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.     

Telomere maintenance through spatial control of telomeric proteins

The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance.     

TRF2-Tethered TIN2 Can Mediate Telomere Protection by TPP1/POT1

The shelterin protein TIN2 is required for the telomeric accumulation of TPP1/POT1 heterodimers and for the protection of telomeres by the POT1 proteins (POT1a and POT1b in the mouse). TIN2 also binds to TRF1 and TRF2, improving the telomeric localization of TRF2 and its function. Here, we ask whether TIN2 needs to interact with both TRF1 and TRF2 to mediate the telomere protection afforded by TRF2 and POT1a/b. Using a TIN2 allele deficient in TRF1 binding (TIN2-L247E), we demonstrate that TRF1 is required for optimal recruitment of TIN2 to telomeres and document phenotypes associated with the TIN2-L247E allele that are explained by insufficient TIN2 loading onto telomeres. To bypass the requirement for TRF1-dependent recruitment, we fused TIN2-L247E to the TRF2-interacting (RCT) domain of Rap1. The RCT-TIN2-L247E fusion showed improved telomeric localization and was fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. These data indicate that when sufficient TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. We therefore conclude that shelterin can protect chromosome ends as a TRF2-tethered TIN2/TPP1/POT1 complex that lacks a physical connection to TRF1.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

