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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to Shelterin Core Hub TIN2 when TPP1 Is Absent

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1–TIN2–TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1–TIN2–TRF2 core complex.     

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.     

TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres

Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telomere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of murine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection.     

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.     

TIN2 stability is regulated by the E3 ligase Siah2

Telomeres are coated by shelterin, a six-subunit complex that is required for protection and replication of chromosome ends. The central subunit TIN2, with binding sites to three subunits (TRF1, TRF2, and TPP1), is essential for stability and function of the complex. Here we show that TIN2 stability is regulated by the E3 ligase Siah2. We demonstrate that TIN2 binds to Siah2 and is ubiquitylated in vivo. We show using purified proteins that Siah2 acts as an E3 ligase to directly ubiquitylate TIN2 in vitro. Depletion of Siah2 led to stabilization of TIN2 protein, indicating that Siah2 regulates TIN2 protein levels in vivo. Overexpression of Siah2 in human cells led to loss of TIN2 at telomeres that was dependent on the presence of the catalytic RING domain of Siah2. In contrast to RNAi-mediated depletion of TIN2 that led to loss of TRF1 and TRF2 at telomeres, Siah2-mediated depletion of TIN2 allowed TRF1 and TRF2 to remain on telomeres, indicating a different fate for shelterin subunits when TIN2 is depleted posttranslationally. TPP1 was lost from telomeres, although its protein level was not reduced. We speculate that Siah2-mediated removal of TIN2 may allow dynamic remodeling of the shelterin complex and its associated factors during the cell cycle.     

The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro

Human TIN2 interacts with the telomeric-DNA-binding protein TRF1, suppresses telomere elongation in telomerase-positive cells, and may control telomere length by modulating telomere structure. To test the latter idea, we developed an in vitro assay, using biotinylated telomeric DNA probes and streptavidin–agarose, to quantify the ability of TRF1 and TIN2 to stimulate interactions of telomeric DNA tracts with each other (probe clustering). This assay revealed that TRF1 alone had weak probe-clustering activity, but TIN2 stimulated activity fivefold to tenfold. A dominant-negative TIN2 mutant protein that increased telomere length in vivo disrupted probe clusters formed by TRF1 and TIN2, suggesting that the ability to stimulate telomeric DNA interactions is important for telomere-length regulation. Unlike TRF1, TIN2 did not form homodimers. We propose that TIN2 alters the conformation of TRF1, which favours a tertiary telomeric structure that hinders telomerase from gaining access to telomeres.     

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K⁺ and in Li⁺, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.     

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.     

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1’s 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼9–17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼2.8–3.6 κ_BT greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This ‘tag-team proofreading’ represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.     

A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix

Telomeres are specialized heterochromatin at the ends of linear chromosomes. Telomeres are crucial for maintaining genome stability and play important roles in cellular senescence and tumor biology. Six core proteins -- TRF1, TRF2, TIN2, POT1, TPP1, and Rap1 (termed the telosome or shelterin complex) – regulate telomere structure and function. One of these proteins, TIN2, regulates telomere length and structure indirectly by interacting with TRF1, TRF2 and TPP1, but no direct function has been attributed to TIN2. Here we present evidence for a TIN2 isoform (TIN2L) that differs from the originally described TIN2 isoform (TIN2S) in two ways: TIN2L contains an additional 97 amino acids, and TIN2L associates strongly with the nuclear matrix. Stringent salt and detergent conditions failed to extract TIN2L from the nuclear matrix, despite removing other telomere components, including TIN2S. In human mammary epithelial cells, each isoform showed a distinct nuclear distribution both as a function of cell cycle position and telomere length. Our results suggest a dual role for TIN2 in mediating the function of the shelterin complex and tethering telomeres to the nuclear matrix.