Here, the cloning of a highly conserved murine homolog of the WRN protein is described. Despite the high degree of sequence identity between these two proteins, the human and mouse WRN homologs do not show similar immunolocalization patterns (12
). Mice bearing a targeted mutation in the catalytic helicase domain of WRN
are viable and fertile, they do not show any histological signs of premature aging, and they are capable of surviving until at least 2 years of age. Splenocytes from these animals proliferate normally in response to a mitogenic stimulus; however, cells from these animals senesce prematurely in cell culture.
mutations accelerate mortality in p53−/−
animals. There are two general possible explanations for this synthetic interaction between the WRN
genes. First, homozygous mutations in WRN
may exacerbate the cancer-prone phenotype of p53−/−
animals. The genome instability reported in cases of WS could be the molecular basis of this interaction. WRN and p53 have recently been shown to interact physically, further suggesting that these proteins may cooperate to maintain genome stability (1
). A second possibility is that the homozygous WRN
mutant animals do have a slightly accelerated aging phenotype. This phenotype might be first evident in the p53−/−
background because of its short life span. In this view, the cancer phenotype itself would be under the control of the aging program of mice. Thus, speeding up this program would advance all of the regulated phenotypes, including cancer in a wild-type or p53−/−
cancer-prone strain. This model predicts that the WRN−/−
animals will also display a slightly shortened life span in the p53
wild-type background. Although some WRN−/−
animals are now over 2 years old, it is still too early to know whether their life span will be shortened compared to that of the wild type.
Lebel and Leder have described a WRN
KO bearing a helicase domain mutation which shows several phenotypes (10
). Mutant ES cells are highly sensitive to camptothecin and show an elevated mutation rate, and late-passage embryonic fibroblasts possess a shortened in vitro life span compared with that of wild-type cells. In addition, mutant animals are born at less than the expected frequency, suggesting that this mutation confers some prenatal lethality. By contrast, mutant embryonic fibroblasts described herein are not hypersensitive to camptothecin. The former difference may stem from biological differences between embryonic fibroblasts and ES cells. WRN−/−
embryonic fibroblasts generated in this work do possess a modestly shortened in vitro proliferative capacity, in accord with the results of Lebel and Leder; however, we find that WRN
mutant mice are born at the expected frequency. Several possible explanations exist for these discrepancies. Modifying loci in ES cells and/or mouse strains may alter the phenotypic consequences of WRN
mutations. The nature of the WRN
alleles generated represents another potential reason for these discrepant results. The allele described herein deletes an exon in the catalytic helicase domain and introduces a frameshift mutation, resulting in no detectable protein expression, as assayed by immunofluorescence (12
) and Western blotting using an anti-C-terminal antibody. As the nuclear localization signal of the human WRN protein lies at the distal C terminus of the protein, it seems likely that this mutation should represent a functional null. By contrast, the mutation described by Lebel and Leder results in the expression of an internally deleted fragment that still has the potential to localize to the nucleus, where it might exert unpredictable effects. Thus, the effects noted by Lebel and Leder might not represent those of a true null allele in the WRN
Several possible explanations exist as to why murine WRN
mutants do not recapitulate the full spectrum of effects seen in human WS patients. Mice may possess more than one WRN
homolog; disruption of the putative second WRN
gene or both genes in the same animal might be required to recapitulate the human phenotype. Several observations argue against this hypothesis. In this study, 22 clones, all derived from the same gene, were isolated via reduced-stringency hybridization of a splenic library. This same gene has been isolated by using degenerate reverse transcriptase PCR (7
). Hence, if there is a second WRN
gene in mice, it must be expressed at much lower levels and/or be significantly diverged in sequence from the one that has been described. The WRN
gene lies in a chromosomal region in the mouse which is syntenic to human chromosome 8p, the location of the human WRN
). Screening of Northern blots at reduced stringency does not reveal any transcripts which might correspond to a second WRN
gene (D. B. Lombard, unpublished data). Finally, antibodies derived against the WRN protein and antibodies against the human WRN protein only recognize the known WRN protein in the mouse (D. B. Lombard and R. Marciniak, unpublished results). Thus, it is unlikely, though still formally possible, that more than one WRN
gene exists in the mouse.
Another possible explanation for the failure to produce a strong WS-like phenotype in the mouse is simply divergence between mice and humans in WRN function and/or, more generally, in DNA repair functions. In humans, the WRN protein is concentrated in the nucleolus, whereas the murine WRN protein is spread diffusely throughout the nucleoplasm (12
). This suggests that some divergence in WRN function may have occurred between mice and humans. It is also possible that murine WRN is functionally redundant with another helicase, either a RecQ family member or perhaps a member of a different helicase family altogether. In addition, mice may show milder effects of a WRN
mutation simply as a result of their smaller size and shorter life span, perhaps not allowing enough time for the full spectrum of effects of WS to manifest themselves.
Another potential reason for the discrepancy between the behavior of WRN
mutants in mice and humans is that the nature of the WRN target may different. One such target of the WRN protein may be the telomeres. In primary human WS cells, telomeres shorten more rapidly than in wild-type cells, though WS cells ultimately senesce with longer telomeres than do wild-type cells (22
). One explanation for the latter observation is that telomeres may be more recombinogenic and unstable in WS cells than in normal cells; hence, there may be more variation in telomere length in WS cells than in wild-type cells. This may occasionally produce a single very short telomere in WS cells which overall retain long telomeres; this could lead to senescence in cells which, for the most part, still possess long telomeres. Data consistent with telomeric instability in WS have been obtained in studies of lymphoblastoid cells (24
). Recent studies in our laboratory suggest that introduction of telomerase into primary WS cells can rescue their premature senescence (B. Johnson, personal communication). Mice, unlike humans, express telomerase constitutively in multiple somatic tissues and possess very long telomeres (9
); thus, if telomeres are an important target of WRN, many of the effects of WS might not be evident in the mouse. One critical test of this model will be to cross WRN
mutant mice with mice lacking the telomerase RNA component to determine whether these double-mutant animals show any synthetic phenotypes. Such experiments are underway.
In summary, we have generated and characterized a murine mutant in the WRN locus. Further studies in both mice and in human cells are necessary to elucidate the role of WRN in normal cellular physiology and its possible role in aging.