Identification of DAF-12 Target Genes by ChIP-chip Figure 1. Generation of DAF-12 transgenic worms. (A) Schematic of daf-12 transcript showing the TAP-tag and region recognized by the anti- DAF-12 antibody in the ligand binding domain (LBD). (B) Expression of the transgene in ALF4 and ALF9 lines was confirmed by Western blot using an anti-DAF-12 antibody. Actin was used as a loading control. The function of the TAP-tagged daf-12 transgene was confirmed by incubation of starved worms with 1% SDS. (C) TAP-tagged DAF-12 bound chromatin was precipitated from ALF9 and ALF4 with an anti-DAF-12 antibody or IgG sepharose. Regions of interest were quantified by qPCR. (D) Binding sites within din-1 and F35G12.2 were visualized in tiling arrays using DNA from three replicas (ALF4) and control (daf-12(0)) with the Integrated Genome Browser software (Affymetrix). Arrows indicate initiation codons. doi:10.1371/journal.pgen.1002179.g001 regulator in C. elegans, via chromatin immunoprecipitation followed by sequencing (ChIP-seq) [32]. Furthermore, approxi- mately 20% of previously identified DAF-12 targets by Shostak et al [33] were also found in our ChIP-chip analysis (not shown), including the lit-1 gene, which was extensively validated in their study. These DAF-12 binding sites tended to cluster in proximal upstream regions with a smaller number inside or downstream of PLoS Genetics | www.plosgenetics.org 3 July 2011 | Volume 7 | Issue 7 | e1002179