Expression of the telomeric repeat binding factor gene NgTRF1 is closely coordinated with the cell division program in tobacco BY-2 suspension culture cells

Telomeres are vital for preserving chromosome integrity during cell division. Several genes encoding potential telomere-binding proteins have recently been identified in higher plants, but nothing is known about their function or regulation during cell division. In this study, we have isolated and characterized a cDNA clone, pNgTRF1, encoding a putative double-stranded telomeric repeat binding factor of Nicotiana glutinosa, a diploid tobacco plant. The predicted protein sequence of NgTRF1 (Mr = 75,000) contains a single Myb-like domain with significant homology to a corresponding motif in human TRF1/Pin2 and TRF2. Gel retardation assays revealed that bacterially expressed full-length NgTRF1 was able to form a specific complex only with probes containing three or more contiguous telomeric TTTAGGG repeats. The Myb-like domain of NgTRF1 is essential, but not sufficient, to bind the telomeric repeat sequence. The glutamine-rich extreme C-terminal region, which does not exist in animal proteins, was additionally required to form a specific telomere-protein complex. The dissociation constant (Kd) of the Myb motif plus the glutamine-rich domain of NgTRF1 to the two-telomeric repeat sequence was evaluated to be 4.5 +/- 0.2 x 10-9 m, which is comparable to that of the Myb domain of human TRF1. Expression analysis showed that NgTRF1 gene activity was inversely correlated with the cell division capacity of tobacco root cells and during the 9-day culture period of BY-2 suspension cells, while telomerase activity was positively correlated with cell division. In synchronized BY-2 cells, NgTRF1 was selectively expressed in G1 phase, whereas telomerase activity peaked in S phase. These findings suggest that telomerase activity and NgTRF1 expression are differentially regulated in an opposing fashion during growth and cell division in tobacco plants. The possible physiological functions of NgTRF1 in tobacco cells are also discussed.     

Tobacco GTBP1, a Homolog of Human Heterogeneous Nuclear Ribonucleoprotein, Protects Telomeres from Aberrant Homologous Recombination

Telomeres are nucleoprotein complexes essential for the integrity of eukaryotic chromosomes. Cellular roles of single-stranded telomeric DNA binding proteins have been extensively described in yeast and animals, but our knowledge about plant single-strand telomeric factors is rudimentary. Here, we investigated Nicotiana tabacum G-strand-specific single-stranded telomere binding proteins (GTBPs), homologs of a human heterogeneous nuclear ribonucleoprotein. GTBPs bound specifically to the plant single-stranded (TTTAGGG)₄ telomeric repeat element in vitro and were associated with telomeric sequences in tobacco BY-2 suspension cells. Transgenic plants (35S:RNAi-GTBP1), in which GTBP1 was suppressed, exhibited severe developmental anomalies. In addition, the chromosomes of 35S:RNAi-GTBP1 cells displayed elongated telomeres, frequent formation of extrachromosomal telomeric circles, and numerous abnormal anaphase bridges, indicating that GTBP1 knockdown tobacco plants experienced genome instability. GTBP1 inhibited strand invasion, an initial step in interchromosomal homologous recombination. We propose that GTBP1 plays a critical role in telomere structure and function by preventing aberrant interchromosomal telomeric homologous recombination in tobacco.     

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1^561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1^561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1^561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1^561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)_n.     

Importance of TRF1 for functional telomere structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.     

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.     

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

A mathematical model of cellular apoptosis and senescence through the dynamics of telomere loss

The shortening of telomeric repeats as a cell replicates has long been implicated as a determinant of cell viability. However, recent studies have indicated that it is not telomere length, but rather whether telomeres have bound a telomere-related protein, which in mammals is TTAGGG repeat binding factor-2 (TRF2), that determines whether a cell undergoes apoptosis (programmed cell death), enters senescence (a quiescent, non-replicative state), or continues to proliferate. When bound to a telomere, TRF2 allows a cell to recognize the telomere as the point where a chromosome ends rather than a break in DNA. When telomeres are not bound by TRF2, the cell can either immediately trigger senescence or apoptosis via the DNA damage response pathway, or indirectly trigger it by attempting to repair the chromosome, which results in chromosomal end joining. We model the ability of telomeres to bind TRF2 as a function of telomere length and apply the resulting binding probability to a model of cellular replication that assumes a homogeneous cell population. The model fits data from cultured human fibroblasts and human embryonic kidney cells for two free parameters well. We extract values for the percent of telomere loss at which cell proliferation ceases. We show, in agreement with previous experiments, that overexpression of TRF2 allows a cell to delay the senescence setpoint. We explore the effect of oxidative stress, which increases the rate of telomere loss, on cell viability and show that cells in the presence of oxidative stress have reduced lifespans. We also show that the addition of telomerase, an enzyme that maintains telomere length, is sufficient to result in cell immortality. We conclude that the increasing inability of TRF2 to bind telomeres as they shorten is a quantitatively reasonable model for a cause of either cellular apoptosis or senescence.     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

