Identification of DAF-12 Target Genes by ChIP-chip Figure 4. DAF-12 regulates heterochronic and miRISC gene expression. (A) Binding sites and ChIP enrichment for DAF-12 relative to ATG within heterochronic and miRISC genes. (B) Promoter regions of corresponding genes were subcloned into pGL3-Luc and luciferase activity was measured in HEK cells upon stimulation for 16 hr with 0.5 mM (25R)-D7–DA or DMSO as a control. (C) mRNA levels from synchronized daf- 12(rh61rh411) or N2 worms were determined by RT-qPCR. Fold difference is shown relative to N2 mRNA levels (dotted line). (D) mRNA levels from synchronized daf-12(rh284) or N2 worms at L3 stage were determined by NanoString analysis for several heterochronic genes (white bars) or miRISC genes (grey bars). Fold difference is relative to N2 mRNA levels (dotted line). Bars denote standard error of mean (SEM). P-values were determined by Student’s t-test. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pgen.1002179.g004 test whether this occurs because daf-12(rh284) still retains some aspect of DAF-12 activity, or because the mis-regulation of these genes is tissue-specific, we created transgenic animals carrying alg- 2 and ain-1 promoter regions upstream of GFP in WT or daf-12(0) mutants. As seen in Figure 5A, daf-12(0) worms at L2 stage show impaired alg-2 expression most notably in the pharynx when compared to WT. Further, expression of ain-1 at L3 stage was also impaired in daf-12(0) worms (Figure 5B). Taken together, these results suggest that DAF-12 controls the expression of miRISC components during reproductive development. daf-12(0) worms are sensitized to genetic mutations Although many genes involved in development were found in our array, in daf-12(0) mutants development usually occurs normally [10]. Based on this observation, we hypothesized that DAF-12 could have a role in developmental robustness. To address this hypothesis, we performed perturbation experiments [50] using the rrf-3 mutant, which is hypersensitive to RNAi [51]. Both rrf-3 and rrf-3;daf-12 were exposed to miRISC gene RNAi, to see whether loss of daf-12 could unmask developmental defects. When rrf-3 worms were exposed to ain-1 RNAi, only 3–4% developed protruding (Pvl) or burst vulva (Bvl) phenotypes. In contrast, approximately 30% of rrf-3;daf-12 animals showed a penetrant vulva phenotype (Figure 6A). No vulva phenotype was observed for rrf-3 or rrf-3;daf-12 animals when exposed to control RNAi (not shown). Treatment of rrf-3 worms with ain-1, ain-2, alg-2 or nhl-2 RNAi produced an increase in body length (Figure 6B and data not shown). This result suggests that the known increased body length of daf-12(0) worms [13] could be due to the down-regulation of miRISC genes. However, when daf-12(0) worms were fed nhl-2 or ain-2 RNAi, a synergistic response was observed (Figure 6B). During development, most hypodermal seam cells undergo a single asymmetric cell division. One daughter cell fuses to the hypodermal syncytium, while the other remains undifferentiated as a seam cell. However, in L2 stage the seam cells V1–4, V6 and H1 undergo a symmetric cell division before the asymmetric division, increasing the number of seam cells from 10 to 16 per side at the L/A switch [52]. Some daf-12 class 1 alleles repeat L2 programs resulting in excessive hypodermal seam cells, but this phenotype is weaker in daf-12(0) animals [10]. As a result, we tested whether miRISC genes interacted with DAF-12 in the control of seam cell division. As shown in Figure 6C, while nhl-2 knock-down induced a modest increase in seam cell number, a Figure 5. DAF-12 regulates expression of miRISC genes. (A) Image of alg-2p::GFP in WT or daf-12(rh61rh411) strains at the L2 stage (left). Graph showing alg-2p::GFP quantification in both strains (right) (p,0.0001). (B) Image of ain-1p::GFP in WT or daf-12(rh61rh411) strains at the L3 stage (left). Graph showing ain-1p::GFP quantification in both strains (right) (p = 0.002). P-values were determined by unpaired t-test. doi:10.1371/journal.pgen.1002179.g005 PLoS Genetics | www.plosgenetics.org 8 July 2011 | Volume 7 | Issue 7 | e1002179