In this study, we provide a guideline for developing regioselective histone acetylating catalysts. At least three factors were critical to determine catalyst-promoted acylation yield of each histone Lys residue; 1) Lys residue's intrinsic reactivity, 2) its distance from the catalytically active site, and 3) its conformational flexibility. BAHA was the more active and selective catalyst motif than DSH. A nucleosome ligand, LANA-DBM, exhibited markedly enhanced binding affinity through bivalent binding to acidic patch and DNA within a nucleosome, compared to a monovalent LANA ligand. The enhanced binding affinity allowed for decreasing the catalyst concentration without diminishing acetylation yield. Furthermore, LANA-DBM provided a unique foothold, the Gly linker moiety, for conjugating the catalyst motif to target low-reactive H2BK43 existing between acidic patch and DNA. MD simulations of the catalystnucleosome complexes assisted the design and optimization of the catalyst structure. We envision that developing new nucleosome ligands that bind bivalently to acidic patch and another locus within nucleosome will further expand regiodivergency of histone acylation catalyst.