Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast

Abstract

Telomerase enables telomere length homeostasis, exhibiting increasing preference for telomeres as their lengths decline. This regulation involves telomere repeat-bound Rap1, which provides a length-dependent negative feedback mechanism, and the Tel1 and Mec1 kinases, which are positive regulators of telomere length. By analysing telomere elongation of wild-type chromosome ends at single-molecule resolution, we show that in tel1Delta cells the overall frequency of elongation decreases considerably, explaining their short telomere phenotype. At an artificial telomere lacking a subtelomeric region, telomere elongation no longer increases with telomere shortening in tel1Delta cells. By contrast, a natural telomere, containing subtelomeric sequence, retains a preference for the elongation of short telomeres. Tethering of the subtelomere binding protein Tbf1 to the artificial telomere in tel1Delta cells restored preferential telomerase action at short telomeres; thus, Tbf1 might function in parallel to Tel1, which has a crucial role in a TG-repeat-controlled pathway for the activation of telomerase at short telomeres.

Figure 1

Single telomere extension analysis at tagged telomere VR in tel1Δ and mec1Δ cells. (A) Methodology of the STEX assay; see text for details. (B) Schematic of tagged telomere VR. The vector-derived sequence is between the ADE2 gene and the telomeric tract ((TG_1–3)_n). (C) Telomere VR sequence analysis of est1Δ/EST1 zygotes. Recipient est1Δ cells were mated with EST1 cells for 3 h. Telomere VR was PCR amplified, cloned, sequenced and analysed for sequence divergence as described in the Methods. Individual telomeres are represented by vertical bars. The red bars indicate the telomeric region that is non-diverging; the blue bars indicate the telomeric region in which the sequence diverges from the sisters. Data are pooled from experiments published in Teixeira et al (2004). (D) Telomere VR sequence analysis of tel1Δ/tel1Δ est1Δ/EST1 zygotes. Recipient tel1Δ est1Δ cells were mated with tel1Δ EST1 cells for 3 h and the sequences of the tagged telomere VR were analysed. Results from four individual matings are pooled. (E) Telomere VR sequence analysis of mec1Δ/mec1Δ tlc1Δ/TLC1 zygotes. Recipient mec1Δ tlc1Δ cells were mated with mec1Δ TLC1 cells for 3 h and the sequences of the tagged telomere VR were analysed. Results from five individual matings are pooled. (F) Frequency of telomere VR extension as a function of telomere length. Sequences obtained from (C) and (D) were ordered according to non-diverging telomere size (as shown in the graphs in (C) and (D)) and pooled into subgroups each containing 15 telomeres. The frequency of elongation in each subgroup was calculated and plotted as a function of telomere length. The grey curve (wild type; wt) describing diverging events at telomere VR in an est1-Δ/EST1 zygote (otherwise wild-type cells) was pre-established (Teixeira et al, 2004). STEX, single telomere extension analysis.

Figure 2

Single telomere extension analysis at native telomere IL in tel1Δ and mec1Δ cells. (A) Schematic of native telomere IL (Tel IL). The telomeric X element combinatorial repeat region contains repeats of the D, C, B and A types, as well as Tbf1-binding sites. (B) Telomere IL sequence analysis of est1Δ/EST1 zygotes. Data are pooled from experiments published in Teixeira et al (2004). Methods and labelling are as described in Fig 1. (C) Telomere IL sequence analysis of tel1Δ/tel1Δ est1Δ/EST1 zygotes. tel1Δ est1Δ cells were mated with tel1Δ EST1 cells lacking the subtelomeric region of telomere IL. Results from four individual matings are pooled. The DNA samples analysed here for telomere IL are the same as in Fig 1C for telomere VR. (D) Telomere IL sequence analysis of mec1Δ/mec1Δ tlc1Δ/TLC1 zygotes. Results from three individual matings are pooled. (E) Frequency of telomere IL extension as a function of telomere length. Analysis was performed as in Fig 1E. STEX, single telomere extension analysis; wt, wild type.

Figure 3

Tethering of the Tbf1 amino-terminal domain to the subtelomere restores preferential elongation of short telomeres on tagged telomere VIIL in tel1Δ cells. (A) Schematic of tagged telomere VIIL. Six UAS_Gs are present between the URA3 reporter gene and the telomeric tract ((TG_1–3)_n). (B) Telomere VIIL sequence analysis of tel1Δ/tel1Δ est1Δ/EST1 diploids expressing Gbd alone. tel1Δ est1Δ pGbd cells were grown and mated with tel1Δ EST1 cells. Diploids were propagated for five generations after initial mating, and then telomeres were analysed for sequence divergence. Results from three individual matings are pooled. (C) Telomere VIIL sequence analysis of tel1Δ/tel1Δ est1Δ/EST1 diploids expressing Gbd-Tbf1N fusion. tel1Δ est1Δ pGbd-Tbf1N cells were grown and mated with tel1Δ EST1 cells. Diploids were propagated for five generations after initial mating, and then telomeres were analysed for sequence divergence. Results from three individual matings are pooled. Gbd, Gal4-DNA-binding domain.