​Discrete regions of the eukaryotic genome assume heritable chromatin structure that is refractory to transcription. In budding yeast, silent chromatin is characterized by the binding of the SilentInformation Regulatory (Sir) proteins to unmodified nucleosomes. Using an in vitro reconstitution assay, which allows us to load Sir proteins onto arrays of regularly spaced nucleosomes, we have examined the impact of specific histone modifications on Sir protein binding and linker DNA accessibility. Two typical marks for active chromatin, H3K79(me) and H4K16(ac) decrease the affinity of Sir3 for chromatin, yet only H4K16(ac) affects chromatin structure, as measured by nuclease accessibility. Surprisingly, we found that the Sir2-4 subcomplex, unlike Sir3, has higher affinity forchromatin carrying H4K16(ac). NAD-dependent deacetylation of H4K16(ac) promotes binding of the SIR holocomplex but not of the Sir2-4 heterodimer. This function of H4K16(ac) cannot be substituted by H3K56(ac). We conclude that acetylated H4K16 has a dual role in silencing: it recruits Sir2-4 and repels Sir3. Moreover, the deacetylation of H4K16(ac) by Sir2 actively promotes the high-affinity binding of the SIR holocomplex.​