Screening Deletion Strains for Genes That Regulate Telomere Length
We carried out a screen of 4,820 MATa
haploid deletion strains representing the majority of non-essential yeast genes to identify loci that contribute to telomere maintenance. DNA was isolated from each strain, digested with XhoI restriction enzyme, and after electrophoresis, analyzed by Southern blotting using a Y′-subtelomeric probe. Most yeast chromosome ends contain one or several Y′-repetitive elements [17
]. The most distal XhoI site on the chromosome is located close to the 3′-end of the Y′-element so that the terminal restriction fragment contains about 900 bp of the subtelomeric Y′-repeat and approximately 350 bp of telomeric TG repeats (A). In addition, the use of Y′-probe resolves two major large restriction fragments derived from the tandem repeats of longer Y′- (6.7 kb) and shorter Y′- (5.2 kb) elements. There are several other minor bands that vary in length due to the presence of other repetitive sequences such as X′-elements. A representative Southern blot from the initial screen is shown in B and the blots for all the deletion strains are provided as supplemental data at http://www.fhcrc.org/labs/bedalov/index.html
. The magnitude of the telomere length-alteration was scored on a 1–3 scale: (1) in short telomere strains corresponding to the reduction of the telomere length by 200, (2) 200–50, or (3) less than 50 bp; and in long telomere strains, (1) an increase by more than 300, (2) 50–300 bp, or (3) less than 50 bp.
Telomere and Subtelomeric Repeats in S. cerevisiae
There were 247 strains in the haploid MATa
set identified as having altered telomere length in the initial screen and further analyzed by re-isolating their DNA and confirming the telomere length-alteration. If confirmed, telomere length in the corresponding homozygous diploid mutant was analyzed. Only mutants that had consistent telomere length- alterations in haploid and homozygous diploids were used for further analysis. As the next step in identifying telomere length-maintenance (TLM)
genes, we carried out allelism analysis of the KanMx
locus and the altered telomere length for all the short telomere mutants and for selected long telomere mutants (mutants that scored 1 and 2). Allelism testing was performed through random spore and tetrad analysis of the progeny derived from the diploids generated by mating mutants with the wild-type strain and confirming the co-segregation of altered telomere length and the KanMx
marker gene. Random spores were obtained through genetic selection for haploid MATa KanMx
progeny using a strategy developed by Tong et al. [18
] and analyzed for telomere length individually or as pools of spores. If the results of random spore analysis suggested lack of co-segregation of the KanMx
and telomere length-alteration, tetrad dissections were carried out and examined for 2:2 co-segregation of the telomere length-change and KanMx
marker. This battery of tests reduced the number of genes that regulate telomere length to 152 genes as listed in .
Genes Whose Deletion Affects Telomere Length and Their Interaction with Telomerase Pathway
Among 4,820 deletion mutants we found 72 tlm
mutants with consistently short telomeres and 80 mutants with long telomeres ( and Supporting Information
). Of these mutants, 64 (42 with short and 22 with long telomeres) corresponded to genes previously identified as telomere maintenance genes including all three ever shorter telomere (EST)
genes, providing validation for our screen. In addition to having the shortest telomeres in the deletion set, mutants in EST
genes also exhibited a large variation in the size of the distal restriction fragment, a consequence of recombination and amplification of subtelomeric Y′-elements. No other tlm
gene deletion exhibited this phenotype suggesting that there are no novel EST
genes in the deletion set. Further validation came from a recent study, Askree et al., who reported a telomere length survey of the deletion set similar to ours [20
]. While there is a significant overlap between the genes found to regulate telomere length in that study with the genes reported here, 94 are unique to our study.
Telomere Blots of Mutants with Short and Long Telomeres
Relationship between TLM Genes and Major Telomere Length-Maintenance Pathways
genes belonged to several functional classes: genes required for POL2
transcription (27 genes) and ribosome biogenesis (22 genes); vacuolar protein sorting (VPS) (15 genes) represented the largest groups of genes. In order to begin to assign specific telomere maintenance roles to the newly identified genes, we examined their relationship with genes that perform known functions at telomeres: telomerase (TLC1)
and telomere-capping functions (YKU70)
Two assays were used to determine the relationship between the novel genes and telomerase. First, we examined whether the mutations affect the ability of yeast to replicate in the absence of telomerase (replicative senescence). Accelerated replicative senescence was interpreted as evidence for non-epistasis with the telomerase pathway. Second, we measured telomere length in double mutants that lack the TLM gene and TLC1, the RNA component of telomerase. The TLM genes that participate exclusively in the telomerase pathway are not expected to affect replicative senescence or increase the rate of telomere loss in the absence of telomerase. Conversely, if misregulation of telomerase activity is responsible for the phenotype in mutants that have increased telomere length, the increased telomere length will be entirely dependent on telomerase.
