Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

Distinct roles of TRF1 in the regulation of telomere structure and lengthening

The telomere is a functional chromatin structure that consists of G-rich repetitive sequences and various associated proteins. Telomeres protect chromosomal ends from degradation, provide escape from the DNA damage response, and regulate telomere lengthening by telomerase. Multiple proteins that localize at telomeres form a complex called shelterin/telosome. One component, TRF1, is a double-stranded telomeric DNA binding protein. Inactivation of TRF1 disrupts telomeric localization of other shelterin components and induces chromosomal instability. Here, we examined how the telomeric localization of shelterin components is crucial for TRF1-mediated telomere-associated functions. We found that many of the mTRF1 deficient phenotypes, including chromosomal instability, growth defects, and dysfunctional telomere damage response, were suppressed by the telomere localization of shelterin components in the absence of functional mTRF1. However, abnormal telomere signals and telomere elongation phenotypes were either not rescued or only partially rescued, respectively. These data suggest that TRF1 regulates telomere length and function by at least two mechanisms; in one TRF1 acts through the recruiting/tethering of other shelterin components to telomeres, and in the other TRF1 seems to play a more direct role.     

Dynamics of TRF1 organizing a single human telomere

Chromosome stability is primarily determined by telomere length. TRF1 is the core subunit of shelterin that plays a critical role in telomere organization and replication. However, the dynamics of TRF1 in scenarios of telomere-processing activities remain elusive. Using single-molecule magnetic tweezers, we here investigated the dynamics of TRF1 upon organizing a human telomere and the protein-DNA interactions at a moving telomeric fork. We first developed a method to obtain telomeres from human cells for directly measuring the telomere length by single-molecule force spectroscopy. Next, we examined the compaction and decompaction of a telomere by TRF1 dimers. TRF1 dissociates from a compacted telomere with heterogenous loops in ∼20 s. We also found a negative correlation between the number of telomeric loops and loop sizes. We further characterized the dynamics of TRF1 at a telomeric DNA fork. With binding energies of 11 k_BT, TRF1 can modulate the forward and backward steps of DNA fork movements by 2–9 s at a critical force of F_1/2, temporarily maintaining the telomeric fork open. Our results shed light on the mechanisms of how TRF1 organizes human telomeres and facilitates the efficient replication of telomeric DNA. Our work will help future research on the chemical biology of telomeres and shelterin-targeted drug discovery.     

TRF1 inhibits telomere C-strand DNA synthesis in vitro

Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomere length in vivo, but the mechanism of regulation remains largely undefined. We have developed an in vitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and synthetic DNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesis catalyzed by DNA polymerase alpha/primase (pol alpha). Inhibition required that TRF1 be bound to duplex telomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibition was shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of the DNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol alpha-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstream of the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but not necessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, we assessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators. In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has no effect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition was observed on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, when bound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussed with respect to the role of TRF1 in telomere length regulation.     

Gene therapy with the TRF1 telomere gene rescues decreased TRF1 levels with aging and prolongs mouse health span

The shelterin complex protects telomeres by preventing them from being degraded and recognized as double‐strand DNA breaks. TRF1 is an essential component of shelterin, with important roles in telomere protection and telomere replication. We previously showed that TRF1 deficiency in the context of different mouse tissues leads to loss of tissue homeostasis owing to impaired stem cell function. Here, we show that TRF1 levels decrease during organismal aging both in mice and in humans. We further show that increasing TRF1 expression in both adult (1‐year‐old) and old (2‐year‐old) mice using gene therapy can delay age‐associated pathologies. To this end, we used the nonintegrative adeno‐associated serotype 9 vector (AAV9), which transduces the majority of mouse tissues allowing for moderate and transient TRF1 overexpression. AAV9‐TRF1 gene therapy significantly prevented age‐related decline in neuromuscular function, glucose tolerance, cognitive function, maintenance of subcutaneous fat, and chronic anemia. Interestingly, although AAV9‐TRF1 treatment did not significantly affect median telomere length, we found a lower abundance of short telomeres and of telomere‐associated DNA damage in some tissues. Together, these findings suggest that rescuing naturally decreased TRF1 levels during mouse aging using AAV9‐TRF1 gene therapy results in an improved mouse health span.     

ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance

Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.     

Cell-cycle-dependent Xenopus TRF1 recruitment to telomere chromatin regulated by Polo-like kinase

Telomeres are regulated by a homeostatic mechanism that includes telomerase and telomeric repeat binding proteins, TRF1 and TRF2. Recently, it has been hypothesized that telomeres assume distinct configurations in a cell-cycle-dependent manner, although direct biochemical evidence is lacking. Here we demonstrated that Xenopus TRF1 (xTRF1) associates with telomere chromatin specifically in mitotic Xenopus egg extracts, and dissociates from it upon mitotic exit. Both the N-terminal TRF-homology (TRFH) domain and the linker region connecting the TRFH domain and the C-terminal Myb domain are required for this cell-cycle-dependent association of xTRF1 with chromatin. In contrast, Xenopus TRF2 (xTRF2) associates with chromatin throughout the cell cycle. We showed that Polo-like kinase (Plx1) phosphorylates xTRF1 in vitro. Moreover, the mitotic xTRF1-chromatin association was significantly impaired when Plx1 was immunodepleted from the extracts. Finally, high telomerase activities were detected in association with replicating interphase chromatin compared with mitotic chromatin. These results indicate that telomere chromatin is actively regulated by cell-cycle-dependent processes, and provide an insight for understanding how telomeres undergo DNA metabolisms during the cell cycle.     

Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1

Continuously dividing cells must be protected from telomeric and nontelomeric DNA damage in order to maintain their proliferative potential. Here, we report a novel telomere-protecting mechanism regulated by nucleostemin (NS). NS depletion increased the number of telomere damage foci in both telomerase-active (TA(+)) and alternative lengthening of telomere (ALT) cells and decreased the percentage of damaged telomeres associated with ALT-associated PML bodies (APB) and the number of APB in ALT cells. Mechanistically, NS could promote the recruitment of PML-IV to SUMOylated TRF1 in TA(+) and ALT cells. This event was stimulated by DNA damage. Supporting the importance of NS and PML-IV in telomere protection, we demonstrate that loss of NS or PML-IV increased the frequency of telomere damage and aberration, reduced telomeric length, and perturbed the TRF2(ΔBΔM)-induced telomeric recruitment of RAD51. Conversely, overexpression of either NS or PML-IV protected ALT and TA(+) cells from telomere damage. This work reveals a novel mechanism in telomere protection.     

Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

The telomere binding protein TRF2 induces chromatin compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.     

Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation

Telomere maintenance is essential for protecting chromosome ends. Aberrations in telomere length have been implicated in cancer and aging. Telomere elongation by human telomerase is inhibited in cis by the telomeric protein TRF1 and its associated proteins. However, the link between TRF1 and inhibition of telomerase elongation of telomeres remains elusive because TRF1 has no direct effect on telomerase activity. We have previously identified one Pin2/TRF1-interacting protein, PinX1, that has the unique property of directly binding and inhibiting telomerase catalytic activity (Zhou, X. Z., and Lu, K. P. (2001) Cell 107, 347-359). However, nothing is known about the role of the PinX1-TRF1 interaction in the regulation of telomere maintenance. By identifying functional domains and key amino acid residues in PinX1 and TRF1 responsible for the PinX1-TRF1 interaction, we show that the TRF homology domain of TRF1 interacts with a minimal 20-amino acid sequence of PinX1 via hydrophilic and hydrophobic interactions. Significantly, either disrupting this interaction by mutating the critical Leu-291 residue in PinX1 or knocking down endogenous TRF1 by RNAi abolishes the ability of PinX1 to localize to telomeres and to inhibit telomere elongation in cells even though neither has any effect on telomerase activity per se. Thus, the telomerase inhibitor PinX1 is recruited to telomeres by TRF1 and provides a critical link between TRF1 and telomerase inhibition to prevent telomere elongation and help maintain telomere homeostasis.     

Essential role for the TRF2 telomere protein in adult skin homeostasis

TRF2 is a component of shelterin, the protein complex that protects the ends of mammalian chromosomes. TRF2 is essential for telomere capping owing to its roles in suppressing an ATM-dependent DNA damage response (DDR) at chromosome ends and inhibiting end-to-end chromosome fusions. Mice deficient for TRF2 are early embryonic lethal. However, the role of TRF2 in later stages of development and in the adult organism remains largely unaddressed, with the exception of liver, where TRF2 was found to be dispensable for maintaining tissue function. Here, we study the impact of TRF2 conditional deletion in stratified epithelia by generating the TRF2^∆/∆-K5-Cre mouse model, which targets TRF2 deletion to the skin from embryonic day E11.5. In marked contrast to TRF2 deletion in the liver, TRF2^∆/∆-K5-Cre mice show lethality in utero reaching 100% lethality perinataly. At the molecular and cellular level, TRF2 deletion provokes induction of an acute DDR at telomeres, leading to activation of p53 signaling pathways and to programed cell death since the time of Cre expression at E11.5. Unexpectedly, neither inhibition of the NHEJ pathway by abrogation of 53BP1 nor inhibition of DDR by p53 deficiency rescued these severe phenotypes. Instead, TRF2 deletion provokes an extensive epidermal cell death accompanied by severe inflammation already at E16.5 embryos, which are independent of p53. These results are in contrast with conditional deletion of TRF1 and TPP1 in the skin, where p53 deficiency rescued the associated skin phenotypes, highlighting the comparatively more essential role of TRF2 in skin homeostasis.     

Nuclear import pathway of the telomere elongation suppressor TRF1: inhibition by importin alpha

Telomere repeat factor 1 (TRF1) regulates the steady-state length of chromosomes, whereby its overexpression results in telomere shortening while dominant negative TRF1 mutations can lead to telomere elongation, which is linked to cell immortalization/transformation. Although present in the nucleus at mammalian chromosomal ends during interphase and mitosis, nothing is known of the mechanism by which TRF1 enters the nucleus or how its nuclear levels may be regulated and the relevance of this, in turn, to telomere length and cell immortalization. Here we examine the nuclear import mechanism of TRF by expressing and purifying a recombinant TRF1-GFP (green fluorescent protein) fusion protein that is functional in terms of being able to bind telomeric DNA specifically as shown using a novel, quantitative double-label gel mobility shift assay. We quantitate the ability of TRF1-GFP to accumulate in the nucleus using real time confocal laser scanning microscopy, showing that the nuclear import pathway of TRF1 is mediated by importin (Imp) beta1 and Ran. Imp beta is shown to bind directly to TRF1 with nanomolar affinity using native gel electrophoretic and fluorescence polarization (FP) approaches; FP experiments also demonstrate that Imp beta residues 1-380 are responsible for TRF1 binding. Intriguingly, when dimerized to Imp beta, Imp alpha was found to inhibit Imp beta-mediated nuclear accumulation, although not affecting Imp beta binding to TRF1. The study represents the first elucidation of the nuclear transport mechanism of TRF1; that its nuclear import is mediated directly by Imp beta but inhibited by Imp alpha may represent a novel regulatory mechanism, with potential relevance to oncogenesis.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

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.