Yeast heterochromatin is a dynamic structure that requires silencers continuously

Transcriptional silencing of the HM loci in yeast requires cis-acting eleme nts, termed silencers, that function during S-phase passage to establish th e silent state. To study the role of the regulatory elements in maintenance of repression, site-specific recombination was used to uncouple preassembl ed silent chromatin fragments from silencers. DNA rings excised from HMR we re initially silent but ultimately reactivated, even in G(1)- or G(2)/M-arr ested cells. In contrast, DNA rings bearing HML-derived sequence were stabl y repressed due to the presence of a protosilencing element. These data sho w that silencers (or protosilencers) are required continuously for maintena nce of silent chromatin. Reactivation of unstably repressed rings was block ed by overexpression of silencing proteins Sir3p and Sir4p, and chromatin i mmunoprecipitation studies showed that overexpressed Sir3p was incorporated into silent chromatin. Importantly, the protein was incorporated even when expressed outside of S phase, during G(1) arrest. That silencing factors c an associate with and stabilize preassembled silent chromatin in non-S-phas e cells demonstrates that heterochromatin in yeast is dynamic.