genes (Figure 2A). The identified DAF-12 target genes are involved in diverse biological functions based on gene ontology associations, including development, metabolism, growth and gene expression (Figure 2B). Examples of these targets include heterochronic genes (Representation Factor = 6.9 p,1.64610210), all identified compo- nents of the miRISC complex (RF = 9.4 p,1.4661026), including regulatory factors such as nhl-2 and xrn-2 [34,35], genes required for autophagy (RF = 7.1 p,3.014e204) and a total of 51 ribosome subunits (rpl and rps) (RF = 1.7 p,2.268e204). Genes involved in development, ribosomal function, mitochondrial function, and aging were found to be particularly over-represented (Table S3) via analysis of the targets with the DAVID program [36]. To examine whether DAF-12 binding sites are gene regulatory regions, we compared DAF-12 binding sites in the miRISC genes alg-2, nhl-2 and ain-1, with RNA Pol II binding sites from L2 stage larva as a proof of principle [32]. As expected, DAF-12 binding is localized to upstream promoter regions and associated with the recruitment of RNA Pol II binding, suggesting that DAF-12 is associated with regions of active transcription (Figure 2C–2E). The peak within the alg-2 promoter contains the DR5 repeat, sequence 59-AGTGCAccgaaAGTGCG-39, a very high affinity DAF-12 binding site [33] comprised of two hexameric DAF-12 binding sites separated by 5 bp (Figure 2C). The nhl-2 promoter does not contain DR repeats but a total of 15 hexameric sequences were found (Figure 2D). The ain-1 promoter contains just a single hexameric sequence, but has multiple M-2 motifs (CACACA), a previously identified DAF-12 binding site (Figure 2E) [37]. When analyzed globally, approximately 30% of DAF-12 target genes are actively transcribed in the L2 stage (not shown), which may reflect target genes being active at stages other than L2 or transcriptional repression instead of activation of a subset of target genes. We then analyzed DAF-12 binding regions to find de novo motifs by MEME and DREME, which gives a minimal MEME- formatted motif [38]. Known DAF-12 binding sites, such as the hexameric sequences identified by Shostak et al [33] and the M-2 motif identified by Ao et al [37] were over-represented (Figure 2F). Together these results confirm that the binding sites found in our array contain known DAF-12 binding sites, which is consistent with them being bound in vivo by DAF-12. The consensus sequence GAGAGA (Figure S1) was also over- represented among binding regions. Interestingly, this GAGA element was also identified as being enriched in PHA-4 binding sites identified by ChIP-seq [32,37]. DAF-12 and PHA-4 have been shown to co-regulate genes involved in pharynx development and we found that this interaction may be more widespread as they share a significant number of target genes (Figure S2). DAF-12–dependent regulation of target genes To test whether binding sites in our array represent DAF-12 response elements, we transfected HEK cells with luciferase reporter constructs along with a myc-tagged DAF-12 expression plasmid [18]. This cell-based assay has the advantage of directly measuring agonist-dependent activation with DA, as opposed to functional assays in S. cerevisiae not involving agonist treatment [33] or worm transgenes, which could be regulated by transcription factors other than DAF-12. Among the targets we identified is the miRISC gene alg-2 whose 2 Kb promoter region is sufficient for agonist-dependent activation (Figure 3A). The alg-2 promoter contains a DR5 sequence that is required for activation (Figure 3A, box). Interestingly, both the WT promoter and the mutant alg-2 promoter that lacks the DR5 domain exhibit weak DAF-12- dependent activation in the absence of DA. This transcriptional activation, which does not require the DR5 domain, could be due to DAF-12 tethering. This activation mechanism was observed for Identification of DAF-12 Target Genes by ChIP-chip other nuclear receptors, such as ER or GR, where adaptor molecules, like SP1 or AP-1 can recruit the nuclear receptor to the DNA and induce transcriptional activation independent of the agonist [39]. Alternatively, over-expressed DAF-12 could bind to low affinity sites, such as incomplete M-2 motifs or GAGA motifs present in the promoter (Figure S1). Regardless, our findings demonstrate that these play a minor role compared to DAF-12 actions through the DR5 domain. Another putative target is the promoter region of the CEOP4404 operon, which contains the atg-7 and let-70 genes. As shown in Figure 3B, a 2 Kb promoter region for atg-7 and let- 70 is strongly regulated by DAF-12 in cells. To address if this activation is due to DAF-12 recruiting a co-activator, we introduced a point mutation (P687S) within the DAF-12 AF-2 domain, which for other nuclear receptors is involved in recruiting co-activators after ligand binding [40]. This mutation is present in the daf-12(rh284) allele and causes a weak Daf-c phenotype at 25uC, perhaps due to impaired DAF-12 transcriptional activity [10]. As seen in Figure 3B, DAF-12 (P687S) is unable to promote transcriptional activity, even though expression of both proteins is similar (Figure 3B, inset). We then analyzed DAF-12 target genes previously reported to be involved in dauer formation (Figure 3C). 2 Kb promoter regions of these genes were subcloned upstream of a luciferase gene reporter and transfected in HEK cells. As seen in Figure 3D, all promoter regions show agonist dependent activation, including the DAF-16 CBP/p300 co-activator CBP-1 [41]. These results suggest that DAF-12 could regulate dauer formation in part by regulation of daf-3, din-1 and daf-16 expression, but also by regulation of daf-16 activity through controlling CBP-1 expression. Furthermore, DAF-12 could also regulate daf-16 activity by controlling the expression of akt-1, akt-2 and pdk-1 [42] (Table S1). When considered together, these results demonstrate that the regions identified by ChIP-chip respond in vivo to DAF-12. DAF-12 regulates post-embryonic development Proper regulation of the heterochronic circuit is essential for normal development [43]. Mutation in these heterochronic genes can cause either a precocious (skipping stage-specific events) or retarded (reiterating stage-specific events in later stages) develop- mental phenotype. In addition, miRISC components, required for mRNA degradation or translational repression are also essential for proper development as miRNAs regulate the heterochronic circuit [20]. As shown in Figure 4A, multiple heterochronic genes and all known miRISC genes contain DAF-12 binding sites, suggesting that DAF- 12 can directly regulate their expression. Regulatory regions of multiple heterochronic genes, such as the F box gene dre-1 [24], the RBCC gene lin-41 [44], the RNA binding gene lin-28 [45] and the circadian clock like genes lin-42A and kin-20 (Figure S3) [46,47] were subcloned upstream of a luciferase reporter. As shown in Figure 4B, all promoter regions tested, except the RNA binding gene lin-28, were activated by DAF-12 in an agonist-dependent fashion. The same analysis was performed for members of the miRISC complex. As shown in Figure 4B, the decapping gene cgh-1 [48] was activated in a DAF- 12-dependent agonist-independent fashion, representing perhaps another example of nuclear receptor tethering (Figure 3A). Also, the argonaute interacting protein, ain-1, shows a modest but reproducible activation (Figure 4B). In addition, alg-2 seems to be a DAF-12 target (Figure 3A), as well as NHL-2, a protein that binds to ALG-1 and ALG-2 to modulate miRNA potency [35] (Figure 4B). We then examined the in vivo effects of daf-12 on heterochronic gene expression. Wild type (N2) and daf-12(0) worms were PLoS Genetics | www.plosgenetics.org 4 July 2011 | Volume 7 | Issue 7 | e1002179