Conversion of a Replication Origin to a Silencer through a Pathway Shared by a Forkhead Transcription Factor and an S Phase Cyclin

Abstract

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 sir1Δ 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 (FKH1hc), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5Δ). FKH1hccaused only a modest increase in Fkh1 levels but effectively reestablished Sir2–4 chromatin at HMR as determined by Sir3-directed chromatin immunoprecipitation. In addition, FKH1hcprolonged 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 clb5Δ. Unexpectedly, and in contrast to SIR1, both FKH1hcand clb5Δ established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRΔE::ARS). HMRΔE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRΔE::ARS1 was reduced by clb5Δ or FKH1hc, whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRΔE::ARS1 did not result from formation of Sir2–4 chromatin because sir2Δ did not rescue origin firing in clb5Δ 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.