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Keywords:

  • aging;
  • cancer;
  • mouse models;
  • shelterin;
  • telomerase;
  • telomeres

Summary

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

Mammalian telomeres are formed by tandem repeats of the TTAGGG sequence bound by a specialized six-protein complex known as shelterin, which has fundamental roles in the regulation of telomere length and telomere capping. In the past, the study of mice genetically modified for telomerase components has been instrumental to demonstrate the role of telomere length in cancer and aging. Recent studies using genetically modified mice for shelterin proteins have highlighted an equally important role of telomere-bound proteins in cancer and aging, even in the presence of proficient telomerase activity and normal telomere length. In this review, we will focus on recent findings, suggesting a role of shelterin components in cancer and aging.


Telomeric DNA and its shield

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

The ends of linear chromosomes are formed by special heterochromatic structure, known as the telomere, which protects them from degradation and repair activities and therefore is essential to ensure chromosomal stability (de Lange, 2005; Blasco, 2007; Palm & de Lange, 2008). Mammalian telomeric chromatin is composed of tandem repeats of the TTAGGG sequence bound by a specialized multiprotein complex known as shelterin (de Lange, 2005; Palm & de Lange, 2008). Telomere repeats can extend to different lengths in different species, and this length can also vary depending on the developmental stage and cell type within a given species (Flores et al., 2008; Marion et al., 2009b). On average, human telomeres span 10–15 Kb, while mouse telomeres are 25–50 Kb long (Blasco, 2005). Telomeres are characterized by having a 150- to 200-nucleotide-long 3′-overhang of the G-rich strand, the G-strand overhang, that can fold back and invade the double-stranded telomeric region forming the so-called T-loop and generating a displacement loop, or D-loop (Fig. 1A,B). The T-loop structure has been proposed to protect chromosome ends from degradation and DNA repair activities as well as from telomerase’s action (Griffith et al., 1999). Some shelterin components have been shown to influence the T-loop formation (de Lange, 2004).

image

Figure 1.  The shelterin complex and telomere structure. (A) Representative image of a mouse metaphasic chromosome stained with DAPI (blue) and the telomeres specifically labeled with PNA probe (yellow). (B) A schematic of the shelterin complex bound to the telomere. Telomeric DNA is bound by TRF1, TRF2, RAP1, TPP1, TIN2 and POT1. The single-stranded overhang (gray strand) invades the doubled-stranded DNA region of the telomere to form a protective telomere T-loop with a displacement D-loop at the invasion site. (C) Six components of the shelterin and their DNA and protein binding abilities are depicted. The telomerase is the enzyme that elongates telomeres. The specific function associated with each shelterin and to the telomerase is highlighted in gray insets.

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The shelterin complex is composed of six core proteins, the telomeric repeat binding factors 1 and 2 (TRF1 and TRF2), the TRF1-interacting protein 2 (TIN2), protection of telomeres 1 (POT1), the POT1-TIN2 organizing protein (TPP1, also known as TINT1, PTOP or PIP1) and repressor/activator protein 1 or RAP1 (de Lange, 2005) (Fig. 1C). TRF1, TRF2 and POT1 bind directly to telomeric DNA repeats, with TRF1 and TRF2 binding to telomeric double-stranded DNA and POT1 to the 3′-singled-stranded G-overhang. TRF1 and TRF2 share a common domain structure consisting of the TRF homology (TRFH) domain and a C-terminal SANT/Myb DNA-binding domain, which are connected through a flexible hinge domain. TRF1 and TRF2 do not interact and bind telomeric DNA independently. In particular, both proteins bind telomeric duplex DNA with a very high specificity for the 5′-YTAGGGTTR-3′ sequence, both as homodimers and oligomeres (Broccoli et al., 1997; Bianchi et al., 1999; Court et al., 2005; de Lange, 2005; Palm & de Lange, 2008). POT1 possesses high specificity for single-stranded telomeric DNA sequence 5′-TAGGGTTAG-3′, thereby binding to the G-strand overhang as well as, most likely, to the displaced G-strand at the D-loop (Baumann & Cech, 2001; Loayza & De Lange, 2003; Lei et al., 2004; Palm & de Lange, 2008). POT1 can bind to TRF1 via protein–protein interactions and this interaction is proposed to affect the POT1 loading on the single-stranded telomeric DNA (Loayza & De Lange, 2003). While human cells contain only one POT1 gene, mouse cells have POT1a and POT1b (He et al., 2006; Hockemeyer et al., 2006; Wu et al., 2006). The two mouse POT1 proteins are highly homologous and can associate with telomeric DNA but they seem to have distinct functions at telomeres (Hockemeyer et al., 2006). TIN2 is able to bind TRF1 and TRF2 through independent domains and to recruit the TPP1-POT1 complex, constituting the bridge among the different shelterin components (Kim et al., 2004; Ye et al., 2004; Chen et al., 2008). TPP1 binds TIN2 and POT1 through its C-terminal and central domains, respectively (Liu et al., 2004; Ye et al., 2004). TPP1 has been proposed to recruit POT1 to telomeres (Chen et al., 2007; Kibe et al., 2010). In addition, the N-terminus of TPP1 contains a telomerase-interacting domain, suggesting a role for TPP1 in the recruitment of telomerase to chromosome ends (Xin et al., 2007). Finally, RAP1 forms a complex with TRF2 and this association is essential for RAP1 binding to telomeres (Li et al., 2000; Li & de Lange, 2003; Celli & de Lange, 2005). RAP1 contains three different domains, (i) a Myb-like domain that is likely to have a role in protein–protein interactions, (ii) a BRCT (BRCA1 C-terminal)- motif and (iii) a C-terminal domain involved in the binding to TRF2 (Li et al., 2000; Hanaoka et al., 2001).

