Establishment of transcriptional silencing in yeast

Transcriptional silencing of the HM loci in Saccharomyces cerevisiae requires cis-acting elements, termed silencers and the Sir2, Sir3, and Sir4 proteins. To examine the role of silencers and chromosomal context in silencing, site-specific recombination was used to excise the HMR locus from the chromosome. The structure of the excised rings was evaluated in terms of DNA supercoiling. I showed that excised rings remain silent indefinitely if they contain silencers. However, rings without silencers initially maintained repressed chromatin but the silent structure was lost at a later time. These results suggested that silencers are required for long-term but not short-term maintenance of silent chromatin structure.; Establishment of silencing in HM loci requires a step during cell-cycle progression which has been commonly assumed to be DNA replication. To directly test if DNA replication is needed for silencing establishment, I tethered Sir1p to a non-replicating DNA ring generated by site-specific recombination in vivo. The transcriptional silencing can be established in this non-replicating DNA ring and is accompanied by proper changes in DNA supercoiling and histone acetylation. Thus, this DNA ring forms a bona fide silenced chromatin structure upon repression. In addition, silencing of this DNA ring requires Sir2p, Sir3p, and Sir4p as well as progression between early S and M phases of cell cycle. The results indicate that DNA replication is not required for establishing silencing and that some other S phase event must account for the cell-cycle dependence of silencing.; It has been proposed that the assembly of silenced chromatin in the HMR locus initiates at the HMR-E silencer and then spreads to other regions of the locus. Although several lines of evidence have been provided to support this spreading model, the process of Sir complex propagating along the chromosome has not yet been demonstrated. To capture the spreading of Sir3p, I used chromatin immunoprecipitation to monitor the association of the Sir3p to the HMR locus during the de novo establishment of silencing. My results showed that binding of Sir3p at the HMR-E silencer can occur in G1-arrested cells. Spreading of Sir3p to distal regions of the locus required subsequent cell cycle progression and was accompanied by transcriptional silencing. Thus, my results demonstrate the real-time spreading of Sir3p during the assembly of silenced chromatin.