Protein expression and purification. The N-terminal bromodomain of BRD4 used for structural studies was expressed and purified as previously described . Briefly, human BRD4 BD1 (residues N44-E168) was subcloned into a pNIC28-Bsa4 vector (N-terminal His6-tag, followed by a TEV protease cleavage site). The expression plasmid was transformed into Escherichia coli BL21(D3)-R3-pRARE2 Rosetta cells. Cells were cultured in Terrific Broth (TB) media at 37 °C to an optical density (OD) of 2.8-3.0, and then expression was induced with 0.5 mM IPTG at 18 °C overnight. Cells were harvested and resuspended in a buffer containing 50 mM HEPES, pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol and subsequently lysed by sonication. The first purification step of the recombinant protein was by Ni 2+ -affinity chromatography. The hexahistidine tag was then removed by TEV protease cleavage overnight, and the cleaved protein was separated by reverse Ni 2+ -affinity purification. The protein was further purified by size exclusion chromatography eluting with a HiLoad 16/600 Superdex