There were three additional categories—inflammatory response, cell cycle, and nucleic acid metabolism—in which genes from at least one but not all three assays were overrepresented. The most notable difference between the PBM2 search from the other assays was an enrichment of genes involved in developmental processes. This is consistent with the known role of HNF4α in early development,34 and could be explained

by the fact that the cells used in the ChIP-chip and RNAi assays are from adult stages, not embryonic stages. In general, the ChIP assay yielded more significant GO terms in all categories, which is most likely a reflection of the more specific nature BMS-777607 cost of this assay and the stringent cutoff values used. In order to more closely compare the three methods of identifying potential target genes, we cross-referenced the PBM2 search results with the HNF4α RNAi and ChIP-chip results. We identified 198 genes that

were positive in all three categories, i.e., bound by HNF4α in ChIP-chip, down-regulated by HNF4α in HepG2 RNAi, and containing one or more verified HNF4α-binding sites in the −2 kb to +1 kb region of the promoter (Fig. 7A). A similar analysis with the SVM2 search yielded 135 genes (Fig. 7B). Among these two categories, there were ∼260 nonredundant genes, PD0332991 order of which ∼240 were not in the original list of HNF4α target genes from the literature (Supporting Table 1A). Several of these genes are new targets within known categories of HNF4α targets Aldol condensation (e.g., homeostasis = solute carrier proteins, SLC genes; lipid metabolism = e.g., ABCC6, DGAT2, hydroxysteroid dehydrogenase

[HSDs] genes), or more recently identified targets of HNF4α (e.g., CREB3L3, NR1I2, NR1H4, DO1).35–38 There were also many genes that, like NINJ1, are in completely new categories of genes not typically associated with HNF4α (e.g., signal transduction, immune response, stress response, apoptosis, cancer related, and cell structure) (Fig. 7C), several of which are reminiscent of the new functional categories identified by GO (Fig. 6). In order to determine whether the ChIP signal overlapped with the PBM or SVM sites in these new targets, all three datasets were visualized using Integrated Genome Browser. Although not all ChIP signals aligned exactly with the PBM or SVM sites, a very large number did; a sampling of these are shown in Fig. 8. Identification of TF binding sites and target genes can be a laborious process. Recent genome-scale technologies such as expression profiling and genome-wide location analysis can greatly expand the repertoire of potential targets with relative ease, although the question remains as to which are direct targets that contain bona fide binding sites. PBMs allow for a high-throughput identification of DNA binding sequences that can then be integrated with the other techniques, and can also be used to predict potential new targets in additional tissues or developmental stages.