Shelterin is proposed to have a fundamental role in protection of chromosome ends (Cosme-Blanco & Chang, 2008; Palm & de Lange, 2008; de Lange, 2009). In addition, several studies suggest that shelterin may modulate telomerase activity at chromosome ends, mainly acting as a negative regulator (Smogorzewska & de Lange, 2004; de Lange, 2005; Palm & de Lange, 2008, although recent findings indicate that some shelterins, such as Tpp 1, are required to recruit TERT to telomeres and to elongate telomeres in vivo (Tejera et al., 2010)). Excessive telomere shortening as a result of telomerase mutations or severe telomere uncapping owing to shelterin dysfunction trigger a DNA damage response (DDR) at chromosome ends, which are then recognized as double-strand breaks (Fig. 2). Subsequent activation of the nonhomologous end-joining pathway results in chromosomal end-to-end fusions, whereas increased homologous recombination may lead to rapid telomere length changes and terminal deletions (Denchi, 2009; de Lange, 2009). A current model for how critically short telomeres activate a DDR is based on the notion that short telomeres harbor insufficient amounts of shelterin to inhibit the ATM and ATR pathways (Fig. 2). In particular, a too-short telomere would fail to recruit the threshold shelterin amount required for repressing the checkpoint activation (Smogorzewska et al., 2000; Palm & de Lange, 2008). This model is supported by the fact that abrogation of certain shelterin components (ie, TRF1) can elicit a DDR in the absence of telomere shortening (Martinez et al., 2009b).

image

Figure 2.  Impact of telomere dysfunction on cancer and aging. A dysfunctional telomere, either because of critically short telomeres or because of uncapping, elicits a DNA damage response (DDR) by the activation of upstream kinases, DNA-PK, ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3 related). Two different outcomes may arise. I. Cancer: upon p53/p21-dependent cell cycle arrest, the damage can either be ‘healed’ by the nonhomologous-end-joining (NHEJ) pathway resulting in chromosomal end-to-end fusions or by the homologous recombination (HR) leading to telomere length changes and terminal deletions. In both cases, chromosomal instability is induced, which may lead to amplification of oncogenes and loss of tumor suppressor genes, increasing the risk of cellular transformation and cancer initiation. II. Aging: activation of the tumor suppressor p53 and/or p21 induces either apoptosis or senescence of cells within, causing tissue degeneration and ultimately organ failure.

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Lessons learned from telomerase-deficient and telomerase over-expressing mice

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

During each cell division cycle, telomeres shorten as a result of the incomplete replication of linear DNA molecules by conventional DNA polymerases, the so-called ‘end-replication problem’ (Watson, 1972; Olovnikov, 1973). Telomerase is a reverse transcriptase (TERT) capable of compensating telomere attrition through de novo addition of TTAGGG repeats onto the chromosome ends by using an associated RNA component as template (Terc) (Greider & Blackburn, 1985). Telomerase is expressed in embryonic stem cells and in most adult stem cell compartments; however, this is not sufficient to maintain telomere length and therefore telomere shortening takes place with age in most tissues (Harley et al., 1990; Blasco, 2007; Liu et al., 2007; Flores et al., 2008). This progressive telomere shortening is proposed to be one of the molecular mechanisms underlying organismal aging (Harley et al., 1990; Rudolph et al., 1999; Collado et al., 2007). Indeed, some diseases characterized by premature loss of tissue renewal and premature death, such as dyskeratosis congenita, as well as some cases of aplastic anemia and idiopathic pulmonary fibrosis, are linked to germline mutations in the Terc and Tert telomerase essential genes (Mitchell et al., 1999; Vulliamy et al., 2001; Yamaguchi et al., 2005; Armanios et al., 2007; Tsakiri et al., 2007). Critical telomere shortening and loss of function of telomere-binding proteins result in the loss of telomere protection, end-to-end chromosome fusions, and cell cycle arrest or apoptosis (Fig. 2) (van Steensel et al., 1998; Karlseder et al., 1999; Goytisolo & Blasco, 2002; de Lange, 2005). Additional evidence for a role of telomerase in tissue renewal and organismal lifespan was obtained from telomerase deficient (Terc-/-) (Blasco et al., 1997) as well as telomerase over-expressing mice in stratified epithelia (K5TERT) (Gonzalez-Suarez et al., 2001; Flores et al., 2005; Tomas-Loba et al., 2008) (Table 1). In particular, longevity is progressively decreased upon successive intercrossing of Terc-deficient mice, which is concomitant with a decreased mobilization of adult stem cell populations and premature organ failure. In this regard, Terc-deficient mice show aging-associated pathologies, such as alopecia, intestinal atrophy, hair graying, infertility, heart dysfunction, bone marrow aplasia, kidney dysfunction, defective bone marrow and proliferative defects of neural stem cells (Lee et al., 1998; Herrera et al., 1999; Samper et al., 2002; Leri et al., 2003; Ferron et al., 2004; Blasco, 2005; Garcia-Cao et al., 2006). Current evidence suggests that short telomeres provoke organismal aging through a p21-dependent induction of cellular senescence (Choudhury et al., 2007; Feldser & Greider, 2007). Thus, mice doubly deficient for Terc and p21 show extended organismal survival and lesser proliferative defects than the single Terc-mutant controls (Choudhury et al., 2007) (Table 1). Rescue of age-related pathologies in the Terc KO model has been also described upon abrogation of PMS2, MSH2 and EXO1 (Table 1) (Schaetzlein et al., 2007; Siegl-Cachedenier et al., 2007; Martinez et al., 2009a). In all these cases, PMS2-, MSH2- and EXO1-deficiency attenuated p21-dependent cell cycle arrest induced by short telomeres. Additionally, p53 ablation in Terc-deficient mice restores stem cell mobilization in these mice, including rescue of hair growth defects, skin renewal and skin wound-healing responses. Interestingly, this is not sufficient to rescue mouse survival, most likely because of the fact that stem cells with dysfunctional telomeres are not able to maintain long-term tissue fitness (Begus-Nahrmann et al., 2009; Flores & Blasco, 2009).

