Alternatively, instead of relying on chromatin structure, the regulation of promoter activities may involve alternative recruitment of specific general transcription factors and specific functional cooperation with enhancers (56,57). C/EBP transcription factors are important for PPAR expression GSK744 (S/GSK1265744) and activities during adipogenesis (7,8,18,19). activated during adipocyte differentiation. Here, we show that PPAR also binds to enhancers already active in preadipocytes as evidenced by an active chromatin state including lower DNA methylation levels despite higher VCL CpG content. These constitutive enhancers are linked to genes involved in the insulin/insulin-like growth factor signaling pathway that are transcriptionally induced during adipogenesis but to a lower extent than lipid metabolism genes, because of stronger basal expression levels in preadipocytes. This is consistent with the sequential involvement of hormonal sensitivity and lipid handling during adipocyte maturation and correlates with the chromatin structure dynamics at constitutive and activated enhancers. Interestingly, constitutive enhancers are evolutionary conserved and can be activated in other tissues, in contrast to enhancers controlling lipid handling genes whose activation is usually more restricted to adipocytes. Thus, PPAR utilizes both broadly active and cell type-specific enhancers to modulate the dynamic range of activation of GSK744 (S/GSK1265744) genes involved in the adipogenic process. == Introduction == White adipose tissue (WAT)5is an endocrine and metabolic organ exerting key functions in glucose and lipid homeostasis (1,2). WAT adapts to energy supply by releasing or storing lipids in response to nutrients and hormonal stimuli. The requirement for supplemental storage capacity is usually provided by both hyperplasic and hypertrophic responses, a process referred to as WAT expandability (3). Moreover, adipocytes are constantly renewed throughout life (4,5), GSK744 (S/GSK1265744) showing that adipocyte differentiation is usually central to WAT development, maintenance, and physiological functions (6). White adipogenesis proceeds from the commitment of mesenchymal stem cells to the adipocyte lineage, followed by differentiation of preadipocytes into adipocytes (7,8). The successive activation of gene networks controlled by, among others, the Wingless-type MMTV integration site, bone morphogenetic protein, and Sonic Hedgehog pathways leads to the formation of preadipocytes. Subsequently, the adipocyte differentiation process results from sequential expression and activation of several transcription factors, such as CCAAT/enhancer binding proteins (C/EBP), which trigger the expression of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) required to reach the mature adipocyte phenotype (7,8). This process also requires PPAR collaboration with another member of the PPAR family,i.e., PPAR/ (9). Recent functional genomics studies, using the murine 3T3-L1 preadipocyte cell line as a model, have improved our understanding of how PPAR promotes differentiation of preadipocytes (1015). The formation of lipid-storing white adipocytes is usually linked to PPAR binding to thousands of transcriptional regulatory elements distinct from gene promoters called enhancers (1015). These enhancers are found in the vicinity of PPAR target genes, which could be activated through chromatin looping between enhancers and promoters as shown for other nuclear receptor family members (16,17). These studies revealed that PPAR genomic binding in adipocytes is usually enriched near genes involved in glucose and lipid metabolism (11,13). PPAR-recruiting enhancers in mature adipocytes are characterized by the typical chromatin structure and epigenetic signature of active enhancers (12,14,15). For instance, these enhancers are found in open chromatin regions showing high sensitivity to DNase I digestion (10) and enrichment by formaldehyde-assisted GSK744 (S/GSK1265744) isolation of regulatory elements (FAIRE) (14). Moreover, they harbor mono- and dimethylated histone H3 lysine 4 (H3K4me1/2) and acetylated H3K27 (H3K27ac) (15). These active enhancer chromatin features have been shown to be established during adipocyte differentiation through sequential recruitment of GSK744 (S/GSK1265744) transcription factors (10,18,19). In this model, early binding of C/EBP initiates chromatin remodeling events that set the ground for subsequent recruitment of PPAR upon adipocyte differentiation (10,18,19). In addition to these enhancers whose activities are induced during adipocyte differentiation (hereafter called activated), PPAR is also recruited to enhancers that are already in an active state in preadipocytes (hereafter called constitutive) (15). However, because they were positively associated with strong transcriptional gene induction, only activated PPAR-recruiting enhancers were deemed to be functionally relevant PPAR-bound regulatory sites during adipogenesis (15). Here, we thoroughly studied the features and functional involvement of these two classes of PPAR-recruiting enhancers in adipogenesis. ==.