Brca1 is an important checkpoint and DNA damage repair gene that is required for maintaining genomic intergrity. We have previously described a mouse model of constitutive DNA damage in which exon 11 of the Brca1 gene has been deleted resulting in the absence of the full length Brca1 isoform. Similar to Brca1 null mice, the Brca1^{Δ11/ Δ11} mice in general die in utero, although this embryonic lethality happens slightly later in development in the Brca1^{Δ11/ Δ11} animals than in the null embryos (Hakem et al., 1997; Ludwig et al., 1997; Xu et al., 2001). Further analysis of Brca1^{Δ11/ Δ11} embryos has demonstrated that there is a significant increase in both spontaneous cell death and cellular senescence (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). Although the observed embryonic lethality of Brca1^{Δ11/ Δ11} mice can be rescued by deletion of one allele of p53, the resulting Brca1^{Δ11/ Δ11} p53^+/- mice exhibit a high rate of spontaneous tumor formation in multiple organs (Xu et al., 2001). Detailed molecular analysis of these malignancies has demonstrated that essentially all of the tumors that arise are accompanied by a loss of heterozygosity in the p53 locus (Brodie et al., 2001; Cao et al., 2006). In addition to this tumor prone phenotype, the Brca1^{Δ11/ Δ11} p53^+/- mice also exhibit many features that are consistent with accelerated aging (Cao et al., 2006; Cao et al., 2003).

Based on our previous observations (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001), we asked whether perturbation of various DDR or cell cycle regulatory components could rescue Brca1^{Δ11/ Δ11} mediated premature senescence. MEFs were prepared from crosses between Brca1^{+/ Δ11} mice and various other animals containing targeted deletions of genes within these identified pathways. As noted in Figure 1B, although Chk2 deletion can rescue the Brca1^{Δ11/ Δ11} embryonic lethality (Cao et al., 2006), Brca1^{Δ11/ Δ11}Chk2^-/- MEFs appear to prematurely senesce at the same elevated rate as Brca1^{Δ11/ Δ11} cells. In contrast, deletion of one allele of p53 resulted in a small reduction in the rate of Brca1^{Δ11/ Δ11} induced senescence, while deletion of both p53 alleles appeared to completely abrogate the observed Brca1^{Δ11/ Δ11} stimulated premature senescence. Such results were not unexpected as p53 deletion abrogates senescence induced by numerous stimuli (Riley et al., 2008; Rodier et al., 2007). Similar analysis with MEFs containing targeted deletions of ATM, Chk1, H2AX, p21, PTEN, Gadd45a, p19^ARF, and Parp1 revealed that none of these gene deletions could rescue premature senescence caused by reduced Brca1 activity (unpublished observations). In contrast, we observed that Brca1^{Δ11/ Δ11} MEFs lacking 53BP1, a DNA damage response and p53 binding protein (Adams and Carpenter, 2006), were seemingly resistant to the observed accelerated senescence (Figure 1B). Similarly, while Brca1^{Δ11/ Δ11} MEFs appeared to have increased levels of p53 and evidence for increased p53 activity (Figure 1C), these biochemical changes were not evident in Brca1^{Δ11/ Δ11}53BP1^-/- MEFs.

To assess whether 53BP1 could also rescue Brca1^{Δ11/ Δ11} mediated senescence in vivo, we took advantage of previous observations that developing Brca1^{Δ11/ Δ11} embryos manifest an intense senescence mediated growth arrest (Cao et al., 2006; Cao et al., 2003). Consistent with those previous reports, Brca1^{Δ11/ Δ11} embryos were smaller than wild type embryos and stained intensely positive for SA-βgal (Figure 1D). This premature embryonic senescence was noticeably absent in Brca1^{Δ11/ Δ11}53BP1^-/- embryos. Interestingly, Brca1^{Δ11/ Δ11} embryos exhibited evidence for constitutive 53BP1 activation (Figure 1E). This response appeared relatively specific as a similar activation was not evident in H2AX^-/- embryos, even though H2AX is also required for the maintenance of genomic stability (Celeste et al., 2002). Previous results have documented that 53BP1 recruitment to DNA requires specific alterations in histone lysine methylation (Botuyan et al., 2006; Huyen et al., 2004). Consistent with these observations, we observed increased staining for histone H4 dimethylated Lys 20 in Brca1^{Δ11/ Δ11} embryos but not in H2AX^-/- embryos (Figure 1E). Other histone modifications known to act as lower affinity binding sites for 53BP1 were also selectively observed in Brca1^{Δ11/ Δ11} embryos (Figure S1).