Table 1.   Mouse models for telomerase and shelterin components and their cancer and aging phenotypes
GenotypeCancer phenotypeAging phenotypeReferences
Terc−/−Reduced incidence of cancerPremature aging: Decreased proliferative potential of adult stem cell populations, alopecia, intestinal atrophy, hair graying, infertility, heart dysfunction, bone marrow aplasia, kidney dysfunction and defective bone marrowBlasco et al., (1997); Lee et al., (1998); Greenberg et al., (1999); Herrera et al., (1999); Gonzalez-Suarez et al., (2000); Samper et al., (2002); Feldser & Greider (2007)
Terc−/−p53−/−Cancer proneIncreased mobilization of adult stem cells with dysfunctional telomeres. Rescue of small body size phenotype. Lower organismal survival.Chin et al., (1999); Artandi et al., (2000); Begus-Nahrmann et al., 2009; Flores & Blasco, 2009)
Terc−/−p21−/−Reduced incidence of cancerRescue of degenerative pathologies and lifespanChoudhury et al., (2007)
Terc−/−PMS2−/−Rescue of PMS2 deficiency–mediated cancer Rescue of degenerative pathologies and lifespanSiegl-Cachedenier et al., (2007)
Terc−/−EXO1−/−Reduced incidence of cancerRescue of degenerative pathologies and lifespanSchaetzlein et al., (2007)
Terc−/−MSH2−/−Cancer proneRescue of aging-associated pathologies. Decreased lifespan because of cancerMartinez et al., (2009a)
K5-TertCancer proneIncreased proliferative response in stratified epitheliaGonzalez-Suarez et al., (2001); Flores et al., (2005)
K5-Tert super-p53 super-p16-p19ARFResistant to cancer40% increase of median longevity, reduction in aging-associated pathologies, improved neuromuscular coordination, increased glucose tolerance and a better fitness of epithelial barriers.Tomas-Loba et al., (2008)
TRF1-koEmbryonic lethalKarlseder et al., (2003)
K5-TRF1Slightly increased susceptibility to skin carcinogenesis protocolsPremature skin deterioration, hyperpigmentation, alopeciaMunoz et al., (2009)
TRF1Δ/ΔK5-Cre (conditional)Rapid development of preneoplastic lesions at 1-3 days of agePerinatal death, epithelia degenerative pathologies, hyperpigmentation hyperkeratosis, defective hair follicle and sebaceous gland developmentMartinez et al., (2009b)
TRF1Δ/ΔK5-Cre p53−/−Increased incidence of spontaneous squamous cell carcinomasRescue of survival, of hair development and of skin defectsMartinez et al., (2009b)
TRF2-koEmbryonic lethalCelli & de Lange (2005)
Mx1-TRF2 (conditional)Not reportedNot reportedLazzerini Denchi et al., (2006)
K5-TRF2Increased susceptibility to spontaneous and induced skin cancerPremature skin deterioration, hyperpigmentation, alopeciaMunoz et al., (2005)
K5-TRF2 Terc−/−Accelerated skin carcinogenesisSevere premature skin deterioration, hyperpigmentation, alopeciaBlanco et al., (2007)
TPP1-koEmbryonic lethalKibe et al., (2010)
Acd (hypomorphic)Not reportedHigh perinatal death, developmental defects, hyperpigmentation, alopecia, infertility, adrenocortical dysplasia and malformations of the skeletal and genitourinary systemKeegan et al., (2005)
Acd p53−/−Increased carcinoma incidenceRescue of survival and of the acd phenotypes except for the germ cell atrophyElse et al., (2009)
TPP1Δ/ΔK5-Cre (conditional)Epithelia dysplasiaPerinatal death, epithelia degenerative pathologies, hyperpigmentation hyperkeratosis, defective hair follicle and sebaceous gland developmentTejera et al., (2010)
POT1a-koEmbryonic lethalWu et al., (2006)
POT1b-koNot reportedHe et al., (2006); Hockemeyer et al., (2006)
POT1b-ko Terc+/−Not reportedhyperpigmentation and fatal bone marrow failureHockemeyer et al., (2008)
TIN2-koEmbryonic lethalChiang et al., (2004)
Rap1-koNot reportedviableSfeir et al., (2010)
Rap1D/D K5-CreNot reportedShow early onset of hyper pigmentation and female obesityMartinez et al., (2010)

Conversely, over-expression of the telomerase reverse transcriptase in Tert over-expressing mice results in an augmented proliferative response in the skin, including increased skin carcinogenesis, that is dependent on an active telomerase complex (depends on the Terc RNA component) (Gonzalez-Suarez et al., 2001; Cayuela et al., 2005; Flores et al., 2005). However, the impact of telomerase over-expression on organismal aging was not addressed until recently because of the cancer-promoting activity of telomerase. This has been recently addressed by telomerase over-expression in mice genetically engineered to be cancer resistant by means of enhanced expression of the p53, p16 and p19ARF tumor suppressors, which showed a 40% increase of the median lifespan (Table 1). In this mouse model, the percentage of mice reaching extremely old ages was significantly increased, demonstrating an anti-aging activity of Tert in the context of a mammalian organism (Tomas-Loba et al., 2008). Concomitantly, these mice showed a reduction in aging-associated pathologies and exhibited an improved neuromuscular coordination, increased glucose tolerance and a better fitness of epithelial barriers. The improved fitness of these mice was accompanied at the molecular level by higher serum levels of IGF1 and a remarkable reduction in the burden of telomere-associated DNA damage (Tomas-Loba et al., 2008). These findings suggested that telomerase exerts its anti-aging activity by counteracting telomere attrition and by preserving the proliferative capacity of stem cells as telomere length influences the ability of epidermal stem cells to mobilize and regenerate tissues (Flores et al., 2005; Sarin et al., 2005). In addition, the fact that these mice showed longer telomeres and improved tissue fitness also at young ages suggests that telomere loss normally limits the proliferation of a fraction of cells, including the stem cells and, therefore, telomere maintenance is relevant not only for aging but also for the optimal fitness of young organisms. Although nontelomeric activities of TERT have been also demonstrated (Sarin et al., 2005; Choi et al., 2008; Park et al., 2009), the fact that TERT transgenic expression in a Terc-deficient background did not impact on survival clearly implicates telomere maintenance as the main mechanism underlying the TERT anti-aging effects (Tomas-Loba et al., 2008). Together, these findings raise the possibility of using telomerase activators for the treatment of age-related diseases.

