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

BackgroundThe 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-�� (PPAR��). PPAR�� 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�� 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�� binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPAR�� binding sites in 3T3-L1 preadipocyte cells.Methodology/Principal FindingsCoupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPAR�� 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�� and RXR and that they are functionally capable of driving PPAR�� specific transcription. Our results strongly indicate that PPAR�� is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPAR��/RXR association is enriched within the proximity of the 5�� 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�� 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��/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPAR�� direct targets that have not been previously reported to promote adipogenic differentiation.Conclusions/SignificanceWe have identified in a genome-wide manner the binding sites of PPAR�� and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPAR�� function in the context of adipocyte differentiation.