Identification of DAF-12 Target Genes by ChIP-chip Figure 8. DAF-12 acts as a phenotypic capacitor in worms. (A) Images of WT or daf-12(0) worms carrying the SCM::GFP transgene that were grown at 20uC or cycles of 15uC–25uC (4 hours each) until adulthood. The number of hypodermal seam cells per side is indicated in each figure. (B) Graph showing quantification of no, weak or strong heterochronic phenotypes. Differences were analyzed using Fisher’s exact test with SAS 9.2 software. * p,0.05. doi:10.1371/journal.pgen.1002179.g008 Together these results suggest that we have successfully identified in vivo targets of DAF-12. DAF-12 controls development by a multi-level regulation of gene expression Our finding that multiple heterochronic genes contain DAF-12 binding sites, suggests that this receptor directly regulates the heterochronic circuit and is consistent with previous observations that the gain of function allele daf-12(rh61) shows heterochronic phenotypes [1,10]. DAF-12 also regulates expression of additional genes with heterochronic phenotypes, such as miRNAs (Figure S7) [18,19] and components of the miRISC complex [56]. Our results show that among miRISC genes, the nuclear receptor DAF-12 directly regulates at least the alg-2 and ain-1 promoters, suggesting that DAF-12 could regulate both miRNA expression and activity. Beyond effects on miRNA expression and activity, DAF-12 regulates other genes that may control miRNA maturation. Recent evidence in mammals points to a role for LIN-28 in inhibiting let-7 processing from pri and pre-let-7 [57]. We show here that the RNA-binding gene lin-28 is repressed by DAF-12 from the L2 to L4 stages. Assuming a conserved function in C. elegans, lin-28 repression by DAF-12 could be required for let-7 activity to promote execution of L3 programs. We also found the F box gene dre-1 to be regulated by DAF-12. While no target proteins have been found so far for this ubiquitin ligase, its down- regulation produces heterochronic defects [24], suggesting a key function for DRE-1 in development. Control of dre-1 by DAF-12 could result in effects on protein stability or degradation. DAF-12 can also regulate its own expression, as well as genes that modulate its activity, such as the co-repressor DIN-1. Our results suggest that DAF-12 regulates gene expression by mechanisms including direct regulation of target promoters, miRNA biogenesis, miRNA activity via miRISC expression and perhaps control of miRNA maturation via lin-28 (Figure 9). DAF-12 acts as a phenotypic capacitor Fluctuations in environmental conditions and genetic mutations during development can have an impact on the phenotypic outcome of a given organism. How biological systems can reduce the impact of these stochastic perturbations to ensure a uniform phenotype is termed canalization or robustness [58]. Genes who function in part to reduce the effect of environment or genetic variability are called phenotypic capacitors and they contribute to robustness [26]. During reproductive development, our data shows that DAF-12 is required for proper expression of multiple heterochronic genes. While loss of daf-12 rarely produces penetrant heterochronic phenotypes during development, the phenotypes of many heterochronic genes are augmented by loss of daf-12 (Figure S4). The lack of a strong phenotype and enhancement of other genotypes suggests that DAF-12 may act to minimize the developmental consequence of mutations in multiple developmental genes. This action is consistent with a role as a phenotypic capacitor. When conditions become inappropriate for reproductive development, DAF-12 binds to the co-repressor DIN-1 and together this complex represses the heterochronic circuit to enter diapause. Entry into dauer results in many heterochronic phenotypes being suppressed upon dauer recovery (Figure S6). The ability of dauer arrest to suppress the effects of heterochronic mutations is also consistent with a role for DAF-12 as a capacitor. Besides dauer formation, DAF-12 has been implicated in other phenotypic decisions, such as the development of parasitic larvae [59] or formation of a tooth-like structure in the nematode P. pacificus [60]. Phenotypic capacitors such as DAF-12 can play important roles in evolution by buffering new mutations that could otherwise disrupt development and have allow the expression of new phenotypes in adult life [26]. DAF-12 could ensure robustness during reproductive develop- ment because it may act as a ‘hub’, interacting with a large PLoS Genetics | www.plosgenetics.org 12 July 2011 | Volume 7 | Issue 7 | e1002179