The number of vertebrate repeats can be regulated at yeast telomeres by Rap1-independent mechanisms

Abstract

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells. Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.

Fig. 1. In tel1Δ cells TG_1–3 and T₂AG₃ repeats are counted by Rap1-independent pathways. (A) Schematic representation of the modified VII-L telomere used to determine the counting capacity of the internal sequence. The positions of the EcoRV (E), SapI (P), NaeI (N), HindIII (H) and BamHI (B) restriction sites are indicated. The gray box bracketed by BamHI sites indicates the position of the inserted sequences (see Materials and methods). (B) Genomic DNA of the Y, Ytel1Δ and Hy strains was digested with SapI and NaeI and hybridized with the ura3 probe. The median length of the telomeric restriction fragment was calculated using a set of molecular weight markers (not shown) and the non-telomeric 1481 nt fragment as an internal control. In order to obtain the length of the distal telomeric repeats, 378 nt was subtracted from the size of the measured terminal fragment, as shown below each lane. The inserted sequence is (TG_1–3)₂₃ in lanes a, e and i; (T₂AG₃)₁₀ in lanes b and f; (RAP1)₄ in lanes c, g and j; and NR in lanes d, h and k. (C) Schematic representation of the results.

Fig. 2. Maintenance of Y′ telomere in humanized yeast. (A) Schematic representation of yeast telomeres associated with a Y′ subtelomeric element, the position of the invariable XhoI (X) site is reported. Digestion of genomic DNA by XhoI allows to measure the size of the terminal fragment of all Y′ telomeres in the cells. The different probes used for Southern blot analysis are shown. The length of the distal repeats was estimated by subtracting 1200 nt from the measured length of the terminal XhoI fragment. (B) Example of Y′ telomere Southern blot hybridized successively to a T₂AG₃, TG_1–3 or Y′ probe. The number of generation after germination is indicated for the Hy cells. Lanes a, d and g: Y cells; lanes b, e and h: Hy cells after 50 generations (early passages); lanes c, f and i: Hy cells after 500 generations (late passages). (C and D) The XhoI blots were hybridized with the Y′ probe. wt: wild type for the RAP1, RIF1, RIF2 and TEL1 genes. In (B–D), the molecular weight markers, used to measure the mean telomere lengths, are indicated in nt. In (C) and (D), the length of distal repeats is indicated at the bottom of each lane.

Fig. 3. Maintenance of the T₂AG₃-only telomere. (A) Schematic representation of the modified VII-L telomere used to monitor the length of the wild type and of T₂AG₃-only telomere. A representation of the ura3-1 allele is shown to indicate the existence of a 753 nt fragment hybridizing with the ura3 probe. (B) Genomic DNA was digested with EcoRV and HindIII and hybridized with the ura3 probe. The median length of the telomeric restriction fragment was calculated using a set of molecular weight markers (not shown) and using the ura3-1 753 nt fragment as an internal control. 720 bp was subtracted from this value to give the size of the distal TG_1–3 repeats (Y strains) or the distal T₂AG₃ repeats (Hy strains). wt: wild type for the RAP1, RIF1, RIF2 and TEL1 genes.

Fig. 4. The binding of Rap1 to the T₂AG₃-only telomere is impaired. A myc tag epitope was inserted at the C-terminus of the RAP1 endogenous gene in Y, Y tel1Δ and Hy strains carrying an URA3-tagged telomere. (A) Schematic representation of the three DNA fragments analyzed for Rap1 binding by ChIP and by real-time PCR. (B) Results of the immunoprecipitation are expressed as the ratio of the immunoprecipitated TEL DNA to that of the RPG DNA. The average value for at least three experiments is given in the diagram as well as the estimated standard deviation.

Fig. 5. Multiple pathways regulate the number of vertebrate telomeric repeats at chromosome ends of budding yeast. The symbols are explained at the bottom of the figure. (A) In tel1Δ cells, the Rap1-counting pathway is no more operative although Rap1 stills binds to telomeres, as shown by ChIP assays. Our data indicate that unknown counting factors able to recognize T₂AG₃ repeats set the mean telomere length in tel1Δ cells. Whether the T₂AG₃-binding factor is associated to vertebrate repeats in TEL1 cells is unknown. (B) In humanized yeast, a T₂AG₃-only telomere can be formed and maintained at a regulated mean length. Noteworthy, this regulation can occur both in TEL1 and in tel1Δ cells and can co-habit with a Rap1-dependent regulation pathway when TG_1–3 repeats or Rap1 sites are inserted internally. Whether the T₂AG₃-binding factor(s) involved in length regulation is (are) the same in TLC1 tel1Δ and in tlc1-h cells is not known.