Telomere shortening is envisioned as a potent tumor suppressor mechanism (Gonzalez-Suarez et al., 2000; Blasco & Hahn, 2003; Blasco, 2005; Collado et al., 2007; Cosme-Blanco et al., 2007, 2008; Feldser & Greider, 2007). Mice that lack telomerase activity are resistant to cancer (Greenberg et al., 1999; Gonzalez-Suarez et al., 2000; Feldser & Greider, 2007), with the exception of p53-deficient, MSH2-deficient and TRF2-overexpressing genetic backgrounds (Table 1) (Chin et al., 1999; Artandi et al., 2000; Blanco et al., 2007; Martinez et al., 2009a). In the setting of a competent p53 pathway, critically short telomeres induce a DDR that results in cell cycle arrest, senescence or apoptosis (Deng et al., 2008). However, in the absence of p53, short telomeres contribute to high chromosomal instability, a hallmark of cancer cells. Therefore, p53 loss results in a permissive environment that favors proliferation and survival of DNA-damaged cells and the eventual progression to cancer. The role of the INK4A-RB pathway in mediating the telomere-associated DDR is less clear (Greenberg et al., 1999; Deng et al., 2008). Telomerase activation, in turn, is able to extend the lifespan of cells in culture by maintaining telomeres (Bodnar et al., 1998) and is found activated in the vast majority of human cancers (Shay & Wright, 2006). Indeed, constitutive telomerase expression in several independent Tert-transgenic mouse models increases the incidence of spontaneous cancer (Gonzalez-Suarez et al., 2001, 2002; Artandi et al., 2002; Canela et al., 2004; Cayuela et al., 2005). In the cancer cell scenario, it is conceivable that upon oncogenic stress, cells accelerate their proliferative rate, being telomere length a limiting factor to their cell division capacity. Indeed, telomeres are usually shorter in tumor cells compared to the healthy surrounding tissue. In the absence of the appropriate checkpoints, short telomeres potentiate occurrence of mutations and reactivation of telomerase would then provide to the mutated precancerous cell with the capacity to divide indefinitely, impinging on tumorogenesis. In support of this notion, sequence variants at the CPTM1L-TERT locus on chromosome5p.15.33 have been associated with many cancers, including lung, brain, urinary bladder, prostate, cervix, pancreas and acute myelogenous leukemia (McKay et al., 2008; Wang et al., 2008; Rafnar et al., 2009; Shete et al., 2009; Petersen et al., 2010). Together, these observations suggest that telomerase activation is common to many cancers and that its targeted inhibition could potentially be an effective anti-cancer therapy by triggering critically short telomeres and loss of cell viability in the tumor.

Mouse models to understand the role of shelterin proteins in cancer and aging

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

Recent studies using genetically modified mice for various shelterin components suggest a role for these proteins in cancer susceptibility and aging-related pathologies even in the presence of normal telomerase activity and normal telomere length. In line with this, expression of TRF1, TRF2, TIN2 and POT1 is altered in some human tumors (Blasco, 2005). In particular, a deregulated expression of TRF1, RAP1 and TPP1 has been recently described for patients with chronic lymphocytic leukemia (Poncet et al., 2008). Similarly, mutations in TIN2, TRF2 and TRF1 have been linked to some cases of Dyskeratosis congenita and aplastic anemia (Savage et al., 2006, 2008; Tsangaris et al., 2008; Walne et al., 2008).

In marked contrast to telomerase-deficient mice (Terc and Tert knock out mouse models) that survive to adulthood, complete abrogation of at least TRF1, TRF2, POT1a, TPP1 and TIN2 results in early embryonic lethality (Karlseder et al., 2003) (Celli & de Lange, 2005) (Lazzerini Denchi et al., 2006; Hockemeyer et al., 2006; Wu et al., 2006; Kibe et al., 2010; Chiang et al., 2004), while abrogation of RAP1 does not affect mouse viability (Sfeir et al., 2010; Martinez et al., 2010). Owing to this fact, the role of shelterin components in telomere biology and disease in the context of the mammalian organism has remained unexplored until very recently. The recent availability of several shelterin transgenic mouse models as well as the generation of tissue-specific conditional mouse models has allowed to study the role of shelterin proteins in cancer and aging. In this review, we will focus on the shelterin mouse models and will discuss the implications of shelterin components in cancer and aging.

TRF1

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

Cell-based in vitro studies using over-expression of TRF1 mutant alleles suggested a role for TRF1 as a negative regulator of telomere length (van Steensel & de Lange, 1997; Smogorzewska et al., 2000; Ancelin et al., 2002). Post-transcriptional modification of TRF1 by tankyrases 1 and 2, which poly-ADP-ribosylate TRF1, can regulate its binding to telomeres, thereby influencing telomere length and sister telomere cohesion (Smith et al., 1998; Cook et al., 2002; Hsiao et al., 2006; Donigian & de Lange, 2007; Hsiao & Smith, 2008). More recently, TRF1 over-expression in the context of mouse epidermis (K5-TRF1 mice) was shown to lead to telomere shortening in vivo (Table 1) (Munoz et al., 2009). TRF1-induced telomere shortening was rescued in the absence of the XPF nuclease, suggesting that TRF1 increased expression results in augmented XPF nucleolytic activity at chromosome ends (Munoz et al., 2009). A similar phenotype, albeit more severe, was also described for targeted TRF2 over-expression to mouse epithelia (Munoz et al., 2005) (Fig. 3). These observations suggest that TRF1 and TRF2 act within the same pathway of telomere length control in mammals, whereby resulting in XPF-dependent telomere shortening when over-expressed. Over-expressed TRF1 was also shown to co-localize with the spindle assembly checkpoints proteins BubR1 and Mad2 and to result in aberrant mitosis (Munoz et al., 2009).

