Bromodomain binding of acetyl-lysine residues is a crucial step in many epigenetic mechanisms governing transcription. Nearly half of human bromodomains exist in tandem with at least one other bromodomain on a single protein. The Bromodomain and ExtraTerminal domain (BET) family of proteins (BrdT, Brd2, Brd3 and Brd4) each encode two bromodomains at their N-termini and are important regulators of acetylation-dependent transcription in homeostasis and disease. Previous efforts have focused on identifying protein acetylation sites bound by individual bromodomains. However, the mechanisms through which tandem bromodomains act cooperatively on chromatin are largely unknown. Here, we first used small angle xray scattering combined with Rosetta ab initio modeling to explore conformational space available to BET tandem bromodomains. For Brd4, the flexible tandem bromodomain linker allows for distances between the two acetyl-lysine binding sites ranging from 15 to 157 Å. Using a bioluminescence resonance energy transfer assay, we show a clear distance dependence for Brd4 tandem bromodomain bivalent binding of multiply acetylated histone H4 peptides. However, isothermal titration calorimetry studies revealed Brd4 binding affinity toward multiply acetylated peptides does not correlate with the potential for bivalent binding. We used sucrose gradient assays to provide direct evidence in vitro that Brd4 tandem bromodomains can simultaneously bind and scaffold multiple acetylated nucleosomes. Intriguingly, our bioinformatic analysis of deposited chromatin immunoprecipitation sequencing data indicates that Brd4 colocalizes with subsets of histone acetyl-lysine sites across transcriptionally active chromatin compartments. These findings support our hypothesis that scaffolding of acetylated nucleosomes by Brd4 tandem bromodomains contributes to higher-order chromatin architecture.family of bromodomains (BrdT, Brd2, Brd3 and Brd4) were 49 discovered in 2010 (20, 21) and since have entered clinical trials 50 as potential treatment for cancer (22)(23)(24)(25)(26)(27) and inflammation-51 driven disease (28, 29). However, the molecular mechanisms 52 underlying the potential therapeutic effects of bromodomain 53 inhibitors are poorly understood. As isolated bromodomains 54 harbor relatively weak affinity toward monoacetylated histone 55 tail peptides in vitro (K d = 10 µM -1 mM) (13), multivalency 56 is emerging as an increasingly crucial concept in bromodomain 57 binding of modified chromatin (30, 31). One category of po-58 tential multivalent interactions is the recognition of multiple 59 sites on one histone tail by an individual bromodomain. For 60 instance, the N -terminal bromodomains (BD1) of Brd4 and 61BrdT cooperatively bind two adjacent acetyl-lysine residues 62 (i.e. lysines 5 and 8) on the histone H4 tail (13, 32) with 3-63 to 20-fold tighter affinity compared to either acetylation in 64 isolation (33, 34) suggesting that multiple neighboring acety-65 lation sites are necessary to recruit these bromodomains to 66 D R A F ...