Telomere shortening exposes functions for the mouse Werner and Bloom syndrome genes

Abstract

The Werner and Bloom syndromes are caused by loss-of-function mutations in WRN and BLM, respectively, which encode the RecQ family DNA helicases WRN and BLM, respectively. Persons with Werner syndrome displays premature aging of the skin, vasculature, reproductive system, and bone, and those with Bloom syndrome display more limited features of aging, including premature menopause; both syndromes involve genome instability and increased cancer. The proteins participate in recombinational repair of stalled replication forks or DNA breaks, but the precise functions of the proteins that prevent rapid aging are unknown. Accumulating evidence points to telomeres as targets of WRN and BLM, but the importance in vivo of the proteins in telomere biology has not been tested. We show that Wrn and Blm mutations each accentuate pathology in later-generation mice lacking the telomerase RNA template Terc, including acceleration of phenotypes characteristic of latest-generation Terc mutants. Furthermore, pathology not observed in Terc mutants but similar to that observed in Werner syndrome and Bloom syndrome, such as bone loss, was observed. The pathology was accompanied by enhanced telomere dysfunction, including end-to-end chromosome fusions and greater loss of telomere repeat DNA compared with Terc mutants. These findings indicate that telomere dysfunction may contribute to the pathogenesis of Werner syndrome and Bloom syndrome.

FIG. 1.

Reproductive defects in mice with combination of mutations in Wrn, Blm, and Terc. (A) Indices of fertility for the first four months of mating. The mean number of pups in each litter for the indicated genotypes and generation are graphed with standard errors. Also indicated are the numbers of mating pairs and mean numbers of pups and litters generated per mating pair. WT, wb, t, and wbt indicate wild-type, Wrn^−/− Blm^M3/M3, Terc^−/−, and Wrn^−/− Blm^M3/M3 Terc^−/− genotypes, respectively. Data from G4 and G5 wb mutants were not significantly different and were pooled. (B) Testes-to-body mass ratios (multiplied by 1,000) for mice of the indicated genotypes and generations. Data for G1 and G2 mice were not significantly different and were pooled. The number of mice examined is indicated (N). Withineach generation, mice were of the same mean age, which was 10, 7.5, 6, and 6 months for G1/G2, G3, G4, and G6, respectively. (C) Representative histologic sections of testes from G4 mice of the indicated genotypes. G4 wbt testes have smaller seminiferous tubule cross sections, absence of spermatogenesis in most tubules, and an apparent increase in Leydig cells surrounding the tubules. All data are for mice from the original crosses.

FIG. 2.

Body habitus, mass, and bone density of the mutant mice. (A) Representative 8-month-old G4 males of the indicated genotypes (original crosses) were photographed after anesthesia. Note small size, kyphosis, and rectal prolapse in the wbt mutant. Genotype abbreviations are the same as for Fig. 1. (B) Growth curves for G4 male mice from 3 weeks through 5 months of age. Weekly body weights are plotted versus age for G4 mice of the indicated genotypes (original crosses), and fitted logarithmic curves are shown. The numbers of mice of each genotype are indicated in parentheses. Note that although two wb mice were heavier than the wild-type controls, the mean masses of wb mice were not significantly different from that of wild-type mice. (C) Body weights at 80 days of age for G1 and G3 male mice of the indicated genotypes (new crosses). For each mouse, the weight at 80 days was averaged with those from 1 week earlier and 1 week later. N is the number of mice in each group, mean values and standard errors are shown, and * indicates P < 0.05 in comparison with each other G3 genotype. Genotype abbreviations are the same as for Fig. 1, and in addition w, b, wt, and bt indicate Wrn^−/−, Blm^M3/M3, Wrn^−/− Terc^−/− and Blm^M3/M3 Terc^−/− genotypes, respectively. (D) Vertebral bone mineral density from DEXA scans of 4.5-month-old G3 females of the indicated genotypes (five mice per genotype). Mean values and standard errors are shown, and * indicates P < 0.05 compared with t mice.

FIG. 3.

