Localization of truncated telomeres. (A) Scheme of natural telomeres with lac^op array and truncated telomeres after STEs are replaced by a gene marker (ADE2 for Tel6R and 5R, LEU2 for Tel8L). Bar graph as in Figure 1D, presents the percentage of foci in zone 1 for natural or truncated telomeres 6R, 8L and 5R (GA-1917, GA-2256 and YG-138) in G1 cells. Here asterisks indicate significantly different distributions (P-values reflect a proportional comparison between natural and truncated telomeres). (B) Telomere position was monitored for natural or truncated telomeres in the indicated strain background. (C) Bar graph as in (B) with zone 1 values for natural Tel6R, 8L and 5R in wt (open boxes) or ku70Δ trains (black boxes, GA-1489, GA-1916, GA-195) or for truncated forms in ku70Δ (grey boxes, GA-1948, GA-2804, GA-149). For P-values see Supplementary Table 1.

Tethering activity of Reb1. GA-1461 (Taddei et al, 2004) contains lac^ops and four lexA-binding sites at ARS607 located in the middle of the right arm of Chr 6. GA-1461 bears GFP-lacI, GFP-Nup49 and Nop1-CFP (pGVH45) along with either plexA or plexA-Reb1N. Colocalization of the ARS607 locus with the nucleolar Nop1 signal was scored in confocal sections as distinct (left image) or colocalizing (right image).

Tethering of Reb1, Tbf1 and VP16 at a remote subtelomeric location reduces telomere length in tel1Δ cells. (A) Schematic representation of Tel6R^nat and lexA sites, indicating the XhoI site (X) used to measure telomere length. (B) Examples of Southern blots used for the telomere length quantifications shown in (C). Genomic DNA was cut with XhoI and probed with a 6R or a Y′-specific probe as indicated. Three independent plasmid-bearing transformants are tested for each condition. (C) The plot represents the mean length of the terminal XhoI fragment of the Tel6R^nat (see B and Materials and methods). Values and error rates represent an average of two measurements of three independent clones of isogenic strains: wild-type (GA-1459), tel1Δ (GA-3453) and sir4Δ (GA-1867).

Telomere anchoring variability. (A) Yeast STEs X core, STR and Y′ with their factors and binding sites are shown. ORC binds the ARS consensus. (B) Focal section of two yeast cells bearing one lac^ops array and expressing fusions of GFP-lacI and GFP-Nup49 for visualizing the tagged locus and the NE. Position of the tagged locus is scored in 3D image stacks for three zones of equal surface, such that a stochastic distribution gives rise to 33% in each zone. (C) Telomeres 6R, 7L, 14L, 8L and 5R were tagged by inserting ∼150 lac^ops at 14, 18, 19, 11 and 16 kb from the chromosomal ends, respectively, in GA-1320 (Heun et al, 2001). STEs X core, STR and Y′ (indicated by indicated boxes) remain intact. Composition of STEs according to SGD database (Cherry et al, 1998) is represented for each telomere. (D) Position of telomeres was monitored in G1 cells on synthetic medium. The graph presents the percentage of indicated telomere detected in zone 1 for Tel6R, 7L, 14L, 8L and 5R (GA-1459, GA-2226, GA-1985, GA-1986 and YG-143). The P-values obtained by a proportional t-test (zone 1 comparison to 33%, dotted line), plus the number of G1 phase cells scored are indicated. S-phase data are in Supplementary Table 1. The asterisk indicates a value not statistically different from random.

Role of Reb1 and Tbf1 in telomere anchoring. (A) The indicated LexA fusions were targeted to four lexA-binding sites located ∼23 kb from the end of truncated Tel6R. Reb1 and Tbf1 domains encompass the transactivation domain (aa 1–405). Orc2 is a full-length fusion. Position of Tel6R^tr was monitored in cells expressing lexA alone (white boxes), lexA-Reb1N (grey boxes), lexA-Tbf1N (black boxes) or a lexA-Orc2 fusion (hatched boxes) in G1 cells of isogenic wild-type (GA-1917) or yku70Δ (GA-1918) strains. (B) As in (A) except that where indicated strains bear the native Tel6R (Tel6R^nat; GA-1459) with either sir4Δ (GA-1867) or tel1Δ (GA-3453). Tel6R^tr is also monitored in a tel1Δ train (GA-3052). Stipled bars indicate values for the same telomeres with lexA in a wild-type background. Symbols are as in Figure 2, and S phase and P-values are in Supplementary Table 1.

VP16 counteracts telomere anchoring in cis. (A) Telomeres used in the VP16 targeting assay. (B) Tel6R^tr position was monitored for G1 and S phase cells (GA-1917) expressing a lexA or a lexA-VP16 fusion as indicated. (C) As (B), but for Tel8L^nat (GA-1986), which lacks lexA sites. (D) As (B), but positions of Tel6R^nat (GA-3453) and Tel6R^tr (GA-3052) were monitored in tel1Δ G1-phase cells. Zone 1 percentages are compared and asterisks represent values that are not significantly different from random.

Model for telomere dynamics. (A) Two opposite types of forces are proposed to determine position and dynamics of yeast telomeres. Parallel anchoring pathways involving yKu70/80 and Sir4 can be challenged by STEs that provide binding sites for Reb1 and Tbf1. Other pulling forces may derive from active transcription, but if both anchoring pathways are intact, telomeres remain enriched at the NE. (B) When the antagonistic forces are strong enough to compromise anchoring, telomerase elongates short TG tracts less efficiently. This is detectable only if the Tel1-mediated counting mechanism is compromised, suggesting that this latter compensates for any drop in telomerase efficiency provoked by a change in telomere–NE interaction.