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Vol. 19, Issue 2, 608-622, February 2008

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

Laurieann Casey^*, Erin E. Patterson, Ulrika Müller^*, and Catherine A. Fox^*,

Department of *Biomolecular Chemistry and Laboratory of Genetics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706

Submitted April 10, 2007; Revised September 19, 2007; Accepted November 20, 2007
Monitoring Editor: Orna Cohen-Fix

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 sir1 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 (clb5). 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 clb5. Unexpectedly, and in contrast to SIR1, both FKH1^hc and clb5 established silencing at HMR using the replication origins, ARS1 or ARSH4, as complete substitutes for HMR-E (HMRE::ARS). HMRE::ARS1 was a robust origin in CLB5 cells. However, initiation by HMRE::ARS1 was reduced by clb5 or FKH1^hc, whereas ARS1 at its native locus was unaffected. The CLB5-sensitivity of HMRE::ARS1 did not result from formation of Sir2–4 chromatin because sir2 did not rescue origin firing in clb5 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.

Address correspondence to: Catherine A. Fox (cfox{at}wisc.edu)