Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Engineered telomere degradation models dyskeratosis congenita Dirk Hockemeyer,1,4 Wilhelm Palm,1,5 Richard C. Wang,2 Suzana S. Couto,3 and Titia de Lange1,6 1Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, New York 10065, USA; 2Department of Dermatology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA; 3Pathology and Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4–5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can be modeled in the mouse. [Keywords: Bone marrow failure; dyskeratosis congenita; POT1; shelterin; telomere; telomerase] Supplemental material is available at http://www.genesdev.org. Received March 28, 2008; revised version accepted April 29, 2008. DC is a rare hereditary multisystem disorder (for review, al. 1997). Whereas telomerase is absent from most hu- see Dokal and Vulliamy 2005). In addition to fatal bone man somatic cells, its activity is detectable in certain marrow failure in the first or second decade, the charac- stem cell compartments, including the bone marrow. teristic symptoms of DC are nail dystrophy, reticulated Telomerase is inferred to extend the replicative life span hyperpigmentation of the skin, and leukoplakia of the of hematopoietic stem cells (Allsopp et al. 2003), most buccal mucosa. DC patients can present with various likely by diminishing the net rate of telomere shortening other features including testicular atrophy, pulmonary rather than fully counteracting telomere attrition (Vaziri fibrosis, liver cirrhosis, predisposition to cancer, osteo- et al. 1994). Reduced telomerase activity and exception- porosis, abnormal dentition, and mental retardation. DC ally short telomeres have been documented in peripheral has been proposed recently to be part of a spectrum of blood lymphocytes of DC patients with mutations in related syndromes with diverse clinical manifestations hTR or hTERT (Yamaguchi et al. 2003; Marrone et al. (Yaghmai et al. 2000; Yamaguchi et al. 2003; Yamaguchi 2004; Vulliamy et al. 2004; Armanios et al. 2005; Cerone et al. 2005; Armanios et al. 2007). DC-like syndromes et al. 2005; Alter et al. 2007; Westin et al. 2007). A telo- may therefore be more frequent than previously inferred. mere-based disease mechanism is consistent with the Autosomal forms of DC can be caused by mutations in generational anticipation observed for DC (Vulliamy et the genes encoding the RNA (hTR) or reverse transcrip- al. 2004; Armanios et al. 2005), since affected parents are tase (hTERT) components of telomerase (Vulliamy et al. expected to contribute shorter telomeres to the next gen- 2001; Armanios et al. 2005; Marrone et al. 2007). In eration. The dominant inheritance pattern of DC caused mammals, telomerase is required to counteract the at- by hTR and hTERT mutations can be explained from trition of telomeric DNA, which has been argued to re- haploinsufficiency of these telomerase components sult primarily from nucleolytic processing of the C-rich (Marrone et al. 2004; Vulliamy et al. 2004; Armanios et telomeric DNA strand (Lingner et al. 1995; Makarov et al. 2005). The more severe X-linked recessive form of DC is due to mutations in the dyskerin gene (DKC1) (Heiss et al. Present addresses: 4Whitehead Institute for Biomedical Research, 9 Cam- 1998), and an autosomal recessive form of DC has been bridge Center, Boston, MA 02142, USA; 5Max Planck Institute of Mo- lecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 shown recently to be due to mutations in NOP10 (Walne Dresden, Germany. et al. 2007). Dyskerin and NOP10 bind H/ACA RNAs, 6Corresponding author. which contribute to a wide variety of cellular processes, E-MAIL [email protected]; FAX (212) 327-7147. Article published online ahead of print. Article and publication date are including ribosome biogenesis, pre-mRNA splicing, and online at http://www.genesdev.org/cgi/doi/10.1101/gad.1679208. telomerase biogenesis (Meier 2006). Observations on GENES & DEVELOPMENT 22:1773–1785 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 1773 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Hockemeyer et al. mice with a lesion in the DKC1 locus have been inter- member of the POT1 family of proteins, which are preted as evidence that DC is due to a defect in ribosome single-stranded telomeric DNA-binding proteins within biogenesis rather than telomere dysfunction (Ruggero et shelterin (Hockemeyer et al. 2006), the complex that pro- al. 2003). On the other