Tandem bromodomains of BRD4 cooperatively read poly-acetylated nucleosomes to enhance chromatin engagement and regulate breast cancer phenotypes

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein implicated in transcriptional control and oncogenesis, yet how its tandem bromodomains (BD1-2) contribute to nucleosomal engagement remains unresolved. Here we show that the tandem bromodomains of BRD4 cooperatively engage poly-acetylated histone H4 tails and nucleosomes in vitro and promote chromatin association in human cells. In stringent peptide pull-down and nucleosome-based biolayer interferometry assays, isolated BRD4 bromodomains bind weakly to poly-acetylated histone peptides and nucleosomes, whereas the tandem BD1-2 module binds much more robustly. These results closely mirror our observations in mammalian cells, where truncations lacking either bromodomain or pocket-disrupting mutations in either domain reduced chromatin association, with dual pocket disruption causing the strongest defect. In the BRD4 short isoform (BRD4-S), maximal chromatin association additionally required the region C-terminal to the BD2, which contains the basic residue-enriched interaction domain (BID) and extraterminal domain (ET), consistent with a multivalent chromatin engagement mechanism beyond the bromodomains alone. Functionally, dual pocket disruption attenuated BRD4-S-dependent breast cancer phenotypes, including impaired growth and reduced transwell migration. Together, these findings define how tandem bromodomains and adjacent BRD4-S regions cooperate to stabilize chromatin residence and inform therapeutic strategies aimed at more precisely disrupting BRD4 function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/719657v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@12d03beorg.highwire.dtl.DTLVardef@50cc45org.highwire.dtl.DTLVardef@92e3ecorg.highwire.dtl.DTLVardef@1b1a26d_HPS_FORMAT_FIGEXP M_FIG C_FIG