC (prion-like amino acid composition) pool. (F–I) Immunofluorescence staining of IRF3 (green), p65 (green), and BRD4 (green) in the nuclear (blue) (F), 3C-qPCR analysis of long-distance interactions between ICAM-1 promoter and seven enhancer loci (the positions of different loci were described as Fig. 5A, x-axis means seven enhancer loci of ICAM-1) (G), ChIP analysis of the enrichment of IRF3, p65, and BRD4 at the super-enhancer region of ICAM-1 (H), and ICAM-1 mRNA level (I) in ox-LDL-treated HCAECs combined with MSA-2 or condensate inhibitor 1,6-hexanediol. (J–M) Immunofluorescence staining of IRF3 (green), p65 (green) and BRD4 (green) in the nuclear (blue) (J), 3C-qPCR analysis of long-distance interactions between ICAM-1 promoter and seven enhancers (x-axis means seven enhancer loci of ICAM-1) (K), ChIP analysis of the enrichment of IRF3, p65 and BRD4 at the super-enhancer region of ICAM-1 (L), and ICAM-1 mRNA level (M) in siSTING- and ox-LDL-treated HCAECs combined with STING overexpression or 1,6-hexanediol. Data are shown as mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.