Esc1, a Nuclear Periphery Protein Required for Sir4-Based Plasmid Anchoring and Partitioning

TLDR: The data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery, and that Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1.

Abstract: The silent mating-type loci (HML and HMR) in the yeast Saccharomyces cerevisiae are maintained in a transcriptionally inactive state due to the formation of a specialized chromatin structure analogous to heterochromatin of higher eukaryotes. Silencing of the mating-type loci requires flanking sequence elements, termed silencers, which bind the transcription factors Rap1 and Abf1, as well as the multisubunit origin recognition complex (ORC) (reviewed in references 11, 15, and 27). Together, these proteins recruit the silencing proteins, Sir1, Sir2, Sir3, and Sir4, that participate in the formation of heterochromatin. Genes placed near telomeres are also silenced. Telomeric silencing depends on Rap1, which binds to telomeric TG1-3 repeats and recruits Sir3 and Sir4 (28, 29). A Sir complex consisting of Sir2, Sir3, and Sir4 then spreads from the telomeres to nearby nucleosomes to form silent chromatin (16, 17). Although RAP1 is an essential gene, mutations that delete the 3′ end of the gene (rap1ΔC) are viable but lead to a complete loss of telomeric silencing (22, 26, 28). This is due to the inability of Rap1ΔC to recruit Sir3 and Sir4 to the telomeres. Expressing Sir proteins as GAL4 DNA-binding domain (GBD) hybrids and tethering them to defective silencers in which binding sites for the ORC, Rap1, and/or Abf1 have been replaced by Gal4 binding sites can lead to silencing. This so-called targeted silencing was first demonstrated with GBD-Sir1 but has since been shown with the other Sir proteins as well as with Rap1 and Orc1 (5, 6, 38). Targeted silencing also has been achieved by forming GBD hybrids with endoplasmic reticulum or Golgi proteins and tethering them to a partially defective HMR E silencer (1). Overexpression of such membrane protein hybrids causes them to accumulate in the endoplasmic reticulum (which is contiguous with the nuclear envelope). As a consequence, the DNA-binding domain of Gal4 is in the nucleus but is anchored to the nuclear membrane. In this case it is thought that silencing occurs because the HMR locus, with Gal4 sites at the E silencer, is drawn to the periphery of the nucleus where there is a high concentration of Sir proteins (1). Circular autonomously replicating sequence (ARS) plasmids that lack a mechanism for mitotic segregation are preferentially retained in mother cells, resulting in the generation of plasmid-free daughters. In the absence of selection, these plasmids are lost from logarithmically growing cultures (30). In contrast, ARS plasmids that contain embedded telomeric sequences or the HMR E silencer are segregated efficiently between dividing cells and are stably propagated (8, 19, 23, 24). Plasmid segregation mediated by the HMR E silencer requires the Sir proteins, and segregation mediated by telomeric sequences is improved by the silencing factors. Previously, we showed that tethering a specific domain of Sir4, the so-called partitioning and anchoring domain (PAD4), directly to ARS plasmids also confers efficient mitotic segregation (2). Using a DNA-topology assay that measures axial rotation of intracellular DNA segments, it was shown that the tethered PAD4 domain of Sir4 also immobilizes the DNA to which it is bound (2). The partitioning and DNA immobilization data suggested a model in which the Sir4 domain attaches to a nuclear component, such as a chromosome or the nuclear membrane, that divides symmetrically between cells at mitosis. Here we describe the results of a screen for factors that, when tethered to a telomere, can reestablish silencing at a telomere in a rap1Δc mutant defective in silencing. In this screen we identified three known Sir-interacting proteins, Rap1, Sir1, and Rad7, as well as a novel protein, Esc1, which we show interacts with the PAD4 domain of Sir4. We further show that Esc1 is located at the nuclear periphery and is essential for the partitioning and anchoring of plasmids by the PAD4 domain of Sir4. The results suggest that Esc1 helps recruit Sir4 to the nuclear periphery.