Author Summary In this study, we were interested in learning more about the roles of microRNAs—small segments of RNA that help turn off genes—during early development. By studying mouse embryonic stem cells, a unique cell type that can give rise to all adult tissues, we found that a number of genes related to cell survival appeared to be affected by global microRNA loss. Interestingly, these changes in gene expression did not lead to large increases in cell death during normal cell growth, but rather became apparent when cells were treated with agents that cause DNA damage, like the chemotherapeutic doxorubicin and gamma irradiation. Our results suggest that these micro- RNAs may provide robustness for mammalian develop- ment, ensuring proper development despite variations in blood flow and oxygen tension, known to cause DNA damage. Given that certain cancers share features of the embryonic state, including rapid proliferation and lack of differentiation, our results also suggest that the re- expression of these microRNAs in tumors may confer resistance to chemotherapeutic drugs. been linked to a number of functions in ES cells including maintenance of pluripotency and proliferation. For instance, miR-290-295 miRNAs have been shown to target Rbl2, which controls the expression of Dnmt3a and Dnmt3b [10,11], suggesting a role for this miRNA cluster in regulating de novo DNA methylation. In addition, miR-290-295 miRNAs have been found to accelerate cell proliferation by promoting the G1 to S phase transition through targets such as p21 and Lats2 [12]. However, additional roles for this cluster remain to be elucidated. Using a combination of target prediction data with microarrays of mESCs before (Dcr WT) and after (Dcr KO) miRNA loss, as well as before (295 WT) and after (295 KO) specific deletion of the mir-295 cluster (Medeiros et al., manuscript in preparation), we have identified novel targets of the mir-295 cluster in ES cells. Initial analysis suggested strong enrichment of targets involved in apoptosis, a function that to date has not been linked to ES-cell specific miRNAs. Through gain and loss of function studies, we show that miR-290-295 miRNAs indeed serve a protective function in preventing mESC apoptosis during exposure to genotoxic stress. This protective effect appears to be mediated in part by direct repression of two novel targets, Caspase 2 and Ei24. As activation of these targets is dependent on DNA damage, we propose that their regulation may be particularly relevant during physiological stress in embryonic development. In addition, given prior indications that these two genes act as tumor suppressors, misexpression of this cluster in the context of cancer may promote resistance to standard genotoxic therapeutics. Results Predicted targets of the mir-295 cluster are enriched in pathways regulating apoptosis In order to identify additional endogenous targets of mESC miRNAs, we performed expression profiling of mESCs following Cre recombinase-mediated Dcr deletion using a previously characterized floxed Dcr mESC line [13,14,15]. As Dcr deletion leads to slower proliferation [6,12], acute loss was examined in a polyclonal population, averaging over potential clonal variants and enriching for initial miRNA-mediated derepression rather than subsequent compensatory changes. Indeed, expression profiling from 3 biological replicates taken 5 days following deletion, a time A Novel Anti-Apoptotic Role for Mir-290-295 point by which cells were predominantly Dcr and a majority of miRNAs were lost (Figure 1A and 1B), showed better clustering than 3 chronic deletion cell lines, as indicated by higher Pearson correlation coefficients (Figure S1). To confirm that targets of the mir-295 cluster show a transcriptome-wide signature in this dataset, we calculated a cumulative density function (cdf) plot comparing expression differences for the set of all mir-295 cluster targets as determined by Targetscan 5.1 [16]. Relative to a control set of genes (control) matched for 3’ UTR length, dinucleotide composition, and expression level, the mir-295 cluster target set (targets) was more derepressed upon Dcr loss (Figure 1C). An even larger derepression was seen for conserved mir-295 cluster targets (conserved targets), suggesting further enrichment of genuine targets in this set (Figure 1C). This observation supports the utility of these expression data for target discovery. To better understand the global effects of miRNA loss in ESCs, we next performed Gene Ontology (GO) analysis on an initial candidate set. Enrichment in specific GO categories was tested for all genes that increased on Dcr loss (defined as $1.2 fold up- regulation). The top statistically significant categories included ‘‘Regulators of Apoptosis’’ and ‘‘Cell Cycle’’ (p = 2.1e28 and p = 5.6e25, respectively). We further refined our candidate list using available array data from the 295 KO line, which also showed cdf plot signature changes for mir-295 cluster targets (Figure 1C) (Medeiros et. al., manuscript in preparation). In all, 807 candidates were identified as Targetscan-predicted targets of the cluster that showed at least a 1.2 fold up-regulation in knockout populations from both datasets (Figure 2A, Table S3). Over 40% of upregulated transcripts were shared between the Dcr KO and 295 KO lines, consistent with the finding that the mir-295 cluster contributes around half of all miRNAs in ES cells. The fact that this overlap is not even greater may be due to direct and indirect effects of non-AAGUGC seeds, as there is significantly more overlap – in fact, closer to 70% – between the two data sets when considering only those genes that are Targetscan-predicted AAGUGC targets (p,0.001, Fischer’s exact test). Several candidate target genes were selected for further examination based on their degree of upregulation in Dcr KO and 295 KO cells, as well as their functional annotations. The tested targets span a range of biological functions and processes, from cell cycle regulators Lats2 and p21 to immunological signal transduction components Irf9 and Irak3. Their 3’ UTRs were cloned into luciferase constructs, and expression levels between Dcr WT and Dcr KO cells were evaluated (Figure 2B). All candidates tested displayed at least mild repression relative to a control construct lacking miRNA target sites, ranging from strong (,5-fold) to modest (,30%) down-regulation. The magnitude of repression for the previously identified miR-295 targets Lats2 and p21 was comparable to that observed previously [12]. Additional transfection studies confirmed that repression could be conferred specifically by miR-295 in a Dcr KO background (Figure S2). These in vitro results support the enrichment of our candidate list for true miR-295 targets. Caspase 2 and Ei24, key apoptotic mediators, are direct targets of the mir-295 cluster We chose to more closely examine one of the most strongly down-regulated reporter targets, Caspase 2 (Casp2), along with Ei24, as these targets could provide a novel link between ESC- specific microRNAs and cell survival. Casp2, an initiator of apoptosis in response to genotoxic stress [17], has four AAGUGC binding sites in its 3’ UTR. Quantitative RT-PCR analysis demonstrated an approximately 5-fold increase in Casp2 tran- script levels in Dcr KO cells, consistent with the degree of PLoS Genetics | www.plosgenetics.org 2 May 2011 | Volume 7 | Issue 5 | e1002054