Abstract: Silent chromatin formation in Saccharomyces cerevisiae begins with the recruitment of silent information regulator (Sir) proteins to silencers at the silent mating-type loci and to telomere ends. Next, Sir2/3/4 proteins propagate across these loci as histones are deacetylated by the NAD(+)-dependent histone deacetylase Sir2p, ultimately resulting in the cessation of transcription and in the loss of SET1- and DOT1-dependent methylation of histone H3 within silent chromatin. We analyzed the effects of modifiable lysine residues on histones H3 and H4 on experimentally defined steps in silencing: recruitment of Sir proteins to silencers, Sir protein spreading, and transcriptional repression. Loss of acetylation, but not methylation, facilitated both Sir recruitment and spreading, and Sir spreading across hypoacetylated chromatin could disrupt SET1- and DOT1-dependent histone methylation without silencing underlying genes. Our data indicate that loss of methylation of K4 and K79 on histone H3 reflects intermediate events during the formation of silent chromatin, and that retention of a positive charge at a single residue on histone H4 (K16) was both necessary and sufficient to permit Sir spreading beyond sites of their recruitment.