Heterochromatin formation involves changes in histone modifications over multiple cell generations

Stable, epigenetic inactivation of gene expression by silencing complexes involves a specialized heterochromatin structure, but the kinetics and pathway by which euchromatin is converted to the stable heterochromatin state are poorly understood. Induction of heterochromatin in Saccharomyces cerevisiae by expression of the silencing protein Sir3 results in rapid loss of histone acetylation, whereas removal of euchromatic histone methylation occurs gradually through several cell generations. Unexpectedly, Sir3 binding and the degree of transcriptional repression gradually increase for 3-5 cell generations, even though the intracellular level of Sir3 remains constant. Strains lacking Sas2 histone acetylase or the histone methylases that modify lysines 4 (Set1) or 79 (Dot1) of H3 display accelerated Sir3 accumulation at HMR or its spreading away from the telomere, suggesting that these histone modifications exert distinct inhibitory effects on heterochromatin formation. These findings suggest an ordered pathway of heterochromatin assembly, consisting of an early phase, driven by active enzymatic removal of histone acetylation and resulting in incomplete transcriptional silencing, followed by a slower maturation phase, in which gradual loss of histone methylation enhances Sir association and silencing. Thus, the transition between euchromatin and heterochromatin is gradual and requires multiple cell division cycles.