title: De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.
abstract: 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 (PPARgamma). PPARgamma 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 PPARgamma have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARgamma binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARgamma binding sites in 3T3-L1 preadipocyte cells.
  
  
  RESULTS
  Coupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARgamma 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 PPARgamma and RXR and that they are functionally capable of driving PPARgamma specific transcription. Our results strongly indicate that PPARgamma is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARgamma/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 PPARgamma 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 PPARgamma/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARgamma direct targets that have not been previously reported to promote adipogenic differentiation.
  
  
  CONCLUSIONS
  We have identified in a genome-wide manner the binding sites of PPARgamma and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARgamma function in the context of adipocyte differentiation.
authors.affiliation: Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore.
authors.forename: Mohamed Sabry
authors.initials: MS
authors.lastname: Hamza
authors.forename: Sebastian
authors.initials: S
authors.lastname: Pott
authors.forename: Vinsensius B
authors.initials: VB
authors.lastname: Vega
authors.forename: Jane S
authors.initials: JS
authors.lastname: Thomsen
authors.forename: Gopalan Srinivasan
authors.initials: GS
authors.lastname: Kandhadayar
authors.forename: Patrick Wei Pern
authors.initials: PW
authors.lastname: Ng
authors.forename: Kuo Ping
authors.initials: KP
authors.lastname: Chiu
authors.forename: Sven
authors.initials: S
authors.lastname: Pettersson
authors.forename: Chia Lin
authors.initials: CL
authors.lastname: Wei
authors.forename: Yijun
authors.initials: Y
authors.lastname: Ruan
authors.forename: Edison T
authors.initials: ET
authors.lastname: Liu
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000502:physiology*; D050156:Adipogenesis / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000502:physiology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D053263:Gene Regulatory Networks; D007004:Hypoglycemic Agents / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D051379:Mice; D046228:Microarray Analysis; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D047488:Retinoid X Receptors / Q000235:genetics / Q000378:metabolism*; D000077154:Rosiglitazone; D045162:Thiazolidinediones / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D047495:PPAR gamma; D034741:RNA, Small Interfering; D047488:Retinoid X Receptors; D045162:Thiazolidinediones; D000077154:Rosiglitazone