Intrinsic specificity of a ‘core’ <scp>TIP60</scp> acetyltransferase complex in <i>Drosophila</i>

The acetylation of histones is a central component of reversible chromatin modification that governs genome regulation. Understanding the complex histone acetylation network requires knowledge about the contributions of individual acetyltransferases. These are not easily determined through perturbation studies in cells, due to indirect effects and limited selectivity of the antibodies that detect site-specific histone acetylation. The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histones H4 and the variant H2A.V, but the precise positions of substrate lysines and their relative acetylation rates were unknown. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V, and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns, and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzyme's mode of action and to evaluate its contribution to histone acetylation patterns in cells.