The gel filtration fractions containing each oligomeric species of HP1 were pooled and concentrated 10-fold by Centricon-20 ultrafiltration

The gel filtration fractions containing each oligomeric species of HP1 were pooled and concentrated 10-fold by Centricon-20 ultrafiltration. to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in are subjected to mosaic repression when juxtaposed to Rabbit Polyclonal to MARK2 heterochromatin by a chromosome rearrangement (for review see Spofford, 1976). This so-called variegated position effect (PEV)1 is formally analogous to the regulation of the mating type genes in budding yeast. These genes are maintained in a repressed state at a pair of silent loci and only become transcriptionally competent when moved to an active mating type locus (for review see Laurenson and Rine, 1992). In both PEV and yeast silencing, a gene that is fully capable of producing a functional product is believed to be inactivated by a heritably stable form of repressed chromatin. Transgenes inserted into heterochromatin are packaged into an unusually highly ordered array of Ispinesib (SB-715992) nucleosomes, and their promoters are resistant to endonuclease digestion (Wallrath and Elgin, 1995). The chromatin of the silent loci is similarly resistant to endonuclease digestion and to enzymes for Ispinesib (SB-715992) DNA repair and methylation (Terleth et al., 1989; Singh and Klar, 1992; Loo and Rine, 1994). Genetic experiments in both yeast and have identified proteins that function in establishing or maintaining the silenced chromatin. In budding yeast, a set of silencing information regulator (Sir) proteins has been identified through mutations that interfere with silencing (Laurenson and Rine, 1992). Biochemical studies have provided evidence for interactions between these proteins, histones, and other chromatin proteins to form the silent state (Kurtz and Shore, 1991; Raff et al., 1994; Granok et al., 1995). Genetic screens for mutations that modify position effect variegation have been used in to identify proteins that affect heterochromatin formation (for review see Grigliatti, 1991). The dose dependence of a large Ispinesib (SB-715992) number of PEV modifiers has led to speculations that Ispinesib (SB-715992) heterochromatin may be similarly composed of a network of interacting proteins (Locke et al., 1988). Heterochromatin protein 1 (HP1) was the first product of a PEV modifier gene shown to have a heterochromatic localization (James and Elgin, 1986; James et al., 1989; Eissenberg, 1990). Because it apparently lacks DNA-binding activity, its localization into heterochromatin is thought to require other proteins. The Sir proteins also have no known DNA-binding activity. The Sir1 protein is the first of these to be localized to silencing nucleation sites through the DNA-binding activity of the origin recognition complex (ORC) (Pillus and Rine, 1989; Chien et al., 1993; Triolo and Sternglanz, 1996; Fox et al., 1997). It then participates in the recruitment of the remaining Sir proteins to the site. The ORC multi-protein complex binds and initiates DNA replication from autonomous replicating sequence (ARS) elements distributed throughout the yeast genome (Bell and Stillman, 1992). It also recruits the Sir1 protein to one of these elements within the silencing nucleation sites. Moreover, mutants for the yeast ORC2 subunit display a silencing defect (Bell et al., 1993; Fox et al., 1995), and this defect can be complemented by the wild-type ORC2 gene (Ehrenhofer-Murray et al., 1995). This finding suggests that the silencing function of ORC is conserved in ORC2 subunit was found to be enriched in centric heterochromatin and specific subunits of the ORC complex (1, 3, and 4) were shown to physically interact with HP1 (Pak et al., 1997). These findings pointed to a role for ORC in recruiting HP1 into heterochromatin. We have.