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Figure 3.  Over-expression of either TRF2 or TRF1 in mouse epithelia leads to Xeroderma pigmentosum-like syndrome in K5-TRF2 and in K5-TRF1 transgenic mice: a model. A structure-specific nuclease involved in the nucleotide excision repair pathway (NER), XPF (green circle) localizes to telomere via interactions with TRF2 (Zhu et al., 2003) and TRF1 (no formal demonstration to date). (A) In wild-type mice, proper homeostasis of shelterin and of the NER components allows telomeric protection and the repair of UV-induced lesions at nontelomeric DNA. (B) Over-expression of TRF1 and TRF2 in mouse epithelia in K5-TRF1 and K5-TRF2 mice leads to aberrant sequestration of XPF at telomeric DNA thereby causing telomere shortening and XPF depletion from nontelomeric DNA causing an enhanced sensitivity to UV damage. In these mouse models, a clear predisposition for skin cancer and premature aging phenotypes are observed.

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Complete TRF1 deletion in mice produces very early embryonic lethality at the blastocyst stage; however, no defects in telomere length or telomere capping were detected at this stage (Table 1) (Karlseder et al., 2003). More recently, by using conditional TRF1 alleles, we and others have shown that TRF1-deleted mouse embryonic fibroblasts (MEFs) show rapid induction of senescence, which is concomitant with abundant telomeric γ-H2AX foci, phosphorylation of ATM and the ATM/ATR downstream checkpoint kinases CHK1 and CHK2 (Martinez et al., 2009b; Sfeir et al., 2009). Abrogation of the p53 and RB pathways bypasses senescence but leads to chromosomal instability, including sister chromatid fusions, chromosome concatenation and occurrence of multitelomeric signals (MTS) (Martinez et al., 2009b). MTS are also elevated in ATR-deficient MEFs or upon treatment with aphidicolin, two conditions known to induce breakage at fragile sites, suggesting that TRF1-depleted telomeres are prone to breakage (Martinez et al., 2009b; Sfeir et al., 2009). In spite of elevated telomere fusions and increased telomere fragility, TRF1-deleted MEFs, however, show normal telomere length, suggesting that TRF1 is not essential for telomere length maintenance but has an important role in telomere protection (Martinez et al., 2009b). In an analogous manner, ES cells conditionally deleted for TRF1 showed normal telomere length but increased telomere fusions (Okamoto et al., 2008).

Importantly, generation of mice conditionally deleted for TRF1 in stratified epithelia has allowed ascertaining the impact of TRF1 abrogation on the context of the organism (Table 1) (Martinez et al., 2009b). These mice die perinatally and show severe skin hyperpigmentation and severe skin morphogenesis defects, including absence of mature hair follicles and sebaceous glands, which are concomitant with the induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. Concomitant with severe atrophies, all stratified epithelia in TRF1-deficient mice developed compensatory preneoplastic lesions (dysplasia and hyperkeratosis) as early as 1–6 days after birth. Importantly, p53 deficiency in p53−/−/TRF1Δ/ΔK5-Cre mice rescues hair follicle stem cell defects, skin hyperpigmentation, as well as mouse survival, indicating that proliferative defects associated with TRF1 abrogation are mediated by p53. Mice doubly deficient for TRF1 and p53 in stratified epithelia develop hyperproliferative epithelial abnormalities, such as oral leukoplakia and nail dystrophy, which are characteristic of human diseases produced by mutations in telomerase-related genes and presence of short telomeres, such as dyskeratosis congenita, aplastic anemia and idiopathic pulmonary fibrosis (Mitchell et al., 1999; Vulliamy et al., 2001; Yamaguchi et al., 2005; Armanios et al., 2007; Tsakiri et al., 2007). Moreover, long-lived TRF1/p53 double null mice spontaneously develop invasive and genomically unstable squamous cell carcinomas (Table 1) (Martinez et al., 2009b). These results suggest that TRF1 normally acts as a tumor suppressor in the context of the organism by preventing telomere-induced genetic instability in proliferating cells.

The TRF1 conditional knockout mice represented the first mouse model in which dysfunction of a telomere-binding protein per se, without having to simultaneously abrogate telomerase expression, leads to the induction of severe telomere damage, in vivo up-regulation of the p53, p21 and p16 cell cycle inhibitors, and subsequent induction of cellular senescence and premature tissue degeneration in the absence of telomere shortening (Blasco et al., 1997; Hockemeyer et al., 2006, 2008; Wu et al., 2006; He et al., 2009). These mice are likely to represent a valuable tool for the study of the molecular mechanisms underlying human telomere–associated diseases.