Given that 53BP1 deletion rescued senescence induced by reduced Brca1 activity, we next asked what role 53BP1 played in other pathways leading to cellular senescence. The growth of MEFs in 20% oxygen results in passage dependent accumulation of senescent cells that in rodent fibroblasts appears to be a stress-mediated, ‘culture shock’ like phenomena (Parrinello et al., 2003). As expected, WT MEFs had a passage-dependent increase in senescence and a corresponding increase in p53 levels and activity under these growth conditions (Figure S2). An identical biological and biochemical response was seen in 53BP1^-/- MEFs. In contrast, p53^-/- MEFs had a complete abrogation of this senescent response (Figure S2). A similar analysis employing other triggers of senescence including oxidative stress with exogenous hydrogen peroxide or gamma-irradiation demonstrated that WT and 53BP1^-/- MEFs responded similarly (Figure 1F). In contrast, in p53^-/- MEFs, the induction of senescence following these stresses was severely compromised or absent.

The above observations suggest that 53BP1 plays a specific role in Brca1^{Δ11/ Δ11} mediated senescence. To begin to try and understand the basis of the specificity, we sought to analyze and compare the cellular response to reduced Brca1 activity, to the response observed with other forms of DNA damage. In MEFs, both irradiation and Brca1 deficiency resulted in activation of 53BP1 and H2AX (Figure 1G). As previously described, in both irradiated cells and in cells treated with hydrogen peroxide (Figure S3), these DNA damage foci also appeared to recruit additional factors including RAD51 and MDC1(van Attikum and Gasser, 2009). Interestingly, the recruitment of both RAD51 and MDC1 was not evident in Brca1^{Δ11/ Δ11} MEFs (Figure 1H). This suggests that the DNA damage foci formed in the setting of reduced Brca1 activity are qualitatively different than what is observed with other DNA damaging stresses.

In addition to inducing cellular senescence, the Brca1^{Δ11/ Δ11} mutation can also trigger programmed cell death and this is particularly evident within the developing embryo (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). We therefore analyzed rates of apoptosis in WT, Brca1^{Δ11/ Δ11} or Brca1^{Δ11/ Δ11} 53BP1^-/- embryos. Similar to our observations regarding Brca1^{Δ11/ Δ11} mediated senescence, deletion of 53BP1 appeared to dramatically rescue Brca1^{Δ11/ Δ11} mediated cell death (Figure 2A and B).

The ability of 53BP1 deletion to rescue in vivo embryonic senescence and apoptosis stimulated by the lack of Brca1 activity suggested that 53BP1 deletion might rescue the overall embryonic lethality caused by decreased Brca1 activity (Cao et al., 2006; Cao et al., 2003; Xu et al., 2001). Indeed, numerous healthy offspring were obtained in the setting of Brca1^{Δ11/ Δ11} along with deletion of 53BP1, while we observed no viable offspring of Brca1^{Δ11/ Δ11} mice containing both copies of 53BP1 and only very rare survival of Brca1^{Δ11/ Δ11} 53BP1^+/- mice (Table 1). Again, 53BP1 appeared unique in this role, as we observed no viable Brca1^{Δ11/ Δ11}H2AX^-/- mice (Table S1).

The survival of Brca1^{Δ11/ Δ11} 53BP1^-/- mice allowed us to assess whether cells from these animals manifested a generalized alteration in their apoptotic threshold. As expected, gamma irradiation triggered a significant increase in apoptosis of WT thymocytes (Figure 2C). This response was unaltered in 53BP1^-/- thymocytes, both in terms of the degree of cell death, as well as the DDR signaling pathway triggered by irradiation (Figure 2D). Similarly, thymocytes obtained from Brca1^{Δ11/ Δ11} 53BP1^-/- mice had similar biological and biochemical responses to irradiation when compared to WT cells (Figure 2C and D). In contrast, numerous previous studies have demonstrated that thymocytes obtained from either p53^-/- or Chk2^-/- mice are significantly impaired in their ability to undergo apoptosis following irradiation (Clarke et al., 1993; Hirao et al., 2000; Lowe et al., 1993). A similar analysis in MEFs demonstrated that 53BP1^-/- cells had intact and in some cases even an augmented apoptotic response following exposure to hydrogen peroxide, doxorubicin or gamma-irradiation (Figure 2E).

One potential explanation for the ability of 53BP1 deletion to rescue mice expressing Brca1^{Δ11/ Δ11} is that 53BP1 somehow altered or reduced the level of DNA damage and genomic instability in Brca1^{Δ11/ Δ11} expressing cells and tissues. To exclude this possibility we analyzed the activation of the DDR pathway in Brca1^{Δ11/ Δ11} or Brca1^{Δ11/ Δ11}53BP1^-/- cells, making use of our previous observation that nuclear foci of γ-H2AX were evident in Brca1^{Δ11/ Δ11} MEFs (Figure 1G). Overall, the degree of H2AX nuclear foci appeared similar when comparing MEFs (Figure 3A and B) or embryonic tissues (Figure 3C-E) derived from either Brca1^{Δ11/ Δ11} or Brca1^{Δ11/ Δ11}53BP1^-/- mice. Similarly, metaphase spreads derived from Brca1^{Δ11/ Δ11}53BP1^-/- adult B cells exhibited a high degree of chromatid and chromosomal breaks (Figure 3F&G).