Telomere Protection by TPP1 Is Mediated by POT1a and POT1b

Mammalian telomeres are protected by the shelterin complex, which contains single-stranded telomeric DNA binding proteins (POT1a and POT1b in rodents, POT1 in other mammals). Mouse POT1a prevents the activation of the ATR kinase and contributes to the repression of the nonhomologous end-joining pathway (NHEJ) at newly replicated telomeres. POT1b represses unscheduled resection of the 5′-ended telomeric DNA strand, resulting in long 3′ overhangs in POT1b KO cells. Both POT1 proteins bind TPP1, forming heterodimers that bind to other proteins in shelterin. Short hairpin RNA (shRNA)-mediated depletion had previously demonstrated that TPP1 contributes to the normal function of POT1a and POT1b. However, these experiments did not establish whether TPP1 has additional functions in shelterin. Here we report on the phenotypes of the conditional deletion of TPP1 from mouse embryo fibroblasts. TPP1 deletion resulted in the release of POT1a and POT1b from chromatin and loss of these proteins from telomeres, indicating that TPP1 is required for the telomere association of POT1a and POT1b but not for their stability. The telomere dysfunction phenotypes associated with deletion of TPP1 were identical to those of POT1a/POT1b DKO cells. No additional telomere dysfunction phenotypes were observed, establishing that the main role of TPP1 is to allow POT1a and POT1b to protect chromosome ends.Mammalian cells solve the chromosome end protection problem through the binding of shelterin to the telomeric TTAGGG repeat arrays at chromosome ends (5). Shelterin contains two double-stranded telomeric DNA binding proteins, TRF1 and TRF2, which both interact with the shelterin subunit TIN2. These three shelterin components, as well as the TRF2 interacting factor Rap1, are abundant, potentially covering the majority of the TTAGGG repeat sequences at chromosome ends (30). TIN2 interacts with the less abundant TPP1/POT1 heterodimers and is thought to facilitate the recruitment of the single-stranded telomeric DNA binding proteins to telomeres (15, 21, 35).Shelterin represses the four major pathways that threaten mammalian telomeres (6). It prevents activation of the ATM and ATR kinases, which can induce cell cycle arrest in response to double-strand breaks (DSBs). Shelterin also blocks the two major repair pathways that act on DSBs: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Removal of individual components of shelterin leads to highly specific telomere dysfunction phenotypes, allowing assignment of shelterin functions to each of its components.The POT1 proteins are critical for the repression of ATR signaling (20). Concurrent deletion of POT1a and POT1b from mouse embryo fibroblasts (POT1a/b DKO cells [12]) activates the ATR kinase at most telomeres, presumably because the single-stranded telomeric DNA is exposed to RPA. POT1a/b DKO cells also have a defect in the structure of the telomere terminus, showing extended 3′ overhangs that are thought to be due to excessive resection of the 5′-ended strand in the absence of POT1b (11-13). The combination of these two phenotypes, activation of the ATR kinase and excess single-stranded telomeric DNA, is not observed when either TRF1 or TRF2 is deleted.In contrast to the activation of ATR signaling in POT1a/b DKO cells, TRF2 deletion results in activation of the ATM kinase at telomeres (3, 16, 20). In addition, TRF2-deficient cells show widespread NHEJ-mediated telomere-telomere fusions (3, 31). This phenotype is readily distinguished from the consequences of POT1a/b loss. POT1a/b DKO cells have a minor telomere fusion phenotype that primarily manifests after DNA replication, resulting in the fusion of sister telomeres (12). In TRF2-deficient cells, most telomere fusions take place in G₁ (18), resulting in chromosome-type telomere fusions in the subsequent metaphase. Chromosome-type fusions also occur in the POT1a/b DKO setting, but they are matched in frequency by sister telomere fusions.The type of telomere dysfunction induced by TRF1 loss is also distinct. Deletion of TRF1 gives rise to DNA replication problems at telomeres that activate the ATR kinase in S phase and leads to aberrant telomere structures in metaphase (referred to as “fragile telomeres”) (28). This fragile telomere phenotype is not observed upon deletion of POT1a and POT1b, and the activation of the ATR kinase at telomeres in POT1a/b DKO cells is not dependent on the progression through S phase (Y. Gong and T. de Lange, unpublished data). Furthermore, deletion of TRF1 does not induce excess single-stranded DNA.These phenotypic distinctions bear witness to the separation of functions within shelterin and also serve as a guide to understanding the contribution of the other shelterin proteins, including TPP1. TPP1 is an oligonucleotide/oligosaccharide-binding fold (OB fold) protein in shelterin that forms a heterodimer with POT1 (32). TPP1 and POT1 are distantly related to the TEBPα/β heterodimer, which is bound to telomeric termini of certain ciliates (2, 32, 33). Several lines of evidence indicate that TPP1 mediates the recruitment of POT1 to telomeres. Mammalian TPP1 was discovered based on its interaction with TIN2, and diminished TPP1 levels affect the ability of POT1 to bind to telomeres and protect chromosome ends (14, 15, 21, 26, 33, 35). Since TPP1 enhances the in vitro DNA binding activity of POT1 (32), it might mediate the recruitment of POT1 through improving its interaction with the single-stranded telomeric DNA. However, POT1 does not require its DNA binding domain for telomere recruitment, although this domain is critical for telomere protection (23, 26). Thus, it is more likely that the TPP1-TIN2 interaction mediates the binding of POT1 to telomeres. However, POT1 has also been shown to bind to TRF2 in vitro, and this interaction has been suggested to constitute a second mechanism for the recruitment of POT1 to telomeres (1, 34).TPP1 has been suggested to have additional functions at telomeres. Biochemical data showed that TPP1 promotes the interaction between TIN2, TRF1, and TRF2 (4, 25). Therefore, it was suggested that TPP1 plays an essential organizing function in shelterin, predicting that its deletion would affect TRF1 and TRF2 (25). Furthermore, cytogenetic data on cells from the adrenocortical dysplasia (Acd) mouse strain, which carries a hypomorphic mutation for TPP1 (14), revealed complex chromosomal rearrangements in addition to telomere fusions, leading to the suggestion that TPP1 might have additional telomeric or nontelomeric functions (9).In order to determine the role of TPP1 at telomeres and possibly elsewhere in the genome, we generated a conditional knockout setting in mouse embryo fibroblasts. The results indicate that the main function of TPP1 is to ensure the protection of telomeres by POT1 proteins. Each of the phenotypes of TPP1 loss was also observed in the POT1a/b DKO cells. No evidence was found for a role of TPP1 in stabilizing or promoting the function of other components of shelterin. Furthermore, the results argue against a TPP1-independent mode of telomeric recruitment of POT1.     

Telomere protection by TPP1/POT1 requires tethering to TIN2

To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the?central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.     

