Identification of DAF-12 Target Genes by ChIP-chip Figure 3. DAF-12 activates target genes in cells. (A) HEK cells were transfected with either pCMV-myc-DAF-12 or pCMV-myc vector, along with pGL3-Luc alg-2 promoter and pRL-TK renilla to normalize for transfection efficiency. Cells were stimulated for 16 hr with 0.5 mM (25R)-D7–DA or DMSO as a control. Mutations within DR5 domain are indicated (box). (B) Luciferase activity was measured for pGL3-Luc let-70/atg-7 promoter as in (A), with either DAF-12 or DAF-12 (P687S) expression plasmids. (Inset) Western blot shows similar expression levels of WT and mutant DAF-12 proteins. (C) Binding sites and ChIP enrichment for DAF-12 relative to ATG within genes involved in transcriptional regulation and dauer formation. (D) Promoter regions of corresponding genes were subcloned into pGL3-Luc and luciferase activity was measured as in (A). 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, n.s. (difference is not statistically significant). doi:10.1371/journal.pgen.1002179.g003 collected at different developmental stages, and heterochronic gene expression was quantified by RT-qPCR. As seen in Figure 4C, daf-12(0) worms show significant differences in expression when compared to N2. Importantly, DAF-12 can either repress or activate the same gene depending on the developmental stage (i.e. lin-41 at L4 or adult stage). Interestingly, the circadian clock gene lin-42, which has been suggested to be important for the molting process [47], is normally repressed by DAF-12 at the adult stage. This could be necessary to prevent extra molting cycles once development is completed. As many heterochronic genes were mis-regulated in L3, we focused on this stage. We compared mRNA levels of different heterochronic and miRISC genes from N2 or daf-12(rh284), a DAF-12 mutant unable to promote transcriptional activity (Figure 3B), using NanoString analysis [49]. As shown in Figure 4D, although all observed differences were not statistically significant, in most cases DAF-12 is necessary for full expression of these targets. However, DAF-12 might be involved in repression of lin-28 as its expression is up-regulated in both daf-12 mutants at the L3 stage (Figure 4C and 4D). Consistent with this, lin-28 mRNA is also up-regulated in din-1(dh149) worms (data not shown). Our findings point to a role for DAF-12 in regulation of heterochronic and miRISC gene expression during post-embryonic develop- ment. Although all known miRISC genes contain DAF-12 binding sites, most of them showed a weak mis-regulation (Figure 4D). To PLoS Genetics | www.plosgenetics.org 6 July 2011 | Volume 7 | Issue 7 | e1002179