Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

The telomeric poly(ADP-ribose) polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor, 182-kDa tankyrase-binding protein (TAB182)

Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase, was originally identified through its interaction with TRF1, a negative regulator of telomere length. Tankyrase 1 ADP-ribosylates TRF1 in vitro, and its overexpression induces telomere elongation in human cancer cells. In addition to its telomeric localization, tankyrase 1 resides at multiple subcellular sites, suggesting additional functions for this protein. Here we identify TAB182, a novel tankyrase 1-binding protein of 182 kDa. TAB182 displays a complex pattern of subcellular localization. TAB182 localizes to the nucleus in a heterochromatic staining pattern and to the cytoplasm, where it co-stains with the cortical actin network. TAB182 coimmunoprecipitates with tankyrase 1 from human cells and serves as an acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in vitro. Like TRF1, TAB182 binds to the ankyrin domain (comprising 24 ankyrin repeats) of tankyrase 1. Surprisingly, dissection of this domain reveals multiple discrete and overlapping binding sites for TRF1 and TAB182. Thus, we demonstrate five well conserved ankyrin repeat clusters in tankyrase 1. Although each of the five ankyrin repeat clusters independently binds to TRF1, only three of the five bind toTAB182. These findings suggest that tankyrase 1 may act as a scaffold for large molecular mass complexes made up of multiple binding proteins. We discuss potential roles for tankyrase 1-mediated higher order complexes at telomeres and at other subcellular sites.     

The role of the poly(ADP-ribose) polymerase tankyrase1 in telomere length control by the TRF1 component of the shelterin complex

Tankyrase1 is a multifunctional poly(ADP-ribose) polymerase that can localize to telomeres through its interaction with the shelterin component TRF1. Tankyrase1 poly(ADP-ribosyl)ates TRF1 in vitro, and its nuclear overexpression leads to loss of TRF1 and telomere elongation, suggesting that tankyrase1 is a positive regulator of telomere length. In agreement with this proposal, we show that tankyrase1 RNA interference results in telomere shortening proportional to the level of knockdown. Furthermore, we show that a tankyrase1-resistant form of TRF1 enforced normal telomere length control, indicating that tankyrase1 is not required downstream of TRF1 in this pathway. Thus, in human cells, tankyrase1 appears to act upstream of TRF1, promoting telomere elongation through the removal of TRF1. This pathway appears absent from mouse cells. We show that murine TRF1, which lacks the canonical tankyrase1-binding site, is not a substrate for tankyrase1 poly(ADP-ribosyl)sylation in vitro. Furthermore, overexpression of tankyrase1 in mouse nuclei did not remove TRF1 from telomeres and had no detectable effect on other components of mouse shelterin. We propose that the tankyrase1-controlled telomere extension is a human-specific elaboration that allows additional control over telomere length in telomerase positive cells.     

Identification of a tankyrase-binding motif shared by IRAP,TAB182, and human TRF1 but not mouse TRF1. NuMA contains this RXXPDG motif and is a novel tankyrase partner

Tankyrase-1 and -2 are closely related poly(ADP-ribose) polymerases that use an ankyrin-repeat domain to bind diverse proteins, including TRF (telomere-repeat binding factor)-1, IRAP (insulin-responsive aminopeptidase), and TAB182 (182-kDa tankyrase-binding protein). TRF1 binding allows tankyrase to regulate telomere dynamics in human cells, whereas IRAP binding presumably allows tankyrase to regulate the targeting of IRAP. The mechanism by which tankyrase binds to diverse proteins has not been investigated. Herein we describe a novel RXXPDG motif shared by IRAP, TAB182, and human TRF1 that mediates their binding to tankyrases. Interestingly, mouse TRF1 lacks this motif and thus does not bind either tankyrase-1 or -2. Using the ankyrin domain of tankyrase as a bait in a yeast two-hybrid screen, we also found the RXXPDG motif in six candidate tankyrase partners, including the nuclear/mitotic apparatus protein (NuMA). We verified NuMA as an RXXPDG-mediated partner of tankyrase and suggest that this interaction contributes to the known colocalization of tankyrase and NuMA at mitotic spindle poles.     

Zinc binding catalytic domain of human tankyrase 1

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.     

Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice

Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.     

PARP1 Is a TRF2-associated poly(ADP-ribose)polymerase and protects eroded telomeres

Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.     

Protein requirements for sister telomere association in human cells

Previous studies in human cells indicate that sister telomeres have distinct requirements for their separation at mitosis. In cells depleted for tankyrase 1, a telomeric poly(ADP-ribose) polymerase, sister chromatid arms and centromeres separate normally, but telomeres remain associated and cells arrest in mitosis. Here, we use biochemical and genetic approaches to identify proteins that might mediate the persistent association at sister telomeres. We use immunoprecipitation analysis to show that the telomeric proteins, TRF1 (an acceptor of PARsylation by tankyrase 1) and TIN2 (a TRF1 binding partner) each bind to the SA1 ortholog of the cohesin Scc3 subunit. Sucrose gradient sedimentation shows that TRF1 cosediments with the SA1-cohesin complex. Depletion of the SA1 cohesin subunit or the telomeric proteins (TRF1 and TIN2) restores the normal resolution of sister telomeres in mitosis in tankyrase 1-depleted cells. Moreover, depletion of TRF1 and TIN2 or SA1 abrogates the requirement for tankyrase 1 in mitotic progression. Our studies indicate that sister telomere association in human cells is mediated by a novel association between a cohesin subunit and components of telomeric chromatin.     

