Molecular mechanism of residue-specific nucleosome acetylation.

The male-specific lethal (MSL) complex catalyzes global acetylation of histone H4 at lysine 16, a paradigm for understanding how histone acetylation is linked to transcription and disease. However, the dynamic yet specific mechanisms governing the function of MSL histone acetyltransferase (HAT) and other HAT complexes remain largely elusive. Here, we report cryogenic-electron microscopy (cryo-EM) structures of the Drosophila MSL complex engaged during active catalysis, revealing how nucleosomes are acetylated in a residue-specific manner. Unlike typical enzyme-substrate complexes, a stable product state is observed, directly visualizing the acetyl group being transferred from coenzyme A to H4K16. The cooperative interplay of MSL subunits acts as a molecular ruler conferring specificity, while recognition of H3K36 trimethylation accelerates enzymatic activity, linking MSL function to transcriptionally active gene bodies. We establish physiological relevance of this mechanism in Drosophila dosage compensation. These findings uncover molecular principles underlying the specificity and regulation of histone acetylation in transcriptionally active chromatin.