Impact of combinatorial histone modifications on acetyllysine recognition by the ATAD2 and ATAD2B bromodomains

Abstract

The ATPase family, AAA domain containing 2B (ATAD2B) protein has a C-terminal bromodomain that functions as a reader of acetylated lysine residues on histone proteins. However, the molecular mechanisms by which it recognizes chromatin with multiple post-translational modifications remain poorly understood. To gain insights into the recognition of acetylated lysines by the ATAD2B bromodomain, we investigated the recognition of combinatorial histone H4 post-translational modifications using structural and functional approaches. Using isothermal titration calorimetry, we assessed the binding affinities of the ATAD2B bromodomain with distinct histone H4 peptides harboring multiple modifications. Our results show that the ATAD2B bromodomain selectively recognizes acetyllysine residues amongst multiple histone H4 modifications. Most of these adjacent modifications are permissive and exhibit distinctive binding affinities, however, some combinations weaken the acetyllysine interaction. Three novel protein-ligand structures reveal a unique binding mode for the ATAD2B bromodomain compared to the ATAD2 bromodomain, and highlight distinctive residues involved in the coordination of acetylated lysines in the context of adjacent post-translational modifications. To investigate the association of ATAD2B with chromatin, we performed CUT&RUN experiments in human breast cancer cells and compared the genome-wide chromatin binding sites to different regulatory histone modifications. Our results support an association of ATAD2B with endogenous acetylated histone H4. Overall, our study highlights how the interplay between various histone modifications, and combinations thereof, impact the reader activity of the ATAD2B bromodomain.