GDP-mannose-4,6-dehydratase is a cytosolic partner of tankyrase 1 that inhibits its poly(ADP-ribose) polymerase activity

Tankyrase 1 is a poly(ADP-ribose) polymerase (PARP) that participates in a broad range of cellular activities due to interaction with multiple binding partners. Tankyrase 1 recognizes a linear six-amino-acid degenerate motif and, hence, has hundreds of potential target proteins. Binding of partner proteins to tankyrase 1 usually results in their poly(ADP-ribosyl)ation (PARsylation) and can lead to ubiquitylation and proteasomal degradation. However, it is not known how tankyrase 1 PARP activity is regulated. Here we identify GDP-mannose 4,6-dehydratase (GMD) as a binding partner of tankyrase 1. GMD is a cytosolic protein required for the first step of fucose synthesis. We show that GMD is complexed to tankyrase 1 in the cytosol throughout interphase, but its association with tankyrase 1 is reduced upon entry into mitosis, when tankyrase 1 binds to its other partners TRF1 (at telomeres) and NuMA (at spindle poles). In contrast to other binding partners, GMD is not PARsylated by tankyrase 1. Indeed, we show that GMD inhibits tankyrase 1 PARP activity in vitro, dependent on the GMD tankyrase 1 binding motif. In vivo, depletion of GMD led to degradation of tankyrase 1, dependent on the catalytic PARP activity of tankyrase 1. We speculate that association of tankyrase 1 with GMD in the cytosol sequesters tankyrase 1 in an inactive stable form that can be tapped by other target proteins as needed.     

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.     

The 68-kDa Telomeric Repeat Binding Factor 1 (TRF1)-associated Protein (TAP68) Interacts with and Recruits TRF1 to the Spindle Pole during Mitosis

The telomere capping protein TRF1 is a component of the multiprotein complex “shelterin,” which organizes the telomere into a high order structure. Besides telomere maintenance, telomere-associated proteins also have nontelomeric functions. For example, tankyrase 1 and TRF1 are required for the maintenance of faithful mitotic progression. However, the functional relevance of their centrosomal localization has not been established. Here, we report the identification of a TRF1-binding protein, TAP68, that interacts with TRF1 in mitotic cells. TAP68 contains two coiled-coil domains and a structural maintenance of chromosome motifs and co-localizes with TRF1 to telomeres during interphase. Immediately after nuclear envelope breakdown, TAP68 translocates toward the spindle poles followed by TRF1. Dissociation of TAP68 from the telomere is concurrent with the Nek2A-dependent phosphorylation at Thr-221. Biochemical characterization demonstrated that the first coiled-coil domain of TAP68 binds and recruits TRF1 to the centrosome. Inhibition of TAP68 expression by siRNA blocked the localization of TRF1 and tankyrase 1 to the centrosome. Furthermore, siRNA-mediated depletion of TAP68 perturbed faithful chromosome segregation and genomic stability. These findings suggest that TAP68 functions in mediating TRF1-tankyrase 1 localization to the centrosome and in mitotic regulation.     

Importance of TRF1 for functional telomere structure

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

Tankyrase polymerization is controlled by its sterile alpha motif and poly(ADP-ribose) polymerase domains

Tankyrases are novel poly(ADP-ribose) polymerases that have SAM and ankyrin protein-interaction domains. They are found at telomeres, centrosomes, nuclear pores, and Golgi vesicles and have been shown to participate in telomere length regulation. Their other function(s) are unknown, and it has been difficult to envision a common role at such diverse cellular locations. We have shown that tankyrase 1 polymerizes through its sterile alpha motif (SAM) domain to assemble large protein complexes. In vitro polymerization is reversible and still allows interaction with ankyrin-domain binding proteins. Polymerization can also occur in vivo, with SAM-dependent association of overexpressed tankyrase leading to formation of large tankyrase-containing vesicles, disruption of Golgi structure, and inhibition of apical secretion. Finally, tankyrase polymers are dissociated efficiently by poly(ADP-ribosy)lation. This disassembly is prevented by mutation of the PARP domain. Our findings indicate that tankyrase 1 has the unique capacity to promote both assembly and disassembly of large protein complexes. Thus, tankyrases appear to be master scaffolding proteins that regulate the formation of dynamic protein networks at different cellular locations. This implies a common scaffolding function for tankyrases at each location, with specific tankyrase interaction partners conferring location-specific roles to each network, e.g., telomere compaction or regulation of vesicle trafficking.     

Mammalian meiotic telomeres: protein composition and redistribution in relation to nuclear pores

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.     

Tankyrase function at telomeres, spindle poles, and beyond

Telomeres have special needs; they require distinct mechanisms for their protection, replication, and separation at mitosis. A dedicated six-subunit protein complex termed shelterin attends to these needs. But shelterin cannot do it alone and often relies on recruits from other cellular locales. One such recruit is tankyrase 1, a poly(ADP-ribose) polymerase that is brought to telomeres by the shelterin DNA binding subunit TRF1, where it functions in telomere length regulation and sister chromatid separation. An understanding of how tankyrase 1 functions at telomeres has been confounded by its complexity; it localizes to multiple subcellular sites, it has many diverse binding partners, and it has a closely related homolog (tankyrase 2) with which it may functionally overlap. This review summarizes our current knowledge of tankyrases focusing on their localization, binding partners, and function.     

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.     

Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells

Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.     

p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.