Growth of telomerase-deficient yeast is characterized by gradual loss of telomeric DNA that is accompanied by loss of viability or senescence. After 60–80 doublings, telomerase-deficient tlc1
cells stop dividing except for a few survivors that have gained the capacity to maintain their telomeres through homologous recombination [6
]. We carried out senescence assays by performing several successive streak-outs of single tlc1
and double tlc1 tlm
mutants. As previously reported, senescence phenotype of tlc1 est1
double mutant was similar to that of tlc1
single mutants. However, double tlc1 yku70
mutants known to be defective in the telomerase-capping functions senesce after approximately 20 divisions in the absence of telomerase ( and unpublished data). We identified 22 tlm
(15 short-telomere and seven long-telomere) genes whose loss accelerated senescence of telomerase-deficient yeast. TLM
genes in this category included those that participate in sister chromatid cohesion (e.g., DCC1, CTF8
), deoxyribonucleotide (dNTP) biosynthesis (MET7, RNR1),
VPS (VPS15, VPS28, VPS23),
transcription (SRB2, SRB5),
and DNA replication and repair (SLX8, ELG1, RAD27, DOA4)
(A and ). Furthermore, mutants in two genes with unknown functions, YDL119
also exhibited a more rapid loss of viability in the absence of telomerase. These results suggest that a subset of tlm
genes carry out functions at telomeres that are not exclusively dependent on telomerase.
TLM Genes That Have Synthetic Phenotypes with the Lack of Telomerase
Because homologous recombination is important for telomere maintenance and cell proliferation in the absence of telomerase [6
], we examined the possibility that TLM
genes affect senescence through interfering with rad52
-dependent homologous recombination pathway. We therefore compared the senescence rates of tlc1 rad52
double and tlc1 rad52 tlm
triple and double mutants for several TLM
genes including YKU70, CTF8, SLX8, DCC1, MET7,
As reported previously [6
], we found that tlc1 rad52
double mutants lose viability after approximately 40 doublings (i.e., most of the tlc1 rad52
double mutants are incapable of forming colonies upon second streak-out) (B). The lack of TLM
genes further accelerate loss of viability, as tlc1 rad52 tlm
triple mutants die as microcolonies following germination (e.g., yku70
) (C) or die at the first streak-out (e.g., ctf8, slx8, dcc1, ypl017c,
) (B and S1
). The observation that the senescence rate effect of TLM
genes is preserved in the absence of RAD52
suggests that homologous recombination is not the primary pathway through which described TLM
genes affect senescence.
To evaluate the relationship between telomerase and other telomere maintenance genes with a more sensitive assay, we compared telomere length in the double tlc1 tlm mutant with the single tlc1 mutant. In agreement with the synthetic interaction in the senescence assay, deletion of sister chromatid cohesion genes (e.g., DCC1, CTF8), dNTP biosynthesis (RNR1, MET7, PRS3), VPS, and two members of mediator complex (SRB2 and SRB5) showed a synthetic telomere phenotype (reduced telomere length) with the lack of TLC1 (A and ). This result confirms that more rapid loss of viability in this subset of double mutants is directly related to accelerated loss of telomeric DNA. However, synthetic interaction was also seen with members of other groups, which did not show an accelerated senescence phenotype, including all of the nonsense-mediated decay (NMD) pathway genes (e.g., NAM7, UPF3, NMD2), telomeric DNA-end-processing (XRS2, RAD50), and two ribosomal genes (RPL13B and RPL34B). In order to directly examine the effect of tlm genes on the rate of telomere loss induced by the absence of telomerase, we employed a galactose-inducible telomerase system. Single tlc1 and double tlm tlc1 strains containing a galactose-inducible TLC1 on a plasmid (GAL-TLC1) were grown in galactose medium (telomerase ON) and then switched to glucose medium (telomerase OFF). After five divisions without telomerase in glucose medium, telomere length of tlc1 strain was indistinguishable from the telomere length of a strain grown in galactose, whereas the telomere length in the double tlc1 tlm mutants was reduced (B). These results demonstrate that the rate of telomere loss in the absence of telomerase is accelerated in a subset of tlm mutants, which further confirms the role of the corresponding TLM genes in telomerase-independent telomere maintenance mechanisms.
The Lack of TLM Genes Accelerates Telomere Loss in the Absence of Telomerase
Similar analyses were carried out in a subset of mutants that confer long telomeres to establish whether their increased telomere length was dependent on telomerase activity (A). Telomere length of the long telomere tlm tlc1 double mutants was indistinguishable from the telomere length of the tlc1 single mutant, indicating that their long telomere phenotype was entirely dependent on the telomerase pathway. This result is consistent with the model where the absence of TLM genes perturbs normal mechanisms that limit telomerase access to telomeres. Interestingly, several of the long telomere mutants exhibited accelerated loss of viability in the absence of telomerase (e.g., rad27, slx8, elg1) (A and ), suggesting that the same defect that increased telomerase-mediated telomere elongation in the presence of telomerase makes chromosome ends more vulnerable in the absence of telomerase.
