Retinoblastoma-binding protein 1 (RBBP1), also named AT-rich interaction domain containing 4A (ARID4A), is a
tumor and
leukemia suppressor involved in epigenetic regulation in
leukemia and Prader-Willi/Angelman syndromes. Although the involvement in epigenetic regulation is proposed to involve its chromobarrel and/or Tudor domains because of their potential binding to methylated
histone tails, the structures of these domains and their interactions with methylated
histone tails are still uncharacterized. In this work, we first found that RBBP1 contains five domains by bioinformatics analysis. Three of the five domains, i.e. chromobarrel, Tudor, and PWWP domains, are Royal Family domains, which potentially bind to methylated
histone tails. We further purified these domains and characterized their interaction with methylated
histone tails by NMR titration experiments. Among the three Royal Family domains, only the chromobarrel domain could recognize trimethylated H4K20 (with an affinity of ∼3 mm), as well as recognizing trimethylated H3K9, H3K27, and H3K36 (with lower affinities). The affinity could be further enhanced up to 15-fold by the presence of
DNA. The structure of the chromobarrel domain of RBBP1 determined by NMR spectroscopy has an aromatic cage. Mutagenesis analysis identified four aromatic residues of the cage as the key residues for methylated
lysine recognition. Our studies indicate that the chromobarrel domain of RBBP1 is responsible for recognizing methylated
histone tails in chromatin remodeling and epigenetic regulation, which presents a significant advance in our understanding of the mechanism and relationship between RBBP1-related gene suppression and epigenetic regulation.