Binding of TPP1 Protein to TIN2 Protein Is Required for POT1a,b Protein-mediated Telomere Protection

The single-stranded DNA binding proteins in mouse shelterin, POT1a and POT1b, accumulate at telomeres as heterodimers with TPP1, which binds TIN2 and thus links the TPP1/POT1 dimers with TRF1 and TRF2/Rap1. When TPP1 is tethered to TIN2/TRF1/TRF2, POT1a is thought to block replication protein A binding to the single-stranded telomeric DNA and prevent ataxia telangiectasia and Rad3-related kinase activation. Similarly, TPP1/POT1b tethered to TIN2 can control the formation of the correct single-stranded telomeric overhang. Consistent with this view, the telomeric phenotypes following deletion of POT1a,b or TPP1 are phenocopied in TIN2-deficient cells. However, the loading of TRF1 and TRF2/Rap1 is additionally compromised in TIN2 KO cells, leading to added phenotypes. Therefore, it could not be excluded that, in addition to TIN2, other components of shelterin contribute to the recruitment of TPP1/POT1a,b as suggested by previous reports. To test whether TIN2 is the sole link between TPP1/POT1a,b and telomeres, we defined the TPP1 interaction domain of TIN2 and generated a TIN2 allele that was unable to interact with TPP1 but retained its interaction with TRF1 and TRF2. We demonstrated that cells expressing TIN2ΔTPP1 instead of wild-type TIN2 phenocopy the POT1a,b knockout setting without showing additional phenotypes. Therefore, these results are consistent with TIN2 being the only mechanism by which TPP1/POT1 heterodimers bind to shelterin and function in telomere protection.     

Distinct functions of POT1 at telomeres

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.     

Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.     

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.     

Coordinated Interactions of Multiple POT1-TPP1 Proteins with Telomere DNA

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5′-TTAGGG-3′ in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.     

OB Fold-containing Protein 1 (OBFC1), a Human Homolog of Yeast Stn1, Associates with TPP1 and Is Implicated in Telomere Length Regulation

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.Telomeres are specialized linear chromosome end structures, which are regulated and protected by networks of protein complexes (1–4). Telomere length, structure, and integrity are critical for the cells and the organism as a whole. Telomere dysregulation can lead to DNA damage response, cell cycle checkpoint, genome instability, and predisposition to cancer (5–9). Mammalian telomeres are composed of double-stranded (TTAGGG)_n repeats followed by 3′-single-stranded overhangs (10). In addition to the telomerase that directly mediates the addition of telomere repeats to the end of chromosomes (11, 12), a multitude of telomere-specific proteins have been identified that form the telosome/shelterin complex and participate in telomere maintenance (9, 13). The telosome in turn acts as the platform onto which higher order telomere regulatory complexes may be assembled into the telomere interactome (14). The telomere interactome has been proposed to integrate the complex and labyrinthine network of protein signaling pathways involved in DNA damage response, cell cycle checkpoint, and chromosomal end maintenance and protection for telomere homeostasis and genome stability.Of the six telomeric proteins (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1) that make up the telosome, TRF1 and TRF2 have been shown to bind telomeric double-stranded DNA (15, 16), whereas the OB³ fold-containing protein POT1 exhibits high affinities for telomeric ssDNA in vitro (17, 18). Although the OB fold of TPP1 does not show appreciable ssDNA binding activity, heterodimerization of TPP1 and POT1 enhances the POT1 ssDNA binding (17, 18). More importantly, POT1 depends on TPP1 for telomere recruitment, and the POT1-TPP1 heterodimer functions in telomere end protection and telomerase recruitment. Notably, the OB fold of TPP1 is critical for the recruitment of the telomerase (18). Disruption of POT1-TPP1 interaction by dominant negative inhibition, RNA interference, or gene targeting could lead to dysregulation of telomere length as well DNA damage responses at the telomeres (18–21).In budding yeast, the homolog of mammalian POT1, Cdc13, has been shown to interact with two other OB fold-containing proteins, Stn1 and Ten1, to form a Cdc13-Stn1-Ten1 (CST) complex (22, 23). The CST complex participates in both telomere length control and telomere end capping (22, 23). The presence of multiple OB fold-containing proteins from yeast to human suggests a common theme for telomere ssDNA protection (4). Indeed, it has been proposed that the CST complex is structurally analogous to the replication factor A complex and may in fact function as a telomere-specific replication factor A complex (23). Notably, homologs of the CST complex have been found in other species such as Arabidopsis (24), further supporting the notion that multiple OB fold proteins may be involved in evolutionarily conserved mechanisms for telomere end protection and length regulation. It remains to be determined whether the CST complex exists in mammals.Although the circuitry of interactions among telosome components has been well documented and studied, how core telosome subunits such as TPP1 help to coordinate the cross-talks between telomere-specific signaling pathways and other cellular networks remains unclear. To this end, we carried out large scale immunoprecipitations and mass spectrometry analysis of the TPP1 protein complexes in mammalian cells. Through these studies, we identified OB fold-containing protein 1 (OBFC1) as a new TPP1-associated protein. OBFC1 is also known as α-accessory factor AAF44 (36). Sequence alignment analysis indicates that OBFC1 is a homolog of the yeast Stn1 protein (25). Further biochemical and cellular studies demonstrate the association of OBFC1 with TPP1 in live cells. Moreover, we showed that OBFC1 bound to telomeric ssDNA and localized to telomeres in mammalian cells. Dominant expression of an OBFC1 mutant led to telomere length dysregulation, indicating that OBFC1 is a novel telomere-associated OB fold protein functioning in telomere length regulation.     