Epistatic Analysis of Telomere Length among TLM Genes
The synthetic phenotype of NMD genes and VPS genes with the lack of telomerase raised the possibility that these mutants affect telomere capping. The Ku DNA-end-binding proteins carry on an important telomere-capping role. We therefore evaluated the relationship of KU pathways and NMD and VPS genes by comparing telomere length in single yku70 and double yku70 nmd or yku70 vps strains. The loss of YKU70 exhibited a synthetic phenotype with the loss of NMD genes and was epistatic with the loss of VPS genes (B and unpublished data). This result indicates that VPS genes function in the KU-capping pathway distinct from the NMD pathway. In further support of this idea we observed synthetic telomere phenotypes between VPS and NMD genes (B).
Telomere Length-Variation in Natural Isolates
Our results using the laboratory yeast strain indicate that myriad genes control telomere length. A trait that is controlled by a large number of genes has the potential to exhibit phenotypic variation in genetically diverse populations, and indeed, when we analyzed variation in telomere length in 13 Saccharomyces cerevisiae
strains isolated from the wild [23
], we found that the size of the terminal restriction fragment varied significantly among different strains (A). The difference in size was due exclusively to alterations in the size of telomeric repeats and was not due to the variation in the size of the Y′-DNA as determined by the analysis of the terminal fragment by PCR and DNA sequencing (unpublished data). The size of the telomeric DNA between the isolates varied between 150 and 400 bp. We hypothesized that different telomere lengths between the strains were due to polymorphisms in many loci. To further evaluate this possibility, we analyzed telomere length in the progeny derived from crossing the laboratory strain (BY) and one of the wild isolates (RM11). The telomeres in the haploid RM11 strain are approximately 100 bp shorter than the telomeres in the haploid BY strain. In order to study the underlying genetic basis of this difference in telomere length, we analyzed telomere lengths of 122 haploid progeny from a cross between BY and RM11. The distribution has several interesting features (B). First, the segregants show a continuous broad range of telomere lengths, consistent with a trait that is controlled by many loci. Second, the average length of telomeres among the segregants of 272 bp is shorter than the average length of the parental strains (302 bp), which is suggestive of non-additive interactions among the loci involved. Finally, it is interesting to note that none of the segregants has telomere length longer than BY; while a fraction (27%) have telomere length shorter than RM11. The phenomenon of a trait having progeny values more extreme than either parent, called transgressive segregation, is observed for many traits and indicates the presence of multiple loci that have compensating effects in the parents. Transgressive segregation can occur as a result of non-additive interactions among the loci and is consistent with the above observation. Furthermore, while all the segregants in this study shared the same replication history following germination (having undergone approximately 80 doublings), it is possible that final telomere length-equilibrium had not been achieved, which may skew the distribution of the telomere lengths.
Telomere Length-Analysis in Outbred Yeast Strains
To identify quantitative trait loci- (QTL) mediating variation in telomere length in the segregants, we performed a genome-wide linkage analysis. In this analysis, genetic markers were used to classify each locus in the genome as inherited from the BY or RM11 parent in each segregant. Then, for each locus, the distributions of telomere lengths were compared between segregants inheriting the BY
allele and those inheriting the RM11
allele. A significant difference between these distributions indicates that the tested locus lies near a sequence polymorphism between the two strains that affect telomere length. Specifically, linkage analysis was performed with 3,312 genetic markers that were previously genotyped in all of the segregants using oligonucleotide microarrays [24
]. Telomere length showed evidence of linkage to several loci (C), with significant linkage peaks occurring on Chromosome 12 and Chromosome 13 (). The LOD scores of 7.4 and 4.3 at Chromosome 12 and Chromosome 13 loci were significant (genome-wide corrected p
< 0.01), as we did not observe any scores higher than four in the analysis of 100 permuted datasets. Several genes known to regulate telomere length reside in the mapped regions including EST1, VPS34, RPS28B, ARV1,
]. Polymorphisms that result in amino-acid substitutions in these genes (Table S1
) are likely candidates that control telomere length in the segregants. Alternatively, telomere length might be determined by the polymorphisms in essential genes that reside in these regions (i.e., genes that were not evaluated directly in this study) or by polymorphisms in the regulatory elements. Interestingly, the telomere length-effect of the Chromosome 12 locus was in the opposite direction from the difference between the parents. In other words, the Chromosome 12 RM11
allele conferred longer telomeres and the BY
allele conferred shorter telomeres, even though the RM11 parent has shorter telomeres than the BY parent, which is consistent with transgressive segregation (). The Chromosome 12 and Chromosome 13 loci explained only 25% and 13% of the telomere length-variation among the segregants, respectively, suggesting the presence of several other unidentified loci that contribute to telomere length-variation. The average telomere length of the segregants closer to the shorter telomere length of the RM11 parent is surprising given the finding that the RM11
alleles of the major Chromosome 12 locus confer longer telomeres. Other RM11
alleles from the loci that were not mapped and that confer short telomeres may account for this finding. Other loci with smaller effects on telomere length may not have been mapped due to limited sample size. Furthermore, genes that participate in telomere length-regulation have complex, epistatic relationships, which is consistent with the evidence for non-additive effects from the distribution of telomere lengths and may decrease the statistical power of single-locus linkage analysis. Taken together, our results support the idea that telomere length in genetically diverse yeast strains behaves as a quantitative trait controlled by many genes.
Loci That Control Telomere Length in RM and BY Strains