Functions of Protosilencers in the Formation and Maintenance of Heterochromatin in Saccharomyces cerevisiae

Bi X (2012) Functions of Protosilencers in the Formation and Maintenance of Heterochromatin in Saccharomyces cerevisiae. PLoS ONE 7(5): e37092. doi:10.1371/journal.pone.0037092 Functions of Protosilencers in the Formation and Maintenance of Heterochromatin in Saccharomyces cerevisiae Xinmin Zhang 0 Qun Yu 0 Lars Olsen 0 Xin Bi 0 Laura N. Rusche, Duke University, United States of America 0 1 Department of Biology, University of Rochester , Rochester , New York, United States of America, 2 School of Pharmaceutical Sciences, Jilin University , Changchun, Jilin , People's Republic of China 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. - Funding: This work was supported by National Institutes of Health grant GM62484-10S1 to XB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Transcriptional silencing in Saccharomyces cerevisiae is a form of region specific gene repression that exists at the HML and HMR loci and subtelomeric regions [1]. It is mediated by heterochromatin established via the association of the SIR silencing complex consisting of Sir2 through Sir4 with nucleosomes. Heterochromatin is a stable but dynamic structure [2]. It is relatively refractory to DNA modifying and repair enzymes as well as endonucleases [3 7]. On the other hand, it is permissive to homologous or sitedirected recombination as well as transposon integration [810]. Nucleosomes in heterochromatin are generally regularly ordered and are hypoacetylated compared to those in euchromatin [11 14]. As a reflection of the special structure of heterochromatin, DNA in heterochromatin is more negatively supercoiled than that in euchromatin [8,9]. Formation of heterochromatin at the cryptic mating loci HML and HMR is promoted by small cis-acting elements called the E and I silencers flanking these loci [1]. Silencers each contain two or three recognition sites for ORC (origin recognition complex for DNA replication), Rap1 and Abf1. These silencer-binding proteins can interact with the Sir3 and Sir4 proteins in the SIR complex on their own or through Sir1 thereby recruiting them to the silencers. Sir2 is a histone deacetylase that is responsible for hypoacetylation of heterochromatin [15]. The SIR complex also binds to nucleosomes with a strong preference for unacetylated ones [16 21]. In addition, SIR complex self interacts and is able to form multisubunit chains. The current model for the de novo formation of heterochromatin proposes that SIR complexes recruited to a silencer deacetylate histones in adjacent nucleosomes. The newly deacetylated nucleosomes then bind additional SIR complexes. Through repeated cycles of histone deacetylation and SIR complex recruitment, SIR complexes are believed to spread along a continuous array of nucleosomes during which the primary chromatin structure pertaining to the distribution of nucleosomes along DNA is altered [1,11,12,22]. The spreading model for heterochromatin formation is supported by our finding that nucleosome-excluding structures can block the propagation of heterochromatin [23]. The function of a silencer is affected by other silencers or protosilencers present in its surroundings. Protosilencers are DNA elements that can enhance the activity of a silencer at a distance without the ability to act as bona fide silencers on their own [24]. Single recognition sites for silencer-binding proteins have protosilencer activities [2,2427]. Silencers and protosilencers are collectively referred to as silencing elements. There have been many documented examples of two silencing elements cooperating to promote stronger silencing, but the underlying mechanisms have not been resolved [2,2427]. The fact that two silencers separated by up to several kb are able to cooperate to silence a reporter gene located between them can be explained by assuming that convergent spreading of Sir proteins emanating from the silencers is additive or synergistic, so that heterochromatin established between the silencers is stronger than that formed by either silencer alone [28]. However, this interpretation does not apply to silencer-protosilencer cooperation since a protosilencer is not able to initiate de novo silencing. It is possible that distant silencing elements cooperate by physically interacting with each other or with a common nuclear structure to create a stronger platform for recruiting Sir proteins [24,26]. Alternatively, or in addition, because a protosilencer is actually a binding site for ORC, Abf1 or Rap1 that has the potential of positioning nucleosomes [23,2933], it is conceivable that a protosilencer modulates nucleosome positioning in the region between it and the silencer in a configuration that is more favorable for SIR complex spreading from the silencer [27]. However, definitive evidence for either hypothesis is lacking. In this work, we examined the functions of the ORC, Rap1 and Abf1 binding sites as constituents of the HMR-E silencer, and as protosi (...truncated)