Author Summary In Caenorhabditis elegans many life history decisions, such as interrupting reproductive development to form a dauer and the rate of aging, are under the control of the DAF-12 nuclear receptor. Recent work has started to elucidate the endocrine regulation of DAF-12, but few target genes for this receptor have been identified. In this study, we use chromatin immunoprecipitation to identify in vivo target genes for DAF- 12. Among the targets are multiple genes involved in developmental progression, which are termed heterochronic genes, and subunits of the miRISC, which is required for the control of target genes by microRNA. In daf-12 worms, both heterochronic and miRISC genes are mis-regulated, but development is usually normal. This led us to examine whether DAF-12 normally acts to reinforce commitment to dauer or adult development and prevent disruption by environmental or genetic fluctuations. Consistent with this, we find that daf-12 mutants are hypersensitive to environ- mental fluctuations and mutations in individual target genes. These results suggest a novel role for daf-12 as a phenotypic capacitor that ensures commitment to reproductive or dauer development despite internal and external variations. Previously, Shostak et al identified DAF-12 binding sites in vitro. However, a small number of genes were described and the in vivo relevance of these target genes was not always clear. To identify novel genes regulated by DAF-12, we developed transgenic animals expressing an epitope-tagged daf-12-transgene. These worms were used to perform chromatin immunoprecipitation followed by hybridization of the precipitated DNA to whole genome tiling arrays. From the arrays, we identified 1175 genomic regions bound to DAF-12 and these regions are within 5 Kb of 3179 genes. Importantly, we detected binding sites in multiple heterochronic genes, miRISC (miRNA-induced silencing complex) genes, and a total of 40 miRNAs, suggesting that DAF-12 controls developmen- tal progression through the direct regulation of these targets. Biological systems are subject to mutations and environmental variation during development, but still are able to ensure a stereotyped developmental process. This is possible because organ- isms can buffer many perturbations and produce an invariant output. This capacity is called ‘robustness’ [26]. Since many heterochronic and miRISC genes found in our array were mis-regulated in a daf- 12(0) allele daf-12(rh61rh411), but did not result in abnormal development [10], we hypothesized that DAF-12 could have a role in developmental robustness. Consistent with this idea, we found that daf-12(0) worms can enhance multiple heterochronic phenotypes, indicating that daf-12 worms are sensitized to genetic mutations. Further, we found that daf-12(0) worms are sensitized to environ- mental variability and show stronger heterochronic phenotypes in variable instead of constant conditions. In addition, multiple DAF-12 target genes were repressed in dauer, suggesting that in diapause DAF-12 can ‘reset’ the heterochronic circuit to ensure a stereotyped developmental program. These results illustrate a role for DAF-12 in the commitment to reproductive versus dauer development and suggest a novel function for this nuclear receptor to ensure a stereotyped phenotype despite internal and external variability. Results Use of a daf-12 transgenic worm for chromatin immunoprecipitation To identify target genes for DAF-12, we developed transgenic animals expressing a TAP-tagged transgene in a daf-12(rh61rh411) Identification of DAF-12 Target Genes by ChIP-chip (daf-12(0)) background (Figure 1A). The tandem affinity purifica- tion, or TAP-tag, was previously used successfully to identify nicotinic receptor-associated proteins in C. elegans [27,28]. The TAP-tag was inserted in-frame in daf-12 exon 1 via homologous recombination [29] as insertion of a GFP-tag at this site produced a functional GFP:DAF-12 fusion protein [10]. By biolistic bombardment of ALF3 (daf-12(rh61rh411); unc-119(ed3)), we obtained two integrated lines, ALF4 and ALF9. In each of these strains, DAF-12 is produced solely from the transgene. During the generation of our transgenic lines, a new commercially available DAF-12 antibody was released and this antibody was able to detect the fusion protein (Figure 1B). However, likely due to low DAF-12 expression in N2, endogenous DAF-12 was not detected. This suggests that the daf-12 transgene is over-expressed relative to the endogenous levels. The transgene also rescues the daf-12(0) allele, as starved transgenic worms form SDS resistant dauers (Figure 1B), suggesting that both transgenic lines express a functional daf-12 transgene. We performed pilot ChIP-chip experiments with ALF9 using the TAP tag and precipitation with IgG sepharose. In two replicas, we identified 52 genes as potential DAF-12 targets (data not shown). To identify additional target genes, we then optimized the assay by comparing ALF9 to ALF4 using IgG sepharose that recognized the TAP tag or the anti-DAF-12 antibody. Regions of no binding (cyp-15) and high affinity binding (F35G12.2 or the DAF-12 co-repressor din-1) were quantified in both ALF9 and ALF4. Both the TAP and DAF-12 antibody precipitate the same regions in two separate transgenic strains (Figure 1C). Importantly, the strongest binding to F35G12.2 and din-1 was obtained using ALF4 and the DAF-12 specific antibody. Based on these findings, this combination was used to perform the ChIP-chip studies. We did not use the DAF-12 antibody for ChIP-chip studies with N2 worms, as these seemed less likely to be successful due to the very low level of endogenous DAF-12 expression. To identify binding regions, arrays from three independent worm collections were analyzed and compared with daf-12(0). We chose L2 and adult stages because daf-12 is known to perform important and separate functions at these stages. Its activity at L2 is important for the dauer decision and developmental progres- sion, whereas in the adult it is known to be required for influencing longevity [30]. Chromatin from ALF4 was collected at middle L2 and adult stages and mixed together for further analysis. As shown in Figure 1D, DAF-12 binding to din-1 and F35G12.2 is highly reproducible, as each of the replicas shows similar binding regions and intensity (ChIP enrichment ratio of 16 and 21 respectively). Global analysis of DAF-12 binding sites From the resulting tiling array data, peaks representing DAF-12 binding sites were identified using TAS software (Affymetrix) (see Materials and Methods for a complete description). The data reported in this publication have been deposited in the NCBI’s Gene Expression Omnibus accession number GSE28350 (http:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE28350) [31]. To be considered a legitimate DAF-12 target, we required the peak to be present in all three replicates and not in a daf-12(0) negative control. This resulted in 1175 genomic regions bound to DAF-12. 1155 were within 5 Kb of 3179 genes and just 20 genomic regions (1.7%) were not associated with any known gene (Table S1). We also repeated the analysis looking for genes within 1 Kb and within 2 Kb of the binding sites (Table S1). Also, a table for binding regions when triplicates were positives and daf-12(0) was also positive is found in Table S2. The number of candidate target genes is lower but comparable in magnitude to the 6018 target genes recently identified for PHA-4, another developmental PLoS Genetics | www.plosgenetics.org 2 July 2011 | Volume 7 | Issue 7 | e1002179