TRF2

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

TRF2, an orthologue of TRF1 (Broccoli et al., 1997; Fairall et al., 2001; Chen et al., 2008), has also been suggested to act as a negative regulator of telomere length when over-expressed in mice and humans (Smogorzewska et al., 2000; Ancelin et al., 2002; Zhu et al., 2003; Wang et al., 2004; Munoz et al., 2005; Blanco et al., 2007). Similar to TRF1 transgenic mice, TRF2 over-expressing mice in stratified epithelia show accelerated XPF-dependent telomere shortening (Table 1) (Munoz et al., 2005). In turn, conditional deletion of TRF2 in MEFs leads to massive induction of end-to-end fusions mediated by the nonhomologous end-joining pathway, which lead to severe proliferative defects (Celli & de Lange, 2005). Similar to complete TRF1 abrogation, TRF2-deficient mice are embryonic lethal (Table 1). This lethality is not rescued by p53 deficiency (Celli & de Lange, 2005). In marked contrast to the essential role of TRF2 for mouse embryo development, TRF2 conditional deletion in the liver (Mx1TRF2 mice) did not impact on liver regeneration or mouse viability arguing that TRF2 is dispensable for hepatocyte regeneration (Table 1) (Lazzerini Denchi et al., 2006). In particular, TRF2 deletion in liver hepatocytes leads to telomere damage and increased telomere fusions; however, this is not accompanied by the loss of liver function. A possible explanation for this observation is that upon partial hepatectomy, liver regeneration occurred without cell division by endoreduplication and cell growth, thereby overcoming the chromosome segregation problems associated with telomere fusions (Lazzerini Denchi et al., 2006). The effects of TRF2 deletion on liver carcinogenesis or loss of liver function associated with aging were not addressed in this mouse model (Table 1).

Evidence of a putative role of TRF2 in cancer and aging is suggested by the phenotypes presented by K5-TRF2 transgenic mice. These mice show rapid telomere shortening and loss of the G-strand overhang, as well as increased chromosomal instability including chromosome ends with MTS. These mice show a severe skin phenotype, particularly in the light-exposed areas, consisting of severe skin hyperpigmentation and skin dryness, as well as increased incidence of spontaneous skin cancer, all of which are hallmarks of the human syndrome xeroderma pigmentosum. The short-telomere phenotype of K5-TRF2 mice occurs in the presence of normal telomerase activity, suggesting that it is not because of defects in the telomerase pathway. In support of this notion, telomerase over-expression did not rescue telomere shortening in K5-TRF2 mice, while telomerase deficiency did indeed accelerate telomere attrition (Munoz et al., 2005). As indicated for K5-TRF1 skin (Munoz et al., 2009), telomere shortening in K5-TRF2 epidermis was mediated by XPF, a structure-specific nuclease involved in the nucleotide excision repair pathway (NER) (Fig. 3). Mutations in NER components, including XPF are responsible for the xeroderma pigmentosum syndrome. Interestingly, XPF has been shown to physically interact with TRF2 (Zhu et al., 2003), suggesting a role for XPF at telomeres. Telomere shortening in K5-TRF2 skin was fully rescued by simultaneous XPF deficiency, supporting a functional interaction between NER and telomeres. Also in support of this, K5-TRF2 mice showed an increased susceptibility to spontaneously develop skin tumors and are prone to UV-induced carcinogenesis, which is analogous to the skin phenotypes of mice deficient in components of the NER pathway (Nakane et al., 1995; Sands et al., 1995; Munoz et al., 2005). In support of a role of increased TRF2 expression promoting skin tumorigenesis, TRF2 expression was frequently elevated in human skin carcinomas (Munoz et al., 2005). Interestingly, telomerase deficiency dramatically accelerates TRF2-induced epithelial carcinogenesis, coinciding with a higher chromosomal instability and higher burden of DNA damage. Telomeric recombination and alternative lengthening of telomeres (ALT)–associated PML bodies (APBs) were augmented by TRF2 over-expression (Blanco et al., 2007), suggesting a role for TRF2 in controlling telomere recombination. The data obtained from the study of the K5-TRF2 mouse model reveal that upregulation of TRF2 levels constitutes a potent oncogenic insult in vivo and suggest that telomerase inhibition may not be effective in the treatment of tumors with high levels of TRF2 expression.

Together, these observations lead us to propose a model in which over-expression of TRF2 leads to an aberrant sequestration of XPF at telomeric DNA thereby causing telomere shortening and a concomitant depletion of XPF from nontelomeric DNA leading to an enhanced sensitivity to UV damage (Fig. 3) (Munoz et al., 2005, 2006).

POT1

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

POT1 is proposed to regulate both telomere length and telomere capping (Loayza & De Lange, 2003; Ye et al., 2004; Denchi & de Lange, 2007; Xin et al., 2007). The mouse genome contains two POT1 orthologs, Pot1a and Pot1b. Double knockout cells for these genes show increased DNA damage foci at telomeres, endoreduplication and early induction of senescence (He et al., 2006; Hockemeyer et al., 2006; Wu et al., 2006). Single knockouts revealed that Pot1a and Pot1b have distinct roles. Hockemeyer and co-workers found that Pot1a was required to inhibit a DNA damage signal at telomere, while Pot1b had the ability to regulate the amount of single-stranded DNA at telomeres in a telomerase-independent manner (Hockemeyer et al., 2006). In particular, POT1 is involved in repressing the ATR pathway (Denchi & de Lange, 2007; Guo et al., 2007). Deletion of POT1a or POT1b did not result in telomere length changes (Hockemeyer et al., 2006). Others have however reported that Pot1a-deficient cells exhibit overall telomere lengthening and 3′ overhang elongation (Wu et al., 2006). Deletion of Pot1a resulted in early embryonic lethality, while Pot1b-deficient mice can survive to adulthood and only show diskeratosi congenital-like degenerative phenotypes (skin hyperpigmentation, bone marrow failure) when generated in a telomerase-haploinsufficient background (Table 1) (Hockemeyer et al., 2006, 2008; He et al., 2009).

