The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPAR␥ and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPAR␥:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPAR␥ RXR-heterodimers. Different temporal and compositional patterns of occupancy are observed. In addition, we detect co-occupancy with members of the C/EBP family. Analysis of RNAPII occupancy uncovers distinct clusters of similarly regulated genes of different biological processes. PPAR␥:RXR binding is associated with the majority of induced genes, and sites are particularly abundant in the vicinity of genes involved in lipid and glucose metabolism. Our analyses represent the first genome-wide map of PPAR␥:RXR target sites and changes in RNAPII occupancy throughout adipocyte differentiation and indicate that a hitherto unrecognized high number of adipocyte genes of distinctly regulated pathways are directly activated by PPAR␥:RXR.[Keywords: Peroxisome proliferator activated receptor; nuclear receptor; ChIP-seq; adipocyte differentiation] Supplemental material is available at http://www.genesdev.org. Adipogenesis is one of the best characterized differentiation processes. Several preadipocyte cell culture models have been developed and used to carefully dissect the sequence of molecular events governing the adipogenic process. Among these adipogenic cell lines, the murine 3T3-L1 preadipocyte cell line (Green and Kehinde 1974) represents one of the best characterized models. Upon addition of adipogenic inducers, including glucocorticoids, cAMP elevating agents, and insulin/insulin-like growth factor, these cells undergo one to two rounds of mitotic clonal expansion followed by growth arrest and terminal differentiation. Several gain-and loss-of-function experiments have revealed an intricate interplay of activating and inhibitory signals involved in the regulation of the adipogenic process (MacDougald and Mandrup 2002;Rosen and MacDougald 2006).The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥; NR1C3) is an obligatory key regulator of adipocyte differentiation in vivo as well as ex vivo (Farmer 2006). In addition, PPAR␥ acts as a transcriptional activator of many adipocyte-specific genes involved in lipid synthesis, handling and storage of lipids, growth regulation, insulin signaling, and adipokine production (Lehrke and Lazar 2005). PPAR␥ is also necessary for maintenance of the adipocyte phenotype and for survival of adipocytes in white adipose tissue in vivo

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors which functions as a master regulator of adipocyte differentiation and metabolism. Here we review recent breakthroughs in the understanding of PPARγ gene regulation and function in a chromatin context. It is now clear that multiple transcription factors team up to induce PPARγ during adipogenesis, and that other transcription factors cooperate with PPARγ to ensure adipocyte-specific genomic binding and function. We discuss how this differs in other PPARγ-expressing cells such as macrophages, and how these genome-wide mechanisms are preserved across species despite modest conservation of specific binding sites. These emerging considerations inform our understanding of PPARγ function as well as adipocyte development and physiology.

Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome-wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPβ and -δ) to transcription factor 'hotspots'. Our results demonstrate that C/EBPβ marks a large number of these transcription factor 'hotspots' before induction of differentiation and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP-binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well-established role in activation of PPARγ transcription, may act as pioneering factors for PPARγ binding.

The raison d'etre of the adipocyte is to store energy (in the form of triacylglycerol) for use during periods of caloric insufficiency. Adipocytes first appear late in fetal development preparatory to postnatal life when a substantial energy reserve is needed to survive periods of fasting. Considerable progress has been made during the past few years in our understanding of the adipocyte differentiation program. This review will focus on the roles of the CAAT/enhancer binding protein (C/EBP) 1 and peroxisome proliferator-activated receptor (PPAR) families of transcription factors in the differentiation program. The reader is referred to other recent reviews (1-3) for references too numerous to include in this Minireview.Our understanding of adipocyte differentiation derives largely from studies with preadipose cell lines in culture, notably the C3H10T1/2 and NIH 3T3 fibroblastic cell lines and the 3T3-L1 and 3T3-F442A preadipocyte lines (1). Treatment of multipotent C3H10T1/2 cells with 5-azacytidine gives rise to cells committed to the myogenic, adipogenic, or chondrogenic lineages. This is consistent with the view that the adipose lineage arises from the same multipotent stem cell population of mesodermal origin that gives rise to the muscle and cartilage lineages (1). When appropriately induced with hormonal agents (e.g. glucocorticoid, insulin-like growth factor-1, and cyclic AMP or factors that mimic these agents) committed preadipocytes differentiate into adipocytes in culture. A large body of evidence shows that differentiation of 3T3 preadipocytes faithfully mimics the in vivo process giving rise to cells that possess virtually all of the biochemical and morphological characteristics of adipocytes (2). Following hormonal induction, confluent preadipocytes undergo mitotic clonal expansion, become growth arrested, and then coordinately express adipocyte gene products (1).Several transcription factors have been identified, which act cooperatively and sequentially to trigger the terminal differentiation program (3-5). These include members of the C/EBP and PPAR families. The sequence of expression of certain of these transcription factors during differentiation is outlined in Fig. 1. It should be noted that these patterns differ somewhat depending upon the differentiation protocol and preadipocyte cell line employed, e.g. 3T3-L1 versus 3T3-F442A versus NIH 3T3 cells.

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