Telomeric Protein Distributions and Remodeling Through the Cell Cycle inSaccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres. INTRODUCTIONTelomeres are nonnucleosomal protein-DNA complexes that prevent uncontrolled fusion, degradation, recombination, and elongation of chromosome ends (Muller, 1938;McClintock, 1941;Wright et al., 1992;Sandell and Zakian, 1993;Hande et al., 1999;Smith and Blackburn, 1999;Hackett et al., 2001). In Saccharomyces cerevisiae, terminal telomeric DNA is composed of ϳ350 base pairs of short, degenerate TG 1-3 repeats. One telomeric strand is polymerized by telomerase (Cohn and Blackburn, 1995) and forms an S-phasespecific TG 1-3 overhang (Wellinger et al., 1993(Wellinger et al., , 1996. The conventional DNA polymerase machinery is thought to synthesize the complementary C 1-3 A strand. Duplex telomeric DNA repeats are bound by the sequence-specific binding protein Rap1p, which recruits proteins Rif1p and Rif2p as well as Sir3p and Sir4p via its C-terminal domain (Moretti et al., 1994;Moazed and Johnson, 1996;Moretti and Shore, 2001).Telomeric regions in yeast also contain subtelomeric X-elements, which have only moderate homology to each other and are present at all chromosome ends, and the highly homologous YЈ elements located distal to the X elements (Chan and Tye, 1983) on about half of all chromosome ends. YЈ elements fall into 5.2-and 6.7-kb size classes (Louis and Haber, 1990;Louis and Haber, 1992), encode a helicase (Yamada et al., 1998), and are bounded by short (Ͻ150-base pair) tracts of internal telomeric TG 1-3 sequence DNA. Because YЈ elements often occur in tandem arrays of two to four repeats, these TG 1-3 tracts are found internal to the chromosome ends at distances depending upon the size class and number of YЈ elements present.The Rap1p, Ku, and Sir2-4 proteins are cross-linkable to DNA as far in as 3-15 kb from the chromosome end, consistent with their simultaneous binding to TG [1][2][3] repeat DNA ...

Palmitoylation controls the dynamics of budding-yeast heterochromatin via the telomere-binding protein Rif1

“…2C). This pattern was similar to that observed by immunoflorecense of Rif1 and is reminiscent of other telomerebinding proteins (22,25). In contrast, in pfa4Δ mutant cells, Rif1-GFP fluorescence was more broadly distributed across the nucleus, and only two to four larger, more diffuse foci per nucleus could be identified (Fig.…”

“…Several studies provide evidence that these foci correspond to telomeric clusters (22,25,35). Rif1 binds telomeres via direct interactions with the telomere-binding protein Rap1, and this interaction is relevant to Rif1's molecular roles at telomeres (25,33). However, our data demonstrat that Rap1-Rif1 interactions were insufficient to explain the discrete Rif1-GFP foci that form at the nuclear periphery, and instead suggest that palmitoylation of Rif1 promoted formation of these foci.…”

“…PFA4 was important for Rif1-GFP's localization to discrete foci at the nuclear periphery. Several studies provide evidence that these foci correspond to telomeric clusters (22,25,35). Rif1 binds telomeres via direct interactions with the telomere-binding protein Rap1, and this interaction is relevant to Rif1's molecular roles at telomeres (25,33).…”

“…Rif1 associates with telomere clusters that are themselves anchored to the inner nuclear membrane, forming distinct foci (22)(23)(24)(25). To test whether Rif1's localization to these foci was affected by PFA4, a Rif1-GFP fusion protein was coexpressed in a strain expressing an HDEL-DsRed fusion protein, which is retained in the ER to allow visualization of the contiguous nuclear and ER membranes (26).…”

Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

“…The probe was random-primed S. cerevisiae telomeric DNA or a random-primed PCR fragment from the ARO1 gene (the primer sequences were 5Ј-TGACTG GTACTACCGTAACGGTTC-3Ј and 5Ј-GAATACCATCTGGTAATTCTGTA GTTTTGAC-3Ј). The data analysis was described previously (36).…”