Fig 2: Chem-seq from intact cells or cellular lysates reveals genomic sites bound by the BET bromodomain-targeting drug JQ1.(a) Features of the Chem-seq method in living cells (in vivo, top) and cell lysates (in vitro, bottom). Top (in vivo): cells are treated with a biotinylated drug to allow drug-target binding to take place in the cellular context. Formaldehyde treatment cross-links chromatin-associated proteins to DNA, including drug-target complexes associated with chromatin. Following cell lysis and sonication, DNA fragments bound to the drug-target complex are enriched using streptavidin beads. Sequencing of the enriched DNA fragments permits genome-wide identification of the loci to which the drug target binds. Bottom (in vitro): the biotinylated drug is added to the cell extract, where it binds protein-DNA complexes. Enrichment of DNA fragments and sequencing is carried out as in the in vivo method.(b) Chemical structures of JQ1 and its biotinylated version, bio-JQ1.(c) Effect of JQ1 (black) and bio-JQ1 (red) on MM1.S cell proliferation. Cells were treated with varying concentrations of drug for 72 h.(d) Heatmap representation of binding of the individual BET proteins (ChIP-seq, black) and bio-JQ1 (in vivo and in vitro Chem-seq, red) to the union of all 25,450 regions occupied by BRD2, BRD3, BRD4 and bio-JQ1. Read density surrounds the center (±5kb) of all occupied regions, rank ordered from highest to lowest BRD4 occupancy.(e) Gene tracks showing BRD2, 3, 4 and bio-JQ1 occupancy of a region of chromosome 12. ChIP-seq reads for BRD2, 3 and 4 (black), Chem-seq reads for biotinylated JQ1 (bio-JQ1, red) or DMSO vehicle control (blue) are shown. The genome-wide data is plotted in reads per million per base pair (rpm/bp).(f) Close-up view of gene tracks showing BRD2, 3 and 4 occupancy (ChIP-seq) and bio-JQ1 occupancy (Chem-seq) across the CCND2 gene locus.

Fig 3: LysM‐Cre‐mediated Brd4 deletion partially inhibited LPS‐induced gene expression in BM‐derived macrophages BM‐derived macrophages from WT and KO mice (from Brd4 fl/fl LysM‐Cre) were analyzed for flow cytometry profiles for CD11b and F4/80 expression (left). Cells were harvested on day 7. Comparable macrophage yields from WT and KO mice (right). Values represent the average of 18 WT and Brd4 KO mice ± SD. NS indicates P‐values > 0.05 Significance was determined by two‐tailed unpaired t‐test.Microarray analysis of LPS‐induced genes in WT and KO macrophages. Expressed genes were identified by a cutoff line of log2 FC > 2 difference with P‐value of < 0.05, shown in the Volcano plot. Significance was determined by two‐tailed unpaired t‐test.Venn diagram depicting the number of LPS‐induced genes in WT and KO macrophages. Note the similarity with RNA‐seq data in Fig 3D.qRT–PCR confirmation of representative BRD4‐dependent and BRD4‐independent genes identified by microarray analysis above. Values represent the average of three independent assays ± SD. Significance was determined by two‐tailed unpaired t‐test and is indicated by: *P < 0.05, **P < 0.01, NS: no significance.Immunoblot analysis of BRD2 and BRD3 in WT and Brd4 KO macrophages. Analysis was done from two independent experiments.GO analysis of BRD4‐dependent genes (downregulated by Brd4 KO) using the Enrichr program.