Small intestine pathology in mutant mice. (A) Representative histologic sections of G4 small intestines from 8-month-old mice of the indicated genotypes stained with hematoxylin and eosin (original crosses). Representative apoptotic nuclei are indicated by arrows. (B) Frequency of apoptotic cells in small intestine crypts (new crosses) assessed by examination of nuclear morphology in hematoxylin- and eosin-stained histologic sections (H&E) and by TUNEL staining of histologic sections. All genotypes are for G3 mice except for G1 wbt and G7 t mice, as indicated. Apoptotic nuclei were counted in at least 80 crypts for hematoxylin and eosin and 50 crypts for TUNEL analysis per mouse, and the mean number of apoptotic cells per crypt and standard errors are shown. For hematoxylin and eosin analyses, four female mice (4.5 months old) were analyzed per genotype except for three mice for G7 t, and for TUNEL analyses three mice were analyzed per genotype. P values for pairwise comparisons with all other genotypes are shown. There were no significant differences in the number of mitoses per crypt among the different genotypes (data not shown).

FIG. 4.

Wound healing and dermal fibroblast growth. (A) Wound healing in G3 females (five mice per genotype, new crosses). Two full-thickness 4-mm punch biopsy wounds were made for each mouse in skin overlying the scapulae, and wound areas were measured for the next 7 days. Mean values and standard errors are shown. Compared with all faster-healing genotypes, wt is different at P < 0.05 at day 6, and wbt is different at P < 0.01 at days 6 and 7. Similar results were obtained for mice from the original crosses (data not shown). (B) Culture growth of skin fibroblasts. Fibroblasts cultured from the ears of two G1 siblings per genotype (new crosses) were passaged serially, and cumulative doublings were measured. Mean values and standard errors are indicated.

FIG. 5.

Shortened life spans of G3 wbt and bt mice. Kaplan-Meier analysis of survival in G3 wild-type, wb, t, wt, bt, and wbt mice (new crosses) is shown. w and b single mutants had no shortening of life span for the 12 months examined, and they were omitted for clarity. Only natural deaths were counted in the analyses; data for mice harvested for analytical autopsy were censored in the analysis and are indicated as points on each line that are not associated with a drop in the fraction alive. The numbers of mice in each cohort are indicated in parentheses. By log-rank test, the G3 wbt life span was shorter than that of all other genotypes at P < 0.0001, and G3 bt life span was different from all other genotypes at P ≤ 0.002.

FIG. 6.

Cytogenetic and telomere length analyses. (A) Frequency of chromosome end-to-end fusions (p-to-p and q-to-q) in cultured splenocytes from G1 and G3 mice (new crosses) and G4 mice (original crosses) of the indicated genotypes. N is the number of metaphases analyzed. For original crosses, mice were 7 to 10 months old; for new crosses, mice were 4.5-month-old females, four to six mice per genotype. Means and standard errors are shown. (B) Telomere length of G1 wb, t, and wbt mice (left) and wild-type and G3 and G4 t and wbt mice (right), measured by flow fluorescence in situ hybridization of bone marrow cells (original crosses). The mean ages of the G1 wb, t, and wbt, wild-type, G3 t and wbt, and G4 t and wbt mice were 11, 12, 10, 10, 14, 14, 9, and 7 months, respectively. Means, standard errors, and P values are indicated. The older age of the G3 mice likely explains why their telomeres are not significantly longer than that of the G4 mice. Measurements of the group on the left were performed on a different day than those of the group on the right, and so the values are not directly comparable between the groups. (C) Representative splenocyte metaphase from a G3 wbt mutant stained for DNA with DAPI (blue) and telomeric repeat DNA with an indocarbocyanine-labeled PNA telomere repeat probe (red). The arrowhead indicates a representative signal-free end, and the arrow indicates a p-to-p chromosome end fusion that lacks telomeric DNA at the fusion point. (D) Quantitation of signal-free ends on splenocyte metaphase chromosomes from mice of the indicated genotypes (new crosses). Chromosomes were stained as in C. N is the number of metaphases analyzed, and mean values, standard errors, and the P value for wbt versus t are shown.