DNA packaging into chromatin requires a dynamic control of chromatin structure to allow regulated gene expression, which is achieved by a complex array of histone post-translational modifications (HPTMs). Histone acetylation, one of the most frequent HPTMs, recently celebrated the 50-year anniversary of its discovery, but despite this long history the precise combinatorial regulation of the histone-modification network and the interplay of enzymes that set these marks are far from being understood. Becker and colleagues have now optimized a mass spectrometry–based strategy to allow accurate quantification of HPTM motifs and have applied it to generate a comprehensive inventory of all acetylation sites and many combinatorial motifs for histones H3 and H4 in Drosophila melanogaster KC cells. The abundance of individual motifs differs greatly, suggesting separation of function. Highly abundant histone-acetylation motifs are more likely to perform structural roles, whereas rarer ones may transmit regulatory signals. To determine the contributions of lysine acetyltransferases (KATs) and deacetylases (KDACs) to the histone acetylome, the authors depleted 31 known or putative KATs and KDACs by RNA interference. In contrast to the widespread notion that KATs are fairly promiscuous enzymes, most KATs showed narrow substrate specificity that was influenced by adjacent acetylation and methylation. In contrast, KDACs were characterized by relaxed substrate specificity. Surprisingly, losses in histone acetylation upon ablation of KATs were usually accompanied by gains in other, sometimes unrelated, motifs. For example, loss of acetylated H4 Lys16, a mark with mainly structural functions, is accompanied by increased acetylation of the adjacent Lys12, a response conserved in human cells. These secondary effects can be interpreted as adaptations of a system that strives to compensate for a global change in acetylation state. Depletion of most KATs also led to changes in histone methylation, thus indicating an intricate co-regulated system. Further mining of this valuable data set will allow derivation of quantitative models and testable hypotheses about the functional cross-talk between histone-modifying enzymes, ultimately increasing knowledge of the systemic response of the chromatin-modification network. (Mol. Cell doi:10.1016/j.molcel.2014.12.008, 8 January 2015)