TPP1

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

Conditional deletion of Tpp1 in MEFs has been recently demonstrated to result in a concomitant loss of POT1a and POT1b from telomeres, while the telomeric location of the other shelterin components was not affected (Kibe et al., 2010). Tpp1 deletion elicits a robust DDR at telomeres that is ATR mediated and that results in an excess of single-stranded telomeric DNA (Guo et al., 2007; Deng et al., 2009; Kibe et al., 2010). TPP1 deletion in MEFs also results in an increased incidence of chromosome-type fusions and in an endoreduplication phenotype (Kibe et al., 2010). TPP1-depleted cells seem to recapitulate the telomere dysfunction phenotypes observed in Pot1a/b double knockout cells, in agreement with the proposed role for TPP1 in recruiting POT1a and POT1b to chromosome ends (Kibe et al., 2010). In addition, Tpp1-deficient MEFs show increased chromosomes with MTS, a telomere aberration related to telomere fragility in the context of altered shelterin expression, including TRF2 over-expression and TRF1 loss of function models (Munoz et al., 2005; Blanco et al., 2007; Martinez et al., 2009b; Sfeir et al., 2009), thus pointing to a model where shelterin is important in the prevention of telomere fragility by facilitating DNA replication throughout telomeric repeats.

In addition to these roles in telomere protection and maintenance of telomere integrity, a role for TPP1 in telomerase recruitment and regulation is also emerging. In particular, based on its direct interaction with telomerase, TPP1 has been proposed to regulate telomerase activity at chromosome ends (Xin et al., 2007). In support of this, Tpp1 deletion results in decreased TERT binding to telomeres and accelerated telomere shortening both in MEFs and conditionally deleted Tpp1-deficient mice (Tejera et al., 2010). Moreover, Tpp1-null cells fail to elongate their telomeres when reprogrammed into pluripotent stem cells by using defined factors (Tejera et al., 2010), the so-called induced pluripotent stem (iPS) cells (Takahashi & Yamanaka, 2006), a process that is dependent on telomerase activity (Marion et al., 2009a,b), thus indicating that TPP1 is essential for telomere elongation in vivo. Together, these results suggest a telomere-capping model where TPP1 not only prevents the induction of a DDR at telomeres by preventing fusions and telomere breakage but is also required for telomere elongation by telomerase (Tejera et al., 2010).

The recent analysis of two independent mouse models with defective TPP1 expression has revealed the importance of this shelterin component in cancer and aging (Else, 2009; Tejera et al., 2010). On one hand, a spontaneous recessive mutation in a splice donor site of the Tpp1 gene renders a hypomorphic mouse model with decreased TPP1 levels, the acd (adrenocortical dysplasia) mouse that presents developmental defects in organs derived from the urogenital ridge (Table 1) (Keegan et al., 2005). The analysis of the mutant embryos revealed defects in caudal specification, limb patterning and axial skeleton formation (Keegan et al., 2005). Those mice that survived to adulthood showed severe developmental defects, including growth retardation, skin hyperpigmentation, sparse body hair, infertility, adrenocortical dysplasia and malformations of the skeletal and genitourinary system (Keegan et al., 2005). Interestingly, acd mice show a abnormal morphology of the adrenal cortex that mimics human adrenal hypoplasis congenita (Else, 2009). Additional loss of p53 rescues the acd phenotype in an organ-specific manner, including skin hyperpigmentation and adrenal morphology, but not germ cell atrophy (Else et al., 2009). Survival to weaning age was also significantly increased although tumor-free survival of the Acdacd/acdp53−/− as well as of the Acdacd/acdp53+/− mice was decreased when compared to Acd+/+ mice (Table 1). Similarly to TRF1-deficient mice acd mice show increased carcinomas when in a p53-null background (Else et al., 2009; Martinez et al., 2009b). Cells derived from acd mice show normal telomere length but increased telomere damage and telomere fusions, suggesting a role for TTP1 in telomere protection (Else et al., 2007; Hockemeyer et al., 2007).

However, the fact that complete Tpp1 abrogation results in embryonic lethality (Kibe et al., 2010) had impeded to date the study of the impact of complete Tpp1 abrogation on the context of the organism and thereby its effect on cancer and aging. Recently, our group has generated an analogous mouse to Tpp1-deficient mice by deleting TPP1 in the context of the mouse stratified epithelia (Table 1) (Tejera et al., 2010). In this context, Tpp1 deficiency leads to perinatal death, severe skin hyperpigmentation, defective hair follicle morphogenesis and widespread epithelia dysplasia. In particular, Tpp1 abrogation had a profound negative impact on hair follicle downgrowth, proliferation and differentiation, hindering the establishment of a mature hair bulge SC compartment. Importantly, these defects are rescued by p53 abrogation, supporting a key role of p53 in mediating proliferative arrest in response to persistent telomere damage in vivo (Chin et al., 1999; Feldser & Greider, 2007; Martinez et al., 2009b; Stout & Blasco, 2009). Conditional Tpp1 abrogation also leads to accelerated telomere shortening in the skin, further supporting a role for Tpp1 in telomere maintenance (Tejera et al., 2010). The epithelial pathologies present in Tpp1-deficient mice are more severe than those present in the acd mouse model in agreement with complete abrogation of the TPP1 protein and quite similar to those produced by TRF1 abrogation in the skin (Martinez et al., 2009b; Tejera et al., 2010). Furthermore, epithelial pathologies in Tpp1-deficient mice are reminiscent of epithelial pathologies in human diseases associated with mutations in telomerase-related genes and the presence of dysfunctional telomeres (Mitchell et al., 1999; Vulliamy et al., 2001; Armanios et al., 2007; Tsakiri et al., 2007), making of Tpp1-deleted mice a useful model to understand human disease.

Together, the information currently available on mouse models for TPP1 deficiency indicates that TPP1 has a dual role in telomere protection and telomere elongation, in this way preserving telomere function and preventing the early onset of degenerative pathologies in mice.

