JCB_201609049 3145..3160

In eukaryotic cells, chromatin is sequestered from the cytoplasm by the nuclear envelope (NE), a double-membrane structure composed of an outer nuclear membrane (ONM) exposed to the cytoplasm and an inner nuclear membrane (INM) exposed to the nucleoplasm. Each bilayer houses a unique set of proteins that define ONMand INM-localized functions (Stancheva and Schirmer, 2014). Nuclear pore complexes (NPCs), numbering ∼100 in a haploid yeast NE (Winey et al., 1997), interrupt the NE surface and extend across the NE through pores formed by the fusion of the INM and ONM. The cylindrically shaped NPCs are ∼100 nm in diameter and are composed of multiple copies of ∼30 different proteins termed nucleoporins or Nups (Aitchison and Rout, 2012). The major structural feature of the NPC is the cylindrical core scaffold. Composed of the most highly conserved Nups, the core scaffold is made up of eight spoke structures of identical composition that lie adjacent to the pore membrane and form a flexible framework for the central channel of the NPC. Built on the core are numerous other Nups, including those that bind to the nuclear transport factors and that line the channel or are positioned specifically on the cytoplasmic face of the NPC or the nucleoplasmic face adjacent to chromatin. In addition to their functions in nuclear transport, Nups with access to the nucleoplasm, together with proteins that populate the INM, are positioned to interact with and sequester chromatin along the two-dimensional surface of the INM (Ptak et al., 2014; Czapiewski et al., 2016; Gonzalez-Sandoval and Gasser, 2016; Ptak and Wozniak, 2016). In yeast and metazoan cells, transcriptionally silent heterochromatin is a prominent interacting partner of INM proteins and a few Nups, whereas transcriptionally active chromatin concentrates at NPCs and binds a subset of Nups. Yeast has provided an important model for the study of chromatin interactions with the NE, and among the most well studied of these interactions is the binding of telomeres and subtelomeric chromatin with the NE. In addition to its role in genome organization, telomere tethering is functionally linked to subtelomeric gene silencing, telomere length regulation, and the repair of persistent DNA damage at telomeres (Taddei and Gasser, 2012; Kupiec, 2014; Marcomini and Gasser, 2015; Ptak and Wozniak, 2016). NE-tethering interactions use multiple protein/DNA complexes along telomeric and subtelomeric regions. Among the chromatin-associated factors that contribute to these binding events are the telomerase complex and the yKu complex, which bind chromosome ends; Rap1, which coats the TG repeat region of telomeres and recruits the silent information regulator (SIR) complex; and the SIR complex itself, which binds distinct regions within subtelomeric chromatin (Taddei and Gasser, 2012; Kupiec, 2014). These telomeric and subtelomeric complexes associate with distinct NE-associated proteins including Esc1, which interacts with the SIR complex through Sir4, and the integral membrane protein Mps3, which has been proposed to bind Sir4 and the yKu/ Interactions occurring at the nuclear envelope (NE)–chromatin interface influence both NE structure and chromatin organization. Insights into the functions of NE–chromatin interactions have come from the study of yeast subtelomeric chromatin and its association with the NE, including the identification of various proteins necessary for tethering subtelomeric chromatin to the NE and the silencing of resident genes. Here we show that four of these proteins—the silencing factor Sir4, NE-associated Esc1, the SUMO E3 ligase Siz2, and the nuclear pore complex (NPC) protein Nup170— physically and functionally interact with one another and a subset of NPC components (nucleoporins or Nups). Importantly, this group of Nups is largely restricted to members of the inner and outer NPC rings, but it lacks numerous others including cytoplasmically and nucleoplasmically positioned Nups. We propose that this Sir4-associated Nup complex is distinct from holo-NPCs and that it plays a role in subtelomeric chromatin organization and NE tethering. Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes