e., native full-length receptor in a crude nuclear extract) (Fig. 1). We show that the PBM results are highly reproducible across different species (human and rat) and isoforms (α2 and α8) of HNF4α under a variety of conditions (Figs. 2 and 3). We identify new rules for DNA binding and develop an SVM model to predict additional sites (Figs. 3 and 4). We compare the PBM and this website SVM results to RNAi expression profiling

(Fig. 5) as well as to published ChIP-chip results in order to develop an integrated approach for the identification of human HNF4α target genes. We show that all three systems yield similar overrepresented categories of target genes (Fig. 6), supporting the notion that specific TF binding sites in promoter

regions are a major factor in driving gene expression. Using this integrated approach, we identified ∼240 new, direct targets of HNF4α, many of which are in new functional categories (Figs. 7 and 8). To our knowledge, this is the first such integration of extensive PBM, ChIP-chip, and Y 27632 expression profiling data for any TF. Finally, to facilitate future HNF4α target gene research, we have developed a publicly available web-based tool (HNF4 Motif Finder) based on our PBM results that can be used to search any DNA sequence for potential HNF4α-binding sites (http://nrmotif.ucr.edu). We define direct targets as genes that meet three criteria: contain a functional binding site in a regulatory region (PBM/SVM search), bind in vivo to the promoter (ChIP), and are down-regulated when HNF4α expression is knocked down (RNAi). Applying these criteria, we expand upon the classical roles of HNF4α by identifying additional target genes involved in metabolism (e.g., APOM, LIPC, LPIN1),

solute carrier transport (e.g., SLC7A2, SLC12A7, SLC25A20), protein transport and secretion (e.g., COPA, GOLGB1, GOLGA1), as well as transcription regulation (e.g., HDAC6, MED14, etc.). The integrated approach also identified new 上海皓元医药股份有限公司 HNF4α targets in pathways not previously associated with HNF4α, such as regulation of signal transduction (e.g., TAOK3, NGEF, PRKCZ, FNTB), and inflammation and immune response (e.g., IL32, BRE, LEAP2, IFITM2, BAT3). Perhaps the most intriguing new categories of HNF4α target genes are those involved in apoptosis, DNA repair, and cancer. HNF4α has long been considered a key factor in hepatocyte differentiation3, 4 but there are an increasing number of reports indicating that HNF4α may act as a tumor suppressor.39, 40 This view is supported by the new target genes identified here, such as NINJ1 (Fig. 5), which may play a role in regulating cellular senescence by inducing the expression of p21, a cell cycle inhibitor gene,41 and is consistent with our previous findings that the p21 gene (CDKN1A) itself is a direct target of HNF4α.