Engineered telomere degradation models dyskeratosis congenita

Abstract

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.

Figure 1.

Accelerated telomere shortening in POT1b-deficient cells. (A) Telomere length dynamics and telomeric overhangs of SV40LT-immortalized mTR-proficient and -deficient MEFs before and after deletion of POT1b with Cre. (Top) Size-fractionated MboI-digested DNA hybridized in gel under native conditions with a radiolabeled [CCCTAA]₄-oligo detecting the 3′ overhang. (Bottom) The same gels after in situ denaturation of the DNA and rehybridization with the same probe. Numbers above the lanes reflect population doublings after introduction of pWZL-hygro-Cre (or the empty vector). MWs are indicated in kilobases on the left. (B) Telomere FISH analysis on metaphase chromosome spreads of the cells analyzed in A. PDs are given in C. DAPI-stained chromosomes are false colored the in red, and the telomere hybridization signal is shown in green. Arrows point at chromosome fusions. (C) Quantification of chromosome fusions detected in metaphases as shown in B.

Figure 2.

Curtailed telomerase exacerbates POT1b KO phenotypes. (A) Genotypes of 281 offspring from intercrosses between 15 POT1b^+/−mTR^+/− breeding pairs. (B) Photograph showing the single POT1b^−/−mTR^−/− animal born and its littermates. (C) Scatter plot of the bodyweights of adult (>3 mo) female and male mice with the indicated genotypes. All animals were age-matched and derived from the intercross described in A. Error bars indicate the standard error of the mean. (D) Quantification of testis size of age-matched males of the indicated genotypes. Error bars indicate the standard error of the mean. (E) H&E stained testis sections at two magnifications. Mice as in A. (F) Incidence of testicular atrophy in mice of the indicated genotypes. Grading is based on H&E staining as shown in E. (G) TUNEL assay on H&E-stained sections from the small intestine of age-matched mice of the indicated genotypes. (H) Quantification of apoptotic cells in the small intestines based on TUNEL staining of age-matched mice with the indicated genotypes generated as in A. (POT1b^+/+mTR^+/+, POT1b^+/+mTR^+/−, POT1b^+/−mTR^+/− n = 4; POT1b^−/−mTR^+/+ and POT1b^−/−mTR^+/− n = 5). For each animal >60 crypts were analyzed. Error bars indicate the standard error of the mean.

Figure 3.

Cutanous phenotypes. (A) Photographs of POT1b^S/S and age-matched control animals showing hyperpigmentation on the snout, ears, tail, and paws of POT1b^S/S animals. (B) Hyperpigmentation of the paws of mice with the indicated genotypes and ages. (C) H&E and Fontana-Masson staining of tail sections of 8-mo-old mice with the indicated genotypes. Arrows indicate the increased deposition of melanosomes over the nuclei of the hyperpigmented mouse tail sections. Arrowheads indicate retention of the melanin in the stratum corneum. Enlarged images of the Fontana-Masson staining of tail sections of POT1b^−/− and wild-type mice are shown at right. (D) Photographs showing abnormal nails on one paw of an 11-mo-old G5 POT1^S/S mouse. (Top) The wild-type control is age-matched.

Figure 4.

Bone marrow failure and premature death in POT1b^−/−mTR^+/− mice. (A) Peripheral blood counts of age-matched mice with the indicated genotypes (POT1b^+/+mTR^+/+, n = 6, average age: 149 d; POT1b^+/+mTR^+/−, n = 5, average age: 132 d; POT1b^+/−mTR^+/−, n = 6, average age: 137 d; POT1b^−/−mTR^+/+, n = 9, average age: 138 d; POT1b^−/−mTR^+/−, n = 8, average age: 110 d). Error bars indicate the standard error of the mean. Differences between POT1b^−/−mTR^+/+ animals and POT1b^−/−mTR^+/− mice are significant (P < 0.05, upaired t-test) for all parameters except for the red blood cells counts. (B) H&E staining of bone marrow section of a 121-d-old POT1b^+/−mTR^+/− mouse (left panels) and an 83-d-old POT1b^−/−mTR^+/− mouse (right panels). (C) Immunohistochemical staining of B220 and Ter119 in spleen sections of mice with the indicated genotypes. (age: POT1b^+/+mTR^+/+ and POT1b^−/−mTR^+/+ animals 179 d, POT1^+/−mTR^+/− and POT1b^−/−mTR^+/− animals 121 d). (D) Kaplan-Meyer survival plot of mice derived from a heterozygous intercross of POT1b^+/−mTR^+/− mice (see Fig. 2A). POT1b^−/−mTR^+/− mice die prematurely compared with the controls (P < 0.0001, based on Log rank test).

Figure 5.

Telomere dysfunction in bone marrow of POT1b^−/−mTR^+/− mice. (A) Q-FISH analysis of the metaphases isolated from bone marrow of 3-mo-old littermates with the indicated genotypes. The X axis shows telomere fluorescence hybridization intensity (TFU), the Y axis shows the frequency of chromosomes ends with the indicated TFUs. Average TFU values are indicated in each panel. (B) Telomere FISH analysis of metaphases isolated from the bone marrow of mice (3-mo-old littermates) with the indicated genotypes. DAPI stained chromosomes are false colored in red and the telomere hybridization signal is shown in green. (C) Quantification of chromosome abnormalities detected in metaphases as shown in B. Controls represent pooled data obtained from bone marrow samples of two POT1b^+/− mTR^+/−, two POT1b^+/−mTR^−/−, and two POT1b^−/− mTR^+/+ mice. tel⁺ and tel⁻ indicate fusions with and without telomeric FISH signals at the fusions sites, respectively.

Figure 6.

Telomere length manipulation in the mouse and comparison with DC Comparison of the symptoms found in DC patients with the phenotypes observed in the late generation of telomerase knockout mice and POT1b^−/− and POT1b^−/−mTR^+/− mice.