TIN2

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

TIN2 was identified as a TRF1-interacting protein (Kim et al., 1999, 2003). Over-expression of TIN2 inhibits telomere elongation in human cell lines, whereas expression of dominant negative N-terminal TIN2 deletion results in aberrant telomere elongation (Kim et al., 1999). It has been suggested that the binding of TIN2 to TRF1 induces changes in TRF1 conformation that favor a telomere structure, which is inaccessible to telomerase. The absence of TIN2 would therefore favor telomerase accessibility and telomere elongation (Kim et al., 1999). In mouse, Tin2-deficient ES show dramatic proliferative changes and die rapidly impeding further analysis of telomere length and function (Chiang et al., 2004). Deletion of Tin2 in mice results in early embryonic lethality (Table 1) (Chiang et al., 2004), mirroring that of TRF1- and TRF2-deficient mice (Karlseder et al., 2003; Celli & de Lange, 2005). Mutations in TRF1, TRF2 and TIN2 have been identified in patients with bone marrow failure syndromes, while mutations in POT1, RAP1 and TPP1 have not yet been reported (Carroll & Ly, 2009). Clearly, further analysis of the in vivo function of these proteins will require conditional or tissue-specific knockouts.

RAP1

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

scRap1 is the major binding activity at yeast telomeres where it controls telomere length and the establishment of subtelomeric silencing through recruitment of the Sir proteins (Kyrion et al., 1993; Hecht et al., 1995; Marcand et al., 1997; Tanny et al., 1999; Imai et al., 2000; Carmen et al., 2002). A similar role in subtelomeric silencing has recently been described for TbRap1 in Trypanosoma brucei (Yang et al., 2009). A homolog of scRap1 has been long reported for human cells (Li et al., 2000). Over-expression of human hRap1 was shown to lead to telomere elongation by unknown mechanisms (Li et al., 2000). More recently, a role of mammalian RAP1 in protecting telomeres from nonhomologous end-joining (NHEJ) activities has been reported both in vitro and in the context of severe telomere uncapping induced by TRF2 dysfunction (Bae & Baumann, 2007; Sarthy et al., 2009). Generation of two independent mouse models for Rap1 deficiency suggests that Rap1 is not necessary for mouse viability and for telomere capping but instead has a role in protection from telomere recombination and telomere fragility (Sfeir et al., 2010; Martinez et al., 2010). In addition, Martinez et al. (2010) found that mammalian Rap1 controls gene expression (including silencing of subtelomeric genes) by binding to extra-telomeric sites through the (TTAGGG)2 consensus sequence. Future studies are required to understand the role of Rap1 in cancer and aging.

Future perspectives

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

In recent years, mounting evidence suggests a potentially important role of shelterin components in cancer and aging. The several mouse models for shelterin proteins discussed here strongly support this notion. However, this may be just a first approximation to understanding the complexity of telomere capping structures. To date, essentially nothing is known on the regulation of the different shelterin components during different developmental stages and in pathological conditions. The fact that the expression of several shelterin components, TRF2, TRF1 and TIN2 has been found altered in human cancer raises the possibility of using these components as potential therapeutic targets for cancer. Nevertheless, it must be kept in mind that this altered expression could be a consequence of tumor growth rather than the cause. Our results showing increased spontaneous carcinogenesis in K5-TRF2 transgenic mice and in mice conditionally deleted for TRF1 and p53 in the skin, as well as the Acd mouse model constitute clear examples on how altered shelterin function triggers chromosomal instability, which upon loss of tumor suppressors such as p53 favors neoplastic transformation. It will be of great interest to understand the impact of oncogenic activation, such a K-RAS or H-RAS, on mice with altered shelterin function. Two scenarios could be envisioned. On the one hand, in a tumor suppressor–proficient background, the severe proliferative defects of cells lacking shelterin would mask the replicative stress imposed by the oncogenes. On the other hand, oncogene-induced replicative stress could fuel transformations by successive mitotic cycles of breakage-fusion-bridges thereby augmenting tumorigenesis.

As some shelterin components have been proposed to act either as negative regulators of telomere length (TRF1 and TRF2) or telomerase recruitment factors (POT1, TPP1), it will be of great interest to understand their impact on telomere maintenance during tumorigenesis. In this regard, TPP1 constitutes the best candidate to bring telomerase to chromosome ends, and therefore could be a good target for cancer therapy. Thus, Tpp1-deficient mice should mimic the tumor-resistant phenotype of telomerase-deficient mice, except in a context where p53 is abrogated.

Regulation of shelterin biology during aging is still unexplored. It is well known that telomere length decreases with age; if the telomere-bound shelterin levels were proportional to telomere length, it would then be expected that telomeric shelterin levels would also diminish during aging. Conversely, decreased shelterin amounts with age may be responsible of inducing a DDR and cell cycle inhibition. Indeed, as discussed previously, some cases of premature aging in human syndromes have been linked to shelterin mutations, such as in TIN2, TRF1 and TRF2. We have shown that deletion of TRF1 in mouse epidermis has no effect on telomere length but yet recapitulates pathologies, such as oral leukoplakia and nail dystrophy, which are characteristic of human diseases produced by mutations in telomerase-related genes and the presence of short telomeres. The above-mentioned data clearly emphasize that the length of telomeres matters as long as some of the shelterin components are bound to them.

Acknowledgments

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References

P.M. is a ‘Ramon y Cajal’ senior scientist. M. A. Blasco′s laboratory is funded by the Spanish Ministry of Innovation and Science, the European Union (genica, FP7), the European Research Council (ERC Advance Grants), the Spanish Association Against Cancer (AECC), and the Körber European Science Award to M.A. Blasco. We apologize to all authors whose work has not been cited owing to space limitations.

References

  1. Top of page
  2. Summary
  3. Telomeric DNA and its shield
  4. Lessons learned from telomerase-deficient and telomerase over-expressing mice
  5. Mouse models to understand the role of shelterin proteins in cancer and aging
  6. TRF1
  7. TRF2
  8. POT1
  9. TPP1
  10. TIN2
  11. RAP1
  12. Future perspectives
  13. Acknowledgments
  14. References