contribution of the mir-295 cluster to cell survival. Genetic deletion offers the best insight into physiological function as it avoids overexpression artifacts of exogenous miRNAs or toxicity effects of miRNA inhibitors. This system also avoids confounding by alternative miRNA-independent roles for Dcr itself in cell survival, as have been recently reported in C. elegans [26]. We first re-examined the reporter constructs for Casp2 and Ei24 in the 295 KO ESC line relative to its wild-type counterpart. In this context, the Casp2 reporter was derepressed approximately half as strongly as it was in Dcr KO cells, suggesting that the miR-302 and miR- 467a families of miRNAs incompletely compensate for loss of the mir-295 cluster (Figure 5A). This partial derepression in the mir- 295 cluster deletion probably reflects the quantitative change in the total level of AAGUGC seed miRNAs, as exogenous miR-295 could further repress Casp2 protein levels (Figure 5B). We next exploited 295 KO ES cells to determine whether these cells had an increase in apoptosis upon exposure to DNA damaging agents. 295 WT and KO ES cells were irradiated and the level of cleaved Casp3 activity was measured 0 and 24 h after treatment (Figure 5C, Figure S3B). As expected, 295 KO cells were much more sensitive to irradiation than their WT counterparts (Figure 5C). Again, overexpression of two miRNAs in the cluster, miR-290-3p and miR-295, reduced the rate of apoptosis (Figure 5D, Figure S7A and S7B). In addition, knockdown of the validated targets Casp2 or Ei24, or the pathway component Bim, partially rescued cells from apoptosis caused by irradiation (Figure 5D, Figure S7A and S7B). We repeated these experiments with 100 nM doxorubicin as before, obtaining similar results (Figure 5E and 5F, Figure S7C and S7D). Therefore, deletion and restoration of mir-295 cluster miRNAs recapitulate the modula- tion of apoptosis rates seen in a Dcr context. Discussion Here, we provide the first demonstration that the mir-295 cluster can suppress apoptosis in mESCs following exposure to the genotoxic stressors ionizing irradiation and doxorubicin. Initially suggested by an informatic comparison of global expression data following Dicer loss, the link between ESC miRNAs and cell death may act in part through the novel targets Casp2 and Ei24. In the case of Casp2, this appears to occur through multiple seed match sites in the 3’ UTR leading to a roughly 5 fold reduction in expression, while for Ei24, targeting is achieved through just a single complementary site conferring approximately a 2 fold repression. Although the exact functions of these two mediators are still emerging, multiple lines of evidence suggest that they are important in cell survival. Initial studies of Casp2 knockout mice showed increased numbers of oocytes suggesting resistance to cell death, which was confirmed by their decreased sensitivity to doxorubicin [27]. Subsequent studies have extended this pro- survival phenotype of Casp2 loss to include a number of tissues and DNA damaging agents [28]. Ei24, which was originally identified in a screen for etoposide-induced transcripts, has been shown to promote cell death by binding and sequestering Bcl2 [19]. Interestingly, these genes as well as several previously identified miR-295 family targets are known to be directly or indirectly associated with p53. Indeed, Pathway Analysis of well-character- ized miR-295 targets brought up a single significant network (p = 10214), ‘‘Cell Death, Cell Cycle, Cellular Function and Maintenance,’’ which prominently featured p53 (Figure 6). Activation of Casp2 can occur through a protein complex in which it associates with the p53 target PIDD [28]. Ei24 itself is a A Novel Anti-Apoptotic Role for Mir-290-295 p53 transcriptional target, identified as one of 14 genes induced by adenoviral transfection of p53 into a p53-null colon cancer cell line [19]. We additionally confirmed two previously identified miR- 295 targets, p21 (also known as Cdkn1a) and Lats2 [29]. In the case of p21, direct activation by p53 promotes cell cycle arrest at the G1/S phase [30]. Lats2, while also induced by p53, exists in a positive feedback loop with p53 in which it binds and inhibits Mdm2, thereby activating p53 [31]. Thus, miR-295 family miRNAs target a number of p53 associated genes and in all cases antagonizing p53 activation, consistent with the protective effect we have identified here. Like p53, the mir-295 cluster affects both arms of cellular proliferation, namely cell death and cell cycle progression [12,29]. Unlike cell cycle progression, however, the anti-apoptotic role is likely to have the greatest developmental consequences following DNA damage, induced physiologically by oxidative stress or metabolites. Interestingly, even simply ex vivo cell passage may be sufficient to induce a low level of stress, as evidenced by the slightly higher apoptotic rate of Dicer cells under basal culture. In their pro-survival capacity, these miRNAs may confer robustness during embryonic development, as has been demonstrated in Drosophila. For instance, miR-7 has been shown to participate in a complex network of feedback loops to ensure proper photorecep- tor cell development despite temperature fluctuations in develop- ment [32]. Similarly, miR-263a/b appear to prevent patterning defects in bristle formation, again consistent with the notion that they promote the fidelity of developmental trajectories [32,33]. Early phenotypic data from mir-290-295 KO mice suggests an incompletely penetrant gestational phenotype (Medeiros et. al., manuscript in preparation), supporting the model that loss of this cluster is tolerated in certain developmental scenarios, perhaps including those with limited stressors during gestation. Beyond regulating development, the miRNAs described here may also have important consequences for cancer, as both Casp2 and Ei24 are considered tumor suppressors. In the case of Casp2, this has been best demonstrated in the Eu-myc lymphoma model, where loss of even a single copy of Casp2 can accelerate malignant transformation [34]. Similarly, Ei24 is found in a region that shows frequent loss-of-heterozygosity in solid tumors, and its loss has been associated with increased breast cancer invasiveness [19]. In addition, knockdown of Ei24 in mouse fibroblasts or human breast cancer cell lines leads to increased resistance against etoposide- induced apoptosis [19,35]. Consistent with these findings, the mir- 295 cluster itself has been speculated to be an ‘‘oncomir’’ cluster, as overexpression of its human homolog, the mir-371-373 cluster, has been found in various human tumors [36,37,38] and may promote malignant transformation [39]. Given our findings, we hypothesize that these miRNAs may have a survival promoting function with dual effects, helping cells navigate physiological stresses during development, and helping cancer cells maintain viability in the face of genotoxic chemotherapeutic agents. In conclusion, these data expand our understanding of ESC miRNA function, linking the ES cell specific miR-295 family to key players in cell death. Further, this analysis reveals a complex relationship between embryonic stem cell miRNA regulation and p53 activation. Materials and Methods ES cell culture Feeder-free Dicer1flox/flox and Dicer12/2 mouse embryonic stem cells (mESCs) were generated and maintained on gelatin as described previously [40]. mESCs cells containing a floxed and excised mir-295 cluster were generated in a similar manner and PLoS Genetics | www.plosgenetics.org 6 May 2011 | Volume 7 | Issue 5 | e1002054