used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65uC, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR (QPCR) reactions were carried out in triplicate on specific genomic regions using SYBR Green Super- mix (Bio-Rad), site-specific primer pairs, and an aliquot of ChIP DNA. The resulting signals were normalized for primer efficiency by carrying out QPCR for each primer pair using Input DNA. ChIP and Input DNAs were amplified by whole-genome amplification (WGA) using the GenomePlex WGA Kit (Sigma). The resulting amplified DNAs were purified, quantified, and tested by QPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions. Amplified DNAs were fragmented and labeled using the DNA Terminal Labeling Kit from Affymetrix, and then hybridized to Affymetrix GeneChip C. elegans Tiling 1.0R arrays at 45uC overnight. Arrays were washed and scanned, and the resulting CEL files were analyzed using Affymetrix TAS software that computed for each probe estimates of fold enrichment (in linear scale) over hybridization with input DNA. At the same time, TAS calculated for each probe a p-value by applying a Wilcoxon signed rank test. A threshold of 2.5 was selected, which corresponds to probe intensities approximately 2.56 stronger on the ChIP array than on the Input array. Additional TAS threshold parameters were MinRun = 180 bp, MaxGap = 300 bp. TAS analysis showed that the selected threshold of 2.5 corresponds approximately to a p-value of 0.01. In addition, the false discovery rate (FDR) was estimated as previously described [67]. At a threshold of 2.5, the FDRs for the 4 samples were estimated to be: AA86 = 3.17%, ALF4(1) = 0.25%, ALF4(2) = 0.34% and ALF4(3) = 2.33%. The TAS-generated BED files (containing the peak intervals) were analyzed using Genpathway (now Active Motif) proprietary software that provides comprehensive information on genomic annotation, peak metrics and sample comparisons for all peaks. Graphs of genomic regions with peak enrichment values were generated using the Affymetrix Integrated Genome Browser. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [31] and are accessible through GEO Series accession number GSE28350 (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc = GSE28350). Western blotting Protein was extracted from N2, ALF3, AA86 or transgenic worms by boiling in LDS loading buffer (Invitrogen). After sonication, samples were run on a 10% polyacrylamide gel and transferred to PVDF. Membranes were probed with 1:750 a-DAF- 12 (ce-267, Santa Cruz), and 1:3000 actin (sc-1616R, Santa Cruz) followed by IRDye 680 goat anti-rabbit (1:10000) (Li-Cor Biosciences). For detection of DAF-12 expressed in HEK cells, a-myc (9E10, Covance) was used at a dilution of 1:1000. Computational analysis Using the Affymetrix TAS software (Affymetrix), we identified all genes associated with probes that were positive in transgenic Chromatin IPs, but negative in the control and plotted the distance between the bound probe and the ATG proximal to each gene. We next downloaded available GO annotations for all genes associated with bound probes (WormMart, 04/09/2010) and classified them using the GO.db library (Version 2.2.0) in R (http://www.r-project.org/). To find de novo DAF-12 binding sites, we identified all sequence intervals associated with genes that are active in experimental samples, but not in the control. We Identification of DAF-12 Target Genes by ChIP-chip submitted 600 unique sequences of 100 nt each, centered on the strongest DAF-12 binding signals to MEME (http://meme.mbcr. net) after masking repeat sequences using RepeatMasker (http:// www.repeatmasker.org). As a negative control, we also likewise analyzed 600 unique sequences of 100 nt each from intervals with the weakest DAF-12 binding signals associated with genes that did not score positive in the control or experimental samples 1 and 2. RNA preparation, RT-qPCR, and NanoString analysis Eggs from N2 and mutant strains were obtained by hypochlorite treatment and hatched overnight in S-Basal with shaking. The arrested L1 larvae were put on NGA plates to resume development. Samples were collected by floatation at the indicated developmental stages, rinsed in milliQ water and snap-frozen. Dauer larvae were isolated by a 15% Ficoll 400, 0.1 M NaCl gradient. RNA extraction and qPCR was performed as previously described [68]. For Nanostring analysis, RNA samples from 6 worm collections were analyzed by Expression Analysis (Durham, NC). Plasmid DNAs The DAF-12 A1 cDNA was PCR amplified from C. elegans cDNA and subcloned as a SfiI-BglI fragment into pCMV myc (Clonetech). The DAF-12 rh284 mutant was made with the Quick- Change mutagenesis kit (Stratagene). For luciferase reporter genes, fragments between 2 Kb and 3.5 Kb upstream the ATG were PCR amplified and subcloned into pGL3 Luc Promoter (Promega). For GFP reporter strains, promoter regions were subcloned in pPD95.75 vector (A gift of Dr. Andy Fire). Oligos sequences are available in Figure S8. The unc-119 rescue fragment was introduced by recombination as previously described [69]. The lin-42::GFP promoter mutant was synthesized and subcloned into pPD95.75 vector (Celtek Bioscience). Cell culture and reporter assay HEK 293T cells were cultured and transfected (Fugene, Roche) in 24well plates, with DMEM and 10% charcoal FBS treated (Hyclone). Briefly, 225 ng of either pCMV myc DAF-12 A1 or pCMV myc vector were co-transfected with 12.5 ng of pRL-TK (Renilla luciferase) and 25 ng pGL3 Luc promoter with the promoter of interest (Promega). The following day, cells were stimulated with 0.5 mM (25R)-D7-DA [70] or DMSO for 16 hr. At this concentration, no difference in activity was observed between the (25R)- D7-DA and (25S)- D7-DA [71] (not shown). Luciferase was measured using Dual-Luciferase Reporter Assay System (Promega). Each experiment was repeated three times and fold activation is relative to the non stimulated control transfected with pCMV vector, unless indicated otherwise. Digital imaging Transgenic worms were mounted for digital photography using an Olympus BX51 upright microscope. All photos within a panel were taken on the same day with identical camera settings to allow direct comparison. Quantification of the GFP reporters was performed with image J software and analyzed with an unpaired t- test. RNAi experiments L3 worms were transferred to RNAi plates at 16uC for approximately 72 hr. 10 adults were transferred to new RNAi plates and left for 3 hr to lay eggs before being removed. Analysis was performed with the adult F1 progeny. PLoS Genetics | www.plosgenetics.org 14 July 2011 | Volume 7 | Issue 7 | e1002179