Rap1–Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast

Abstract

In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner. Several proteins are involved in telomeric heterochromatin structure including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4. By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms. The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin. In this study we describe how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome. We found that close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus. In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin. These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome. In complex eukaryotes, such as Drosophila melanogaster, heterochromatin at centromeres and telomeres is cytologically condensed throughout the cell cycle, located near the nuclear periphery, and represses gene activity in an epigenetic manner. Such repression occurs by the spreading of the heterochromatin complex into adjacent genes (Henikoff 1990;Wakimoto 1998). Yeast, Saccharomyces cerevisiae, also contains heterochromatin-like regions that include telomeres and the silent matingtype loci (HML␣ and HMRa). These regions, that are found near the nuclear periphery, use a number of known proteins that include Rap1; Silencing Information Regulators Sir2, Sir3, Sir4; and histones H3 and H4 to form heterochromatin (Laurenson and Rine 1992;Grunstein 1997;Cockell and Gasser 1999). Among these proteins Rap1 has a special role in the initiation of heterochromatin because it directly binds cis-acting DNA elements such as C 1-3 A repeat sequences at telomeres and the E and I silencers at the silent HM mating-type loci. Because of the interaction of Rap1 with both Sir3 and Sir4 in two-hybrid experiments and in vitro (Moretti et al. 1994), and interactions in cell extracts and in vitro among Sir3-Sir4-Sir2 (Moazed et al. 1997;Strahl-Bolsinger et al. 1997), it has been proposed that Rap1 may initiate the formation of heterochromatin by recruiting the Sir complex (Moretti et al. 1994;Hecht et al. 1996). The yKu70/yKu80 heterodimer, the yeast homolog of mammalian Ku70/Ku80 that binds to the ends of doublestranded DNA with high affinity (Mimori and Hardin 1986), is also present at the telomeres and is required for the integrity of telomeric heterochromatin (Laroche et al. 1998;Mishra and Shore 1999). Recent studies have shown that the yKu70/yKu80 heterodimer participates in silencing by counteracting Rif1, a Rap1-bindi...

Long-Range Communication between the Silencers of HMR

“…Besides playing a role in tethering the HMR domain to telomeric foci, the role of the silencers would also be to nucleate the spread of Sir proteins (41,51). Our quantitative analysis demonstrating increased concentrations of Sir proteins at silencers with reduced levels of these proteins the further one traverses from the silencer would be consistent with this role for the silencers, though other models are equally possible.…”

Chromatin Domain Boundaries Delimited by a Histone-binding Protein in Yeast

“…In addition Sir3 interacts with histones H2A and H2B (20). Sir4 can interact with Rap1 at telomere proximal positions in the absence of Sir2 and Sir3 proteins, but Sir4 binding at telomere-distal heterochromatin is strongly dependent on other Sir proteins (21). These interactions and the discovery of the NAD-dependent histone deacetylase activity of Sir2 (22)(23)(24) has led to the current model for the establishment of telomeric silencing.…”

Histone H3 Lysine 36 Methylation Antagonizes Silencing in Saccharomyces cerevisiae Independently of the Rpd3S Histone Deacetylase Complex

“…Unlike the initial nucleation event, the spreading of the Sir complex along chromatin appears to be independent of DNA sequence context (for review, see Moazed 2001). The spread of a complex containing Sir2, Sir3, and Sir4 along DNA is dependent on the histone deacetylase activity of Sir2 (Hoppe et al 2002;Luo et al 2002;Rusche et al 2002). Sir3 and Sir4 have greater affinity for H3 and H4 that are hypoacetylated on their N-terminal tails (Carmen et al 2002).…”