We next sought to analyze the overall biological consequences of 53BP1 deletion in the setting of the Brca1^{Δ11/ Δ11} expression. After weaning, Brca1^{Δ11/ Δ11} 53BP1^-/- as well as 53BP1^-/- mice appeared outwardly healthy (Figure 4A), although by three months of age, mice deficient in 53BP1 exhibited a very modest but significant weight reduction compared to WT mice (Figure S4). Similarly, at a year of age, Brca1^{Δ11/ Δ11}53BP1^-/- mice continued to weigh and to appear essentially indistinguishable from WT mice (Figure S5). In contrast, Brca1^{Δ11/ Δ11} animals rescued by haploinsufficiency of p53, weighed only 70% as much as WT mice at one month of age and approximately 50% of WT animals at 7 months of age (Cao et al., 2006; Cao et al., 2003).

In contrast to Brca1^{Δ11/ Δ11}53BP1^-/- mice, by seven months of age, Brca1^{Δ11/ Δ11}p53^+/- mice exhibited clear evidence of accelerated aging (Figure 4A). This included among other signs, the development of marked kyphosis, as well as changes in the animal’s coat and overall physical activity. We also observed increased senescence in Brca1^{Δ11/ Δ11}p53^+/- tissues when compared to WT or Brca1^{Δ11/ Δ11}53BP1^-/- tissues (Figure 4B). Similarly, assessment of the rates of spontaneous apoptosis in rapidly dividing organs such as the intestine revealed that Brca1^{Δ11/ Δ11} expression triggered significantly increased cell death in p53^+/- rescued mice, but not in Brca1^{Δ11/ Δ11}53BP1^-/- mice (Figure 4C). Consistent with this increase in cell death, Brca1^{Δ11/ Δ11}p53^+/- cells had a constitutive activation of p53 not observed in cells obtained from either 53BP1^-/- or Brca1^{Δ11/ Δ11}53BP1^-/- mice (Figure S6).

Given that accumulation of senescent cells and increased cell death are both thought to mediate the accelerated aging phenotypes seen in models of chronic DNA damage, we next sought to assess the rate of aging in Brca1^{Δ11/ Δ11} mice that were rescued by either 53BP1 deletion or by haploinsufficiency of p53. As noted above, the latter mice exhibited increased tissue senescence and apoptosis triggered by Brca1^{Δ11/ Δ11} expression, while in 53BP1^-/- rescued mice, these responses were largely absent. Analysis of skin thickness (Figure 4D) and bone density (Figure S7) revealed that the changes associated with accelerated aging present in Brca1^{Δ11/ Δ11}p53^+/- mice, were not evident in Brca1^{Δ11/ Δ11}53BP1^-/- animals. This point is reinforced by the analysis of overall lifespan. As opposed to Brca1^{Δ11/ Δ11}p53^+/- mice, whose maximal lifespan is roughly one year, nearly 80% of Brca1^{Δ11/ Δ11}53BP1^-/- were still alive at 20 months (Figure 4E).

There is a growing realization that the accumulation of senescent and apoptotic cells might contribute to organismal aging, as well as serving as a barrier to tumor formation (Bartkova et al., 2005; Bartkova et al., 2006; Braig et al., 2005; Chen et al., 2005; Collado et al., 2005; Di Micco et al., 2006; Gorgoulis et al., 2005; Michaloglou et al., 2005). One of the interesting observations from the current study is the extended lifespan and near wild type appearance of Brca1^{Δ11/ Δ11} 53BP1^-/- mice. The relative absence of various progeriod phenotypes (e.g. kyphosis, reduced skin thickness, osteoporosis, etc.), as well as the extended overall lifespan, suggests that blocking cellular senescence and cell death mitigates the accelerated aging phenotype seen in this model of DNA damage. Perhaps more surprising are the observations that Brca1^{Δ11/ Δ11}53BP1^-/- mice exhibit constitutive high levels of genomic instability, yet still do not manifest a marked increase in tumor formation over the first 20 months of life. In previous examples where the Brca1^{Δ11/ Δ11} mice have been rescued from embryonic lethality, there was an extraordinary high rate of subsequent malignancies. These high rates of tumor incidence include a near 100% incidence in Brca1^{Δ11/ Δ11} p53^-/- mice by 3 months of age (Bachelier et al., 2003; Brodie et al., 2001; Xu et al., 2001) and a greater than 70% incidence of breast cancer in female Brca1^{Δ11/ Δ11}Chk2-/- mice by 16 months of age (Cao et al., 2006). One potential explanation for the relatively low levels of tumor formation in the Brca1^{Δ11/ Δ11} 53BP1^-/- mice compared to previous results is that Brca1^{Δ11/ Δ11} 53BP1^-/- cells appear to maintain an intact senescent and apoptotic response to various other stresses. In contrast, following exposure to a wide range of DNA damaging stresses, mice deficient in either Chk2 or p53 are known to have a generalized and widespread impairment in their apoptotic and\or senescent response.

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