The 3′ Overhangs at Tetrahymena thermophila Telomeres Are Packaged by Four Proteins,Pot1a,Tpt1, Pat1, and Pat2

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3′ overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.     

Tetrahymena Pot2 Is a Developmentally Regulated Paralog of Pot1 That Localizes to Chromosome Breakage Sites but Not to Telomeres

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.     

Distinct requirements for Pot1 in limiting telomere length and maintaining chromosome stability

The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.     

HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence

The telomere integrity is maintained via replication machinery, telomere associated proteins and telomerase. Many telomere associated proteins are regulated in a cell cycle-dependent manner. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a single-stranded oligonucleotide binding protein, is thought to play a pivotal role in telomere maintenance. Here, we identified hnRNP A1 as a novel substrate for vaccinia-related kinase 1 (VRK1), a cell cycle regulating kinase. Phosphorylation by VRK1 potentiates the binding of hnRNP A1 to telomeric ssDNA and telomerase RNA in vitro and enhances its function for telomerase reaction. VRK1 deficiency induces a shortening of telomeres with an abnormal telomere arrangement and activation of DNA-damage signaling in mouse male germ cells. Together, our data suggest that VRK1 is required for telomere maintenance via phosphorylation of hnRNP A1, which regulates proteins associated with the telomere and telomerase RNA.     

Tetrahymena POT1a regulates telomere length and prevents activation of a cell cycle checkpoint

The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3' strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.     

Tetrahymena mutants with short telomeres.

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation.     

Polypeptide Components of Telomere Nucleoprotein Complex

Chromosome telomeres of humans and many model organisms contain a structure called a t-loop, which is maintained by TERF, TINF2, Pot1, and other proteins. Increase in TERF1 concentration prevents telomere elongation by telomerase. Decrease in TERF2 concentration (preventing t-loop formation) is accompanied by blockade of proliferation and appearance of other signs of cellular senescence in experiments. Natural regulation of TERF1 involves tankyrase, ATM protein kinase, and fluctuations of the protein level across a cell cycle. The telomere nucleoprotein complex also interacts with various polypeptide macromolecules (e.g., Sir2, PinX1, Rap1, Ku, Rad50/Mre11/Nbs1) responsible for heterochromatin formation, modulation of telomerase activity, DNA repair, and signaling to other cell compartments about telomere state. Study of structure and functioning of telomere nucleoprotein complex may contribute to elucidation of poorly understood mechanisms of aging and processes of tumor transformation of cells.     

Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b^−/− mTR^+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.     

Cell cycle localization, dimerization, and binding domain architecture of the telomere protein cPot1

Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.     

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View, and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.     

The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

Single-stranded DNA binding factor AtWHY1 modulates telomere length homeostasis in Arabidopsis

Telomere homeostasis, a process that is essential for the maintenance of chromosome integrity, is regulated by telomerase and a collection of associated proteins. By mass spectrometry we have identified a new telomeric protein encoded by the AtWHY1 (Arabidopsis thaliana Whirly 1) gene in Arabidopsis. AtWHY1 specifically binds the single-stranded plant telomeric DNA sequences, but not double-stranded telomeric DNA. To gain insights into the function of AtWHY1 in telomere biogenesis, we have identified two Arabidopsis lines harboring T-DNA insertions in AtWHY1. These lines exhibit neither growth nor developmental defects. However, AtWHY1-deficient plants show a steady increase in the length of telomere tracts over generations. This telomere elongation is correlated with a significant increase in telomerase activity. On the contrary, transgenic plants expressing AtWHY1 show a decreased telomerase activity and shortened telomeres. The evidence presented here indicates that AtWHY1 is a new family of telomere end-binding proteins that plays a role in regulating telomere-length homeostasis in Arabidopsis.