Expanded Roles of the Origin Recognition Complex in the Architecture and Function of Silenced Chromatin in Saccharomyces cerevisiae

Abstract

The silenced chromatin at the cryptic mating-type loci (HML and HMR) of Saccharomyces cerevisiae requires a cell cycle event between early S phase and G 2 /M phase to achieve repression. Although DNA replication per se is not essential for silencing, mutations in many of the proteins involved in DNA replication affect silencing. Each of the four silencers, which flank the silenced loci, includes an origin recognition complex (ORC) binding site (ACS). ORC directly interacted with Sir1 and recruits Sir1 to the silencers. This study describes additional roles for ORC in the architecture of silenced chromatin. Using chromatin immunoprecipitation (ChIP) analysis, we found that ORC physically interacts throughout the internal regions of HMR as well as with silencers. This interaction depended on the presence of Sir proteins and, in part, on the HMR-I silencer. ORC remained associated with the internal regions of HMR even when these regions were recombinationally separated from the silencers. Moreover, ORC could be recruited to the silencers lacking an ACS through its Sir1 interaction.One mechanism for silencing in eukaryotes involves the formation of heterochromatin, which blocks transcription in a region-specific and non-gene-specific fashion. Once established, silencing in such regions is stably maintained and inherited through multiple cell divisions despite the potentially disruptive effects of DNA replication, recombination, and repair. In Saccharomyces cerevisiae, heterochromatin is found in three locations: telomeres (24), ribosomal DNA (rDNA) (54), and the silent mating-type loci HML and HMR (51), which contain functional copies of MAT␣ and MATa genes, respectively. Silencing of HML and HMR is crucial for proper haploid cell identity (26).Transcriptional silencing of HML␣ and HMRa is controlled by the E and I silencers that flank HML and HMR loci (7). Silencers contain binding sites for the origin recognition complex (ORC), Rap1p, and/or Abf1p, which together recruit Sir1, Sir2, Sir3, and Sir4 proteins, which are essential for initiating and spreading heterochromatin (8,42,48,51). Deletion of SIR2, SIR3, or SIR4 completely abolishes silencing; however, deletion of SIR1 results in a population of cells in which HML and HMR are silenced in some cells, but not in others. Both states of HML and HMR are heritable in sir1 mutants. This and other observations led to the view that Sir1 is required primarily for the establishment of transcriptional silencing (44), whereas Sir2 to Sir4 are required for both the establishment and maintenance of silencing (4, 41). At HML and HMR, Sir1 associates mostly with chromatin at the silencers, whereas Sir2, Sir3, and Sir4 associate with chromatin throughout the silenced region (51). Sir2 is the only protein among the Sir proteins with both structural and enzymatic roles in silencing (30,31,35). Once recruited to the silencers via multiple interactions between the silencer binding proteins and other Sir proteins, Sir2 deacetylates the N-terminal tails of histones H3 and H4 of the n...

Transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication

“…For example, the second histone-binding domain in the C terminus of Sir3 (20) or the modification of the ATPase domain to bind the Sir2 product O-acetyl-ADP ribose (54) could have arisen after duplication. It is also possible that even after subfunctionalization, Orc1 retained some ability to propagate along chromatin, as recently suggested (55).…”

Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing

“…RNA analysis was performed exactly as described (51). cDNA was analyzed by quantitative PCR as described in SI Materials and Methods.…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…Here the experimental record has been equivocal. Some studies have found Sir1 limited to silencers (Zhang et al 2002), whereas others have found the protein and ORC subunits distributed throughout the HM loci (Gardner and Fox 2001;Rusche et al 2002;Ozaydin and Rine 2010). Whether Sir1 and ORC function throughout silent chromatin domains or whether they simply hitchhike onto silent chromatin as passengers of Sir4 is not yet clear.…”