Conversion of a replication origin to a silencer through a pathway shared by a Forkhead transcription factor and an S phase cyclin.
Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator (Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2-4. However, silencing is semistable even in sir1Delta cells, indicating that SIR1-independent establishment mechanisms exist. Furthermore, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of FKH1 (FKH1(hc)), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Delta). FKH1(hc) caused only a modest increase in Fkh1 levels but effectively reestablished Sir2-4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1(hc) prolonged the cell cycle in a manner distinct from deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5Delta. Unexpectedly, and in contrast to SIR1, both FKH1(hc) and clb5Delta established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRDeltaE::ARS). HMRDeltaE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRDeltaE::ARS1 was reduced by clb5Delta or FKH1(hc), whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRDeltaE::ARS1 did not result from formation of Sir2-4 chromatin because sir2Delta did not rescue origin firing in clb5Delta cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2-4 chromatin and late-origin firing through opposing regulation of a common pathway.
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