Telomere binding of the Rap1 protein is required for meiosis in fission yeast.

Telomeres are essential for chromosome integrity, protecting the ends of eukaryotic linear chromosomes during cell proliferation. Telomeres also function in meiosis; a characteristic clustering of telomeres beneath the nuclear membrane is observed during meiotic prophase in many organisms from yeasts to plants and humans, and the role of the telomeres in meiotic pairing and the recombination of homologous chromosomes has been demonstrated in the fission yeast Schizosaccharomyces pombe and in the budding yeast Saccharomyces cerevisiae. Here we report that S. pombe Rap1 is a telomeric protein essential for meiosis. While Rap1 is conserved in budding yeast and humans, schemes for telomere binding vary among species: human RAP1 binds to the telomere through interaction with the telomere binding protein TRF2; S. cerevisiae Rap1, however, binds telomeric DNA directly, and no orthologs of TRF proteins have been identified in this organism. In S. pombe, unlike in S. cerevisiae, an ortholog of human TRF has been identified. This ortholog, Taz1, binds directly to telomere repeats [18] and is necessary for telomere clustering in meiotic prophase. Our results demonstrate that S. pombe Rap1 binds to telomeres through interaction with Taz1, similar to human Rap1-TRF2, and that Taz1-mediated telomere localization of Rap1 is necessary for telomere clustering and for the successful completion of meiosis. Moreover, in taz1-disrupted cells, molecular fusion of Rap1 with the Taz1 DNA binding domain recovers telomere clustering and largely complements defects in meiosis, indicating that telomere localization of Rap1 is a key requirement for meiosis.     

铅和硒对端粒长度、端粒酶及端粒结合蛋白的影响

以酿酒酵母细胞为实验材料 ,在分子水平上研究铅 (Pb)和硒 (Se)对端粒长度、端粒酶及端粒结合蛋白的影响。结果发现 :与对照组相比 ,添加 1mg/LPb的培养基中培养 10 0代后的酿酒酵母细胞中端粒DNA的平均长度缩短 ,端粒结合蛋白Rap1p含量减少 ,而且Rap1p蛋白的二级结构受到扰动、端粒酶活性降低、43%的细胞死亡。加 1mg/LSe培养 10 0代后的酿酒酵母细胞与对照组相比 ,细胞中端粒平均长度增加 ,Rap1p蛋白浓度和二级结构保持稳定 ,端粒酶活性增加 ,细胞正常存活。以上结果表明 ,Pb对酿酒酵母细胞端粒有损伤 ,而且其损伤在子代细胞中有累积效应 ;而Se对Pb损伤具有一定程度的修复保护作用 ,适量给机体补Se对抑制细胞损伤和衰老有一定作用。由于端粒的特殊结构特征 ,推断Pb和Se是通过作用于端粒酶及端粒结合蛋白而间接影响端粒长度的     

Hypomethylating drugs efficiently decrease cytosine methylation in telomeric DNA and activate telomerase without affecting telomere lengths in tobacco cells

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.     

Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.     

Human UPF1 interacts with TPP1 and telomerase and sustains telomere leading-strand replication

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5'-to-3' helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis.     

Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction ( approximately 73%) has binding dynamics similar to TRF1 (residence time of approximately 44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of approximately 11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.     

Loss of hPot1 function leads to telomere instability and a cut-like phenotype

The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.     

spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.     

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.     

The Pif1 Helicase,a Negative Regulator of Telomerase,Acts Preferentially at Long Telomeres

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.