Integrated Approach for the Identification of Human Hepatocyte Nuclear Factor 4α Target Genes Using Protein Binding Microarrays

Hepatocyte nuclear factor 4 alpha (HNF4 alpha), a member of the nuclear receptor superfamily, is essential for liver function and is linked to several diseases including diabetes, hemophilia, atherosclerosis, and hepatitis. Although many DNA response elements and target genes have been identified for HNF4 alpha, the complete repertoire of binding sites and target genes in the human genome is unknown. Here, we adapt protein binding microarrays (PBMs) to examine the DNA-binding characteristics of two HNF4 alpha species (rat and human) and isoforms (HNF4 alpha 2 and HNF4 alpha 8) in a high-throughput fashion. We identified similar to 1400 new binding sequences and used this dataset to successfully train a Support Vector Machine (SVM) model that predicts an additional similar to 10,000 unique HNF4 alpha-binding sequences; we also identify new rules for HNF4 alpha DNA binding. We performed expression profiling of an HNF4 alpha RNA interference knockdown in HepG2 cells and compared the results to a search of the promoters of all human genes with the PBM and SVM models, as well as published genome-wide location analysis. Using this integrated approach, we identified similar to 240 new direct HNF4 alpha human target genes, including new functional categories of genes not typically associated with HNF4 alpha, such as cell cycle, immune function, apoptosis, stress response, and other cancer-related genes. Conclusion: We report the first use of PBMs with a full-length liver-enriched transcription factor and greatly expand the repertoire of HNF4 alpha-binding sequences and target genes, thereby identifying new functions for HNF4 alpha. We also establish a web-based tool, HNF4 Motif Finder, that can be used to identify potential HNF4 alpha-binding sites in any sequence. (HEPATOLOGY 2010;51:642-653.)