Telomeres are essential structures that cap the end of chromosomes, which is required for maintenance of chromosomal stability, cell viability and the capacity of cells to proliferate. A

complex of specific telomere-binding proteins (TRF1, TRF2, POT1, TIN2, TPP1 and RAP1), also known as the Shelterin complex, is essential for telomere capping by assisting the formation of

tertiary telomeric structures.1 Gene mutations in components of the Shelterin complex (_hTIN2_, _hPOT1_ and _hTPP1_) lead to bone marrow failure and cancer formation in human genetic

diseases including dyskeratosis congenita (DC), which is caused by Tin2 mutation in 20% of the cases.2, 3 All known _TIN2_ mutations are heterozygous, autosomal-dominant and patients

telomere-binding proteins may also contribute to carcinogenesis in somatic cells and tissues.5, 6 It was shown that Epstein–Barr virus-encoded LMP1 and Epstein–Barr virus-infection itself

induce the downregulation of TRF1, TRF2 and POT1 at the transcriptional and translational level resulting in complex chromosomal aberrations, alternative lengthening of telomeres and the

induction of Hodgkin's lymphoma.7, 8 The causal relation between gene dosage reductions of telomere binding protein and the development of cancer and tissue aging remains elusive. Mouse

knockout studies revealed that homozygous deletions of _Tin2_ or _Trf1_ lead to early embryonic lethality.9, 10 The conditional homozygous deletion of _Trf1_ was shown to provoke severe

defects in tissue maintenance11 and in combination with homozygous _p53_ deletion led to cancer formation in skin.12 However, these models did not address the question of whether moderate

reductions in the gene dose of telomere-binding proteins contribute to tissue aging and/or carcinogenesis. To address this question we followed aging cohorts of mice carrying heterozygous

deletion of _Trf1_ and/or _Tin2_ in comparison with wild-type mice.9, 10 Heterozygous _Trf1__+/−__Tin2__+/−_ knockout mice showed a 40–50% reduction in the mRNA expression level of Trf1 and

Tin2, but had no effect on the mRNA expression profile of other telomere-binding proteins (Figures 1a and b, Supplementary Figures 1a and d). Protein analysis of whole-spleen extracts

revealed an ~50% reduced Tin2 expression in _Tin2__+/−_ mice compared with _Tin2_+/+ mice (Figure 1c, Supplementary Figure 1e). Trusty antibodies for detection of endogenous Trf1 protein in

tissues are still lacking. To monitor the Trf1 protein amounts in _Trf1_ heterozygous mice, a _Trf1_ hemagglutinin (HA)-tag knockin mouse line was generated carrying the HA-tag at the

N-terminus of the endogenous _Trf1_ gene locus. Opposed to the homozygous _Trf1_ knockout mouse, homozygous HA-_Trf1_ knockin mice (_Trf1_ki/ki) are viable, do not exhibit an overt

organismal phenotype and show normal telomere structure indicating that the HA-tag did not interfere with Trf1 function. Trf1 protein from the knockin mice was quantitatively

immunoprecipitated with an anti-HA antibody using equally concentrated lysates. Heterozygous _Trf1_ deletion led to a reduction in Trf1 protein amounts, whereas the heterozygous deletion of

_Tin2_ had no significant impact on Trf1 protein levels (Figure 1d; Supplementary Figure 1f). As Trf1 binds directly to Tin2, the heterozygous _Trf1_ knockout mice (_Trf1__ki/−_ _Tin2_+/+)

also exhibited a reduction in the amount of Trf1-bound Tin2 compared with _Trf1__ki/ki_ _Tin2_+/+ mice (Figure 1d; Supplementary Figure 1f). Immuno-fluorescence _in situ_ hybridization

staining of telomeres and telomere-binding proteins revealed that heterozygous deletion of _Trf1_ reduces the co-localization of Trf1 with telomeric DNA in _Trf1_+/− mice and in

