Mechanistic insights into acetylated histone recognition by the CECR2 bromodomain.

Chromatin remodeling regulator CECR2 (cat eye syndrome chromosome region candidate 2) plays critical roles in neurulation, spermatogenesis, DNA damage response, and cancer metastasis, yet the structural determinants underlying its recognition of acetylated histones remain poorly understood. Here, we characterize the histone-binding specificity and structural determinants of the CECR2 bromodomain using isothermal titration calorimetry, biochemical assays, and structure-guided computational modeling. The CECR2 bromodomain recognizes multiple monoacetylated histone H4 peptides, including H4K5ac, H4K8ac, H4K12ac, and H4K16ac, demonstrating engagement across the H4 N-terminal tail. Combinatorial acetylation differentially modulates binding: while H4K5acK8ac exhibits reduced affinity, H4K5acK12ac and H4K8acK12ac maintain binding comparable to monoacetylated peptides. The bromodomain also recognizes acetylated peptides from other histones, including H3K23ac and H2AK5ac. Mutational analysis identifies conserved pocket residues essential for acetyllysine recognition. Molecular docking suggests that a single acetyllysine occupies the canonical bromodomain pocket, while additional modifications extend along the protein surface, providing a structural basis for selective recognition of combinatorial marks. Comparative analysis with the BET family bromodomain BRD4-BD1 highlights differences in pocket architecture and ligand accommodation, with CECR2 displaying a more permissive binding environment that can accommodate larger acyllysine modifications. Together, these findings provide biochemical and modeled structural insight into histone recognition by the CECR2 bromodomain and establish a foundation for understanding how it contributes to chromatin engagement.