BRD4, the most extensively studied member of the BET family, is an epigenetic regulator that localizes to DNA via binding to acetylated histones and controls the expression of therapeutically important gene regulatory networks through the recruitment of transcription factors to form mediator complexes, phosphorylating RNA polymerase II, and by its intrinsic histone acetyltransferase activity. Disrupting the protein–protein interactions between BRD4 and acetyl-lysine has been shown to effectively block cell proliferation in cancer, cytokine production in acute inflammation, and so forth. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed to human clinical trials. Herein, we summarize the advances in drug discovery and development of BRD4 inhibitors by focusing on their chemotypes, in vitro and in vivo activity, selectivity, relevant mechanisms of action, and therapeutic potential. Opportunities and challenges to achieve selective and efficacious BRD4 inhibitors as a viable therapeutic strategy for human diseases are also highlighted.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-B (NF-B) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-B-dependent genes is unknown. In this study, we used a tetracyclineregulated cell line expressing an NF-B inhibitor to systematically identify NF-B-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-B signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ؎ 3-fold relative to control. Of these, 28 were NF-B-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-B-dependent genes and five genes not previously known to be NF-B-dependent (Gro-␤ and-␥, IB⑀, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-B regulated. Expression of constitutively active enhanced green fluorescent⅐NF-B/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-B sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IB␣/⑀, Gro-␤/␥, and Naf-1 promoters directly bound NF-B/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-B-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappaB (NF-kappaB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappaB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

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