Net1, a Sir2-Associated Nucleolar Protein Required for rDNA Silencing and Nucleolar Integrity

The Sir2 protein mediates gene silencing and repres- Abf1, and the origin recognition complex (ORC) (Shore sion of recombination at the rDNA repeats in budding and Nasmyth, 1987; Halfter et al., 1989; Rhode et al., yeast. Here we show that Sir2 executes these func- 1989; Bell and Stillman, 1992). These proteins act pritions as a component of a nucleolar complex desig- marily to recruit to DNA a second class of proteins, the nated RENT (regulator of nucleolar silencing and telo- dedicated silencing proteins, Sir2, Sir3, and Sir4 (Klar phase exit). Net1, a core subunit of this complex, et al., 1979; Rine and Herskowitz, 1987; Aparicio et al., preferentially cross-links to the rDNA repeats, but not 1991; Moretti et al., 1994; Triolo and Sternglanz, 1996). to silent DNA regions near telomeres or to active In addition, the Sir1 protein is required for silencing at genes, and tethers the RENT complex to rDNA. Net1 is the silent mating–type loci but not telomeres (Pillus and furthermore required for rDNA silencing and nucleolar Rine, 1989; Aparicio et al., 1991). Once recruited to the integrity. During interphase, Net1 and Sir2 colocalize DNA, some of the Sir proteins spread along chromatin to a subdomain within the nucleolus, but at the end and create inactive domains that can extend for several of mitosis a fraction of Sir2 leaves the nucleolus and kilobases from the initiation sites (Hecht et al., 1996; disperses as foci throughout the nucleus, suggesting Strahl-Bolsinger et al., 1997). A third class of proteins that the structure of rDNA silent chromatin changes comprises the histones. The N termini of histones H3 during the cell cycle. Our findings suggest that a pro- and H4 are required for silencing and are thought to tein complex shown to regulate exit from mitosis is be involved in both the initiation and spreading steps also involved in gene silencing.