Structural Insight Into The Recognition Of Histone H3 Acetylated On K56 By The CBP Bromodomain

Histone post-translational modifications(PTMs)are important parts of epigenetic regulatory circuits.PTMs of hisontes regulate chromatin architecture and DNA-templated process,including DN A replication,gene transcription and DN A repair.As one of the most abundant histone PTMs,lysine-N-acetylation introduces a hydrophobic acetyl group and neutralizes the positive charge of lysine side chain.These two effects weaken the interactions between histones and DNA,loose the chromatin architecture and further facilitate gene transcription.Histone acetylation can also influence DNA replication,chromatin assembly and the chromatin high-order structure.Most research in the past decades has been focused on acetylation of the intrinsically disordered and flexible N-terminal histone tails,but little is known about the core globular region,which is expected as the key to mediate higher order chromatin structures.Bromodomain is an important protein interaction module that exclusively recognizes lysine acetylation,through the hydrogen bonding and hydrophobic interaction between acetylated lysine and the hydrophobic residues in the pocket,which is formed by four ? helices(?z,?A,?B,?c)and variable loop regions(ZA and BC loops)of bromodomain.Lysine 56 of histone H3(H3K56)resides at the entry-exit point of nucleosomal DNA superhelix,which may influence nucleosome stability by acetylation.H3K56ac enhances the unwrapping of the DNA at the entry-exit site of nucleosome and keeps these regions accessible to effector proteins,which are recruited to specific genes to promote gene transcription.Previous studies have revealed that the bromodomain of CBP/P300 binds to H3K56ac with a high affinity,but the molecular mechanism of the recognition remains unknown,and it has not been determined whether the bromodomain of CBP/P300 recognizes H3K56ac in the context of nucleosome or free histone.Here,we solved the 1.45 A crystal structure of the CBP bromodomain in complex with acetylated H3K56 peptide(PDB ID:5GH9),which uncovered the molecular mechanism under the recognition of H3K56ac by CBP bromodomain.The combined binding assay and structural analysis revealed that the bromodomain prefers an aromatic residue at-2 and an arginine at-4 position from the acetylated lysine.Subsequently,we express and purify H3K56 acetylated histone H3 using a genetically installing site-specific acetylation method.We demonstrated CBP bromodomain can not recognize H3K56ac in the context of H3/H4 tetramer and nucleosome through GST pull-down and ITC assay using the reconstituted H3K56-acetylated H3/H4 tetramer,nucleosome and oligonucleosome extracted from 293T cell.Our results indicate that CBP bromodomain alone recognizes H3K56ac in free histone but not in nucleosome context due to steric hindrance.