hand, dyskerin binds hTR, pro- tects telomeres from the DNA damage response (de motes telomerase biogenesis, and is associated with ac- Lange 2005; Palm and de Lange 2008). Among the two tive human telomerase (Mitchell et al. 1999; Mochizuki mouse POT1 proteins, POT1a is essential, probably be- et al. 2004; Cohen et al. 2007). Furthermore, diminished cause in its absence, telomeres induce an ATR-depen- telomerase activity and telomere length defects have dent DNA damage response (Hockemeyer et al. 2006; been demonstrated for DC cases due to mutations in Wu et al. 2006; Lazzerini Denchi and de Lange 2007). In dyskerin and NOP10 (Mitchell et al. 1999; Wong et al. contrast, mice lacking POT1b are viable but have telo- 2004; Wong and Collins 2006; Walne et al. 2007). meres with unusually long 3Ј overhangs, suggesting ex- An additional complication in the context of a telo- cessive C-strand degradation (Hockemeyer et al. 2006). mere-based etiology of DC is that telomerase has been We argued that if POT1b protects the 5Ј ends of telo- proposed to have functions independent of telomere meres from degradation, POT1b-deficient cells and mice maintenance, including in stem cell regulation (Zhu et might experience accelerated telomere shortening and al. 1999; Stewart et al. 2002; Sarin et al. 2005). So far, could thus provide an alternative approach to modulat- mouse models have not yielded a decisive answer to the ing telomere length in the mouse. Here we show that question whether DC is a disease of telomere dysfunc- loss of POT1b results in telomere degradation in mouse tion because mice lacking components of telomerase cells and use POT1b KO mice to study the phenotypic have failed to show the phenotypes typical of DC. Due to consequences of this type of telomere length modula- the large telomere reserve of Mus musculus musculus, tion. mTR and mTERT null mice have no discernable pheno- types in the first generations (Blasco et al. 1997; Nikaido et al. 1999; Liu et al. 2000). When the telomeres become Results shortened in the later generations, the telomerase KO POT1b protects telomeres from C-strand degradation mice show prominent premature aging phenotypes con- sistent with stem cell depletion in highly proliferative To test whether the extended overhangs in POT1b-defi- organs (Lee et al. 1998; Rudolph et al. 1999, 2000). The cient mouse cells are caused by excessive nucleolytic phenotypes include testicular and ovarian atrophy, alo- degradation of the C-rich telomeric strand, we deter- pecia, hairgraying, ulcerative skin lesions, and atrophic mined the effect of POT1b loss on telomere shortening. villi in the duodenum of older mice. Late generation As reported previously, POT1b was inactivated either telomerase KO mice are also less tolerant to exogenously through insertion of a STOP cassette before exon 3 induced stress, such as treatment with 5-FU, skin lac- (POT1bS/S) or through deletion of exon 3 (POT1b−/−) eration, and liver injury. Nonetheless, the hallmark of (Hockemeyer et al. 2006). Both genotypes represent ap- DC, progressive bone marrow failure, is not observed in parent null alleles of POT1b and the phenotypes of the late generation telomerase KO mice. Although the mice cells and mice were indistinguishable. The alleles used have slightly reduced levels of hematopoietic progeni- in this study are identified in each experiment. tors, they do not show histological signs of defects in the Telomere dynamics were studied in SV40-LT immor- bone marrow, spleen, or thymus; they are not immuno- talized mouse embryo fibroblasts (MEFs) carrying condi- deficient or anemic; and they have normal peripheral red tional alleles of POT1b (POT1bF/− or POT1bF/F). As ex- and white blood cell counts and blood chemistry. Late pected, Cre-mediated deletion of POT1b induced an in- generation telomerase knockout mice also lack the hy- crease in single-stranded telomeric DNA in telomerase- perpigmentation and nail dystrophy that are typical of proficient (mTR+/+) and telomerase-deficient (mTR−/−) DC, and their life span is only moderately reduced (18 cells (Fig. 1A; Supplemental Fig. 1A). In addition, POT1b mo vs. 24 mo). Even when the initial telomere length is deletion induced telomere shortening. Telomere short- curbed by using M. m. castaneus, telomerase deficiency ening was apparent from examination of the bulk telo- did not give rise to overt DC phenotypes (Hao et al. meres as well as the higher-molecular-weight (MW) spe- 2005).