_Trf1_+/−_Tin2_+/− mice compared with _Trf1_+/+ control and _Tin2_+/− mice (Figures 1e and f). Since Trf1 mediates binding of Tin2 at telomeres, heterozygous deletion of _Trf1_ also led to a

reduced binding of Tin2 protein at telomeres compared to wild-type controls (Figures 1e and g). Heterozygous deletion of _Tin2_ reduced Tin2 localization at telomeres compared to wild-type

mice but in agreement with the fact that Trf1 binds directly to telomeres _Tin2_ deletion did not affect Trf1 expression at telomeres (Figures 1e and f). The heterozygous gene deletions of

_Tin2_ and/or _Trf1_ did not affect the Rap1 localization at telomeres, which is known to bind directly to telomeres in mammalian cells or through its interaction with Trf2 (Supplementary

Figures 1g and h). Together, the data on protein expression showed that the heterozygous deletion of _Trf1_ and/or _Tin2_ lead to reduction of Trf1 and/or Tin2 expression and to reduced

localization of the proteins at telomeres in murine cells and tissues. Cohorts of single heterozygous knockout mice (_Tin2_+/−, _n_=21 and _Trf1_+/−, _n_=32), double heterozygous knockout

mice _Trf1_+/−_Tin2_+/− (_n_=33) and wild-type mice (_Tin2_+/+, _Trf1_+/+, _n_=26) were weekly monitored during aging and exhibited no overt premature aging phenotype (Figure 1h). Analysis

of the bone marrow of a cohort of mice at the age of 14–16 months did not reveal evidence for bone marrow failure. Specifically, the _Tin2_ and/or _Trf1_ gene status did not affect the

number of hematopoietic stem and progenitor cells in bone marrow (Figure 1i; Supplementary Figure 1i). Mice were humanly killed when showing >15% of weight loss during aging or other

apparent abnormalities (wounds and tumors). Knockout animals exhibited increased incidence of tumors infiltration of spleen, liver and lymph nodes. Kaplan–Meier analysis of tumor-free

survival revealed a significant increase in tumor formation in the three cohorts of heterozygous and double-heterozygous knockout mice compared with the wild-type cohort (Figure 1j;

Supplementary Figure 1j). Specifically, the analysis indicated that tumor formation in the heterozygous knockout cohorts was accelerated and occurred significantly earlier in life compared

with the wild-type cohort. Overall, 83.3% of the aged wild-type mice were free of tumors when humanly sacrificed due to aging characteristics. Histological analysis of macroscopic tumors

revealed that in the wild-type cohort 13.3% of the mice had malignant tumors and 3% carried hyperplastic tumor nodules (Figure 1k). In contrast, 68% (60–76%) of the mice from single or

double heterozygous knockout cohorts developed macroscopic tumors including malignant tumors in 54% of the mice (52–56%) and the percentage of hyperplasia was increased to an average of 14%

(6.7–20%, Figure 1k). Histological analysis of five representative malignant tumors of _Trf1_+/−_Tin2_+/− mice indicated that the majority of the malignant tumors were T- or B-cell lymphoma

(Supplementary Figure 1k). Aside from the increased overall frequency, there was no significant shift in the spectrum of tumors in heterozygous or double-heterozygous knockout mice compared

with the wild-type cohort. Studies on telomerase deficient mice indicated that telomere shortening leads to an increase in chromosomal instability and tumor initiation.13 To analyze whether

such mechanisms were involved in accelerating tumor formation in response to heterozygous deletion of telomere-binding proteins, we studied telomere stability and DNA damage at telomeres in

bone marrow derived metaphase spreads of 14–16-month-old mice. Telomere length analysis showed shortening of telomeres from double heterozygous _Trf1_+/−_Tin2_+/− mice compared with

wild-type mice (Figure 2a). In addition, telomeric fluorescence _in situ_ hybridization analysis revealed increases in (i) broken telomeres (Figures 2b and c) and (ii) multi-telomeric

signals (Figures 2b and d), both are markers of fragile telomeres and replication stress. Moreover, sister chromatid exchange rates at telomeres (another feature of fragile telomeres) were

increased in _Trf1_+/− and _Tin2_+/− as well as in double heterozygous mouse embryonic fibroblasts compared with wild-type mouse embryonic fibroblasts (Figures 2e and f). To analyze whether

telomere fragility would also lead to an accumulation of DNA damage, the number of 53BP1 DNA damage foci was analyzed in small intestine of 14–16-month-old mice. There was a significant

increase in the number of intestinal epithelial cells in basal crypts harboring 53BP1+ DNA damage foci in the heterozygous mutant cohorts compared with control mice (Figures 2g and h).

