De-Novo Identification of PPARγ/RXR Binding Sites and Direct Targets during Adipogenesis

Background: The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPAR gamma). PPAR gamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPAR gamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPAR gamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPAR gamma binding sites in 3T3-L1 preadipocyte cells. Methodology/Principal Findings: Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPAR gamma and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPAR gamma and RXR and that they are functionally capable of driving PPAR gamma specific transcription. Our results strongly indicate that PPAR gamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPAR gamma/RXR association is enriched within the proximity of the 59 region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPAR gamma as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPAR gamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPAR gamma direct targets that have not been previously reported to promote adipogenic differentiation. Conclusions/Significance: We have identified in a genome-wide manner the binding sites of PPAR gamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPAR gamma function in the context of adipocyte differentiation.