Affinity Map of BRD4 Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1 [Protein Structure and Folding]

February 7th, 2014 by Jung, M., Philpott, M., Muller, S., Schulze, J., Badock, V., Eberspacher, U., Moosmayer, D., Bader, B., Schmees, N., Fernandez–Montalvan, A., Haendler, B.

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histone tails via its tandem bromodomains BD1 and BD2, and forms a complex with the positive transcription elongation factor b which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. We analyzed the binding of BRD4 BD1 and BD2 to different partners and showed that the strongest interactions took place with di- and tetra-acetylated peptides derived from the histone 4 (H4) N-terminal tail. We also found that several H4 residues neighboring the acetylated lysines significantly influenced binding. We generated ten different BRD4 BD1 mutants and analyzed their affinities to acetylated histone tails and to the BET inhibitor JQ1 using several complementary biochemical and biophysical methods. The impact of these mutations was confirmed in a cellular environment. Altogether, the results show that W81, Y97, N140 and M149 play similarly important roles in the recognition of acetylated histones and JQ1. P82, L94, D145 and I146 have a more differentiated role, suggesting that different kinds of interactions take place and that resistance mutations compatible with BRD4 function are possible. Our study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1 binding, and may help in the design of new BET antagonists with improved pharmacological properties.