RPA and POT1: Friends or foes at telomeres?

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.Key words: RPA, POT1, telomere, ATR, checkpointTelomeres, the natural ends of chromosomes, are composed of repetitive DNA sequences and “capped” by both specific proteins and non-coding RNAs.^1–3 One of the critical functions of telomeres is to prevent chromosomal ends from recognition by the DNA damage response machinery. Critically short or improperly capped telomeres lead to telomere dysfunction and are a major source of genomic instability.⁴ While telomeres need to be properly capped to remain stable, they also need to be duplicated during each cell division by the DNA replication machinery. The requirement of these two seemingly competing processes for telomere maintenance suggests that the cell must coordinate DNA replication and capping of telomeres to ensure faithful telomere duplication yet avoid an inappropriate DNA damage response.Telomeric DNA is unique in several ways. The bulk of each human telomere is comprised of double-stranded TTA GGG repeats. At the very end of each telomere, a stretch of single-stranded TTAGGG repeats exists as a 3′ overhang. The TTA GGG repeats in the telomeric single-stranded DNA (ssDNA) allow it to loop back and invade telomeric double-stranded DNA (dsDNA), forming a structure called the t-loop.⁵ At the base of the t-loop, the TTAGGG strand of the telomeric dsDNA is displaced by the invading single-stranded 3′ overhang to form a single-stranded D-loop. Thus, the unique DNA sequence and structures of telomeres confer the ability to bind proteins in both sequence- and structure-specific manners, providing the basis for additional regulations.In human cells, telomere capping is orchestrated by the protein complex shelterin, which contains TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.³ Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to telomeric ssDNA in a sequence-specific manner.^6–8 While the human genome contains only one POT1 gene, the mouse genome contains two POT1-related genes, POT1a and POT1b.^9–11 TIN2 functions to stabilize TRF1 and TRF2 DNA binding and also tethers the POT1-TPP1 heterodimer to the rest of the shelterin complex on telomeric dsDNA.^12,13Unlike the properly capped telomeres, double-stranded DNA breaks (DSBs) with ssDNA overhangs are known to activate the ATR checkpoint kinase.^14,15 In a complex with its functional partner ATRIP, ATR is recruited to ssDNA by RPA, a non-sequence-specific ssDNA-binding protein complex.¹⁶ In addition to the ATR-ATRIP kinase complex, several other checkpoint proteins involved in ATR activation are also recruited in the presence of RPA-ssDNA.¹⁵ The structural resemblance between DSBs and telomeres and the presence of ssDNA at telomeres raise the important question as to how ATR activation is repressed at telomeres.     

Genome-wide YFP fluorescence complementation screen identifies new regulators for telomere signaling in human cells

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ～12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.     

TRF2 promotes dynamic and stepwise looping of POT1 bound telomeric overhang

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.     

Functional dissection of human and mouse POT1 proteins

The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5'-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5'-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5'-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.     

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA‐binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1–TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1–TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1–TPP1 increased the translocation efficiency. A template‐mutant telomerase that synthesizes DNA that cannot be bound by POT1–TPP1 exhibited increased processivity only when the primer contained at least one POT1–TPP1‐binding site, so a single POT1–TPP1–DNA interaction is necessary and sufficient for stimulating processivity. The POT1–TPP1 effect is specific, as another single‐stranded DNA‐binding protein, gp32, cannot substitute. POT1–TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1–TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.     

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.     

Structure,dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD⁺ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.