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

Abstract 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. Competing Interest Statement MRM, IKP and BDS own shares in EpiCypher with BDS also a board member of same. The other authors do not have conflicts of interest with the content of this article.