Identification of DAF-12 Target Genes by ChIP-chip Figure 9. Multi-level control of genes involved in developmental decisions by DAF-12. The nuclear receptor directly regulates the heterochronic circuit, miRNA biogenesis and miRISC expression at the transcriptional level. Other regulators of miRNA activity, such as lin-28 are repressed by DAF-12. Together the miRNA and miRISC regulate the levels and translation of many heterochronic genes. DAF-12 also promotes dauer entry by regulating dauer loci. Finally, DAF-12 regulates its own expression and is also a target for miRNA. doi:10.1371/journal.pgen.1002179.g009 number of genes to create a network of mutually regulating genes. Such networks have been shown to be important for robustness [61]. Also, DAF-12 could secure robustness by positive regulation of dauer loci, such as daf-3, daf-16 or hbl-1 and by repression of genes that inhibit dauer formation, such as lin-42 [54,62]. In addition, the system can buffer perturbations because gene products, such as miRNAs or DIN-1, modulated by DAF-12 can also regulate DAF-12 activity (Figure 9) [19]. Materials and Methods Strains ALF3 strain (daf-12(rh61rh411), unc-119(ed3)) was generated by crossing DP38 (unc-119(ed3)) and AA86 (daf-12(rh61rh411)). The unc-119 phenotype was verified by behavior and daf-12 allele was verified by PCR. AA86, AA82 (daf-12(rh284)), AA34 (daf- 12(rh61)), JR667 (unc-119(e2498::Tc1)III; wIs51) and DR1568 (daf- 2(e1371)) were obtained from the CGC which is supported by NIH funding. MM5438 (elt-2::GFP, rol-6) was kindly provided by Dr Morris F. Maduro (UC Riverside). Generation of a TAP tagged daf-12 transgene The daf-12:TAP transgene was generated from the fosmid WRM0611cB06 (Source Bioscience, Nottingham, UK) by homologous recombination in the SW106 E. coli strain as previously described [29]. Briefly, in the first step the galK gene was inserted into the fosmid between amino acids 26 and 27 in exon 1 using a PCR product generated from pMOD4 galK-GT using oligos containing 50 bp regions of fosmid homology (Dataset S1). Then, the galK gene was replaced with the TAP tag using a PCR product generated from pBS1761 with the same oligos [63]. The unc-119 marker was then added to this fosmid via cre-loxP recombination with pLoxP unc-119 [29]. The EPI300 strain (Epicentre Biotechnologies) was then used to amplify the fosmid for purification with the FosmidMAX DNA purification kit (Epicentre). The modified fosmid was then sequenced to verify the tag sequence and location. Transgenic worms ALF4 and ALF9 strains were obtained by biolistic bombard- ment of ALF3 with the TAP-tagged daf-12 fosmid as previously described [64]. ALF62 and ALF63 strains were generated by biolistic bombardment of the ALF3 strain. ALF62 (unc-119 (ed3), bafIs62 (lin-42p::GFP)) was generated by integration of an extra chromosomal array by TMP/UV irradiation [65]. ALF63 (unc- 119 (ed3), bafIs63 (lin-42pmut::GFP)) was directly obtained from bombardment. ALF62 and ALF63 were then crossed with HT1593 (unc-119(ed3)) to generate daf-12(+) strains. ALF70 (unc- 119 (ed3), bafIs70 (alg-2p::GFP)) and ALF72 (unc-119 (ed3), bafIs70 (ain-1p::GFP)) were generated by biolistic bombardment of the DP38 strain (unc-119(ed3)). These were crossed with ALF3 to place the reporters in a daf-12(0) background (ALF73 and ALF74 respectively). Sample preparation and ChIP-chip Synchronized worms at mid-L2 stage and young adults were collected by floatation, rinsed with milliQ water (DEPC treated) and frozen at 280uC. Whole C. elegans were simultaneously broken up and fixed by brief sonication in PBS containing 1% formaldehyde. Total fixation time was 30 minutes at room temperature, after which the reaction was quenched by the addition of glycine to a final concentration of 0.125 M. Preparation of chromatin and ChIP was carried out essentially as described [66]. In brief, lysates were sonicated and the DNA sheared to an average length of 300– 500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipita- tion. Pellets were resuspended and the resulting DNA was quantified on a Nanodrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantification of the total chromatin yield. Equal amounts of L2 and adult chromatin (20 mg each) were mixed together and samples were pre-cleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using an antibody against DAF-12 (Santa Cruz, ce-267). After incubation at 4uC overnight, protein A agarose beads were PLoS Genetics | www.plosgenetics.org 13 July 2011 | Volume 7 | Issue 7 | e1002179