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

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Molecular biology of the cell (Impact Factor: 4.47). 03/2008; 19(2):608-22. DOI: 10.1091/mbc.E07-04-0323
Source: PubMed

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 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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Available from: Catherine Anne Fox, Sep 29, 2015
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    • "Cells were grown at 23°C for all experiments and synchronized in late G1, early S, and G2/M by incubation for 3 h in 7.5 nM α-factor (Sigma, T6901), 200 mM hydroxyurea (Sigma, H8627), or 10 µg/mL nocodazole (Sigma, M1404), respectively. ChIP-chip experiments were performed as described previously [42], with the following modifications and reagents: chromatin was sheared to an average size of 300 bp using a Covaris S2 instrument; immunoprecipitations were performed with 9E10 (Covance, MMS150) at 1∶100 followed by pull-down with Protein G Dynabeads (Invitrogen, 10004D), or with anti-Fkh1/2 polyclonal antibody [43], which was pre-crosslinked to protein A-Sepharose 4B beads (Invitrogen, 10–1041), at 1∶40 (packed bed volume). Up to 10 ng immunoprecipitated (IP) and total DNA samples were subjected to whole genome amplification (Sigma, WGA2), followed by primer extension labeling with Cy5 and Cy3 end-labeled random nonamers, as described previously [42]. "
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    ABSTRACT: Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating mating-type silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycle-regulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome.
    PLoS ONE 02/2014; 9(2):e87647. DOI:10.1371/journal.pone.0087647 · 3.23 Impact Factor
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    • "ARS1 contains an ORC-BS and an Abf1-BS required for replication origin function. Under a special circumstance (high copy expression of FKH1) ARS1 in place of HMR-E silencer has been shown to mediate HMR silencing (together with HMR-I silencer) [65]. In this report, we showed that ARS1 inserted at HML locus makes HML heterochromatin more resistant to cell cycle-dependent disruption (Fig. 5). "
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    ABSTRACT: In Saccharomyces cerevisiae, transcriptionally silent heterochromatin at HML and HMR loci is established by silencers that recruit SIR complex and promote its propagation along chromatin. Silencers consist of various combinations of two or three binding sites for origin recognition complex (ORC), Abf1 and Rap1. A single ORC, Abf1 or Rap1 site cannot promote silencing, but can enhance silencing by a distant silencer, and is called a protosilencer. The mechanism of protosilencer function is not known. We examine the functions of ORC, Abf1 and Rap1 sites as components of the HMR-E silencer, and as protosilencers. We find that the Rap1 site makes a larger and unique contribution to HMR-E function compared to ORC and Abf1 sites. On the other hand, Rap1 site does not act as a protosilencer to assist HML-E silencer in forming heterochromatin, whereas ORC and Abf1 sites do. Therefore, different mechanisms may be involved in the roles of Rap1 site as a component of HMR-E and as a protosilencer. Heterochromatin formed by ORC or Abf1 site in collaboration with HML-E is not as stable as that formed by HMR-E and HML-E, but increasing the copy number of Abf1 site enhances heterochromatin stability. ORC and Abf1 sites acting as protosilencers do not modulate chromatin structure in the absence of SIR complex, which argues against the hypothesis that protosilencers serve to create a chromatin structure favorable for SIR complex propagation. We also investigate the function of ARS1 containing an ORC site and an Abf1 site as a protosilencer. We find that ARS1 inserted at HML enhances heterochromatin stability, and promotes de novo formation of a chromatin structure that partially resembles heterochromatin in an S phase dependent manner. Taken together, our results indicate that protosilencers aid in the formation and maintenance of heterochromatin structure.
    PLoS ONE 05/2012; 7(5):e37092. DOI:10.1371/journal.pone.0037092 · 3.23 Impact Factor
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    • "Not only were the relocated ARSs showing reduced replication and late firing, they were now boosting the epigenetic silencing of the nearby genes, contrary to their activities in their native chromosomal locations. A fine twist to these phenomena is that the replicator ARSs did not make perfect silencers (Casey et al., 2008) or proto-silencers (Rehman et al., 2009), reflecting the fact that silencer ARSs sometimes do not make perfect replicators (Chang et al., 2008; Palacios DeBeer et al., 2003). It is not clear how the new chromatin environment of the relocated ARSs has contributed to their functional conversions. "
    DNA Replication-Current Advances, 08/2011; , ISBN: 978-953-307-593-8
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