Immunohistochemical staining against phosphorylated H2AX (γH2AX—another marker for DNA breaks) confirmed these results (Figures 2i and j). Co-labeling of telomeres and γH2AX foci revealed a

significant increase in telomere-induced DNA damage foci (TIFs) in the heterozygous knockout cohorts compared with wild-type controls (Figures 2k and l). Together, the current study provides

the first experimental evidence that heterozygous gene deletion reduces the binding of Trf1 and Tin2 proteins at telomeres resulting in telomere fragility, DNA damage accumulation and

enhanced lymphoma formation in aging mice, but not in premature aging _per se_. These results indicate that a tight control of the expression level of telomere-binding proteins is important

to avoid tumor formation, but premature failure in organ homeostasis of patients carrying heterozygous _TIN2_ mutations likely involves gain of function or dominant negative effects of the

mutant alleles as drivers of organ failure. Recent findings on reduced expression of telomere-binding proteins, telomere uncapping and increases in sister chromatid exchange rates in human

lymphoma and in response to Epstein–Barr virus infection of human B-lymphocytes suggest that the results from this study are relevant for the development of human hematopoietic

malignancies.7, 14, 15 Alterations in the expression level of telomere-binding proteins could represent an alternative, telomere length independent route to telomere dysfunction, which in

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2010; 29: 503–515. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We gratefully acknowledge Titia de Lange (Rockefeller University, NY, USA) for kind donation of

antibodies and TRF1 deficient mice, Jeffrey Chiang and Richard Hodes for providing Tin2 knockout mice, as well as Leticia Quintanilla-Fend (University of Tübingen, Germany) for assistance

with the lymphoma classification. This work was supported by the DFG (Ru745-10, RU-745-12), the European Union (ERC-2012-AdG 323136), the BMBF (GerontoSys—SyStaR 315894), the state of

Thuringia, and intramural funds from the Leibniz association. AUTHOR CONTRIBUTIONS KLR and KH designed the research; KH, AI, FL and AB performed the research; KH, AI and FL analyzed the

data; KH and KLR wrote the paper. AUTHOR INFORMATION Author notes * A Illing Present address: 5Current address: Department of Internal Medicine, University Hospital of Ulm, Ulm, Germany., *

A Baisantry Present address: 6Current address: Department of Kidney, Liver and Metabolic Diseases, Children’s Hospital, Hannover Medical School, Hannover, Germany., AUTHORS AND AFFILIATIONS

* Cooperation Group of the Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI) Jena and Ulm University (UULM), Ulm, Germany K Hartmann, A Illing, A Baisantry & K L Rudolph

* Department of Pathology, University Hospital of Ulm, Ulm, Germany F Leithäuser * Institute of Pathology, University Hospital Tübingen, Tübingen, Germany L Quintanilla-Martinez * Faculty

of Medicine, Research Group on Molecular Aging, University Hospital Jena (UKJ), Friedrich-Schiller-University (FSU), Jena, Germany K L Rudolph Authors * K Hartmann View author publications

You can also search for this author inPubMed Google Scholar * A Illing View author publications You can also search for this author inPubMed Google Scholar * F Leithäuser View author

publications You can also search for this author inPubMed Google Scholar * A Baisantry View author publications You can also search for this author inPubMed Google Scholar * L

Quintanilla-Martinez View author publications You can also search for this author inPubMed Google Scholar * K L Rudolph View author publications You can also search for this author inPubMed 

Google Scholar CORRESPONDING AUTHOR Correspondence to K L Rudolph. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Supplementary

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A., Leithäuser, F. _et al._ Gene dosage reductions of _Trf1_ and/or _Tin2_ induce telomere DNA damage and lymphoma formation in aging mice. _Leukemia_ 30, 749–753 (2016).

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