Temporal recruitment of CCAAT/enhancer-binding proteins to early and late adipogenic promoters in vivo

The CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators is critically important for the activation of adipogenic genes during differentiation. The C/EBP beta and delta isoforms are rapidly induced upon adipocyte differentiation and are responsible for activating the adipogenic regulators C/EBP alpha and peroxisome proliferator activated receptor (PPAR)gamma 2, which together activate the majority of genes expressed in differentiating adipocytes. However, mitosis is required following the induction of adipogenesis, and the activation of C/EBP alpha. and PPAR gamma 2 gene expression is delayed until cell division is underway. Previous studies have used electromobility shift assays to suggest that this delay is due, at least in part, to a delay between the induction of C/EBP beta protein levels and the acquisition of DNA binding capacity by C/EBP beta. Here we used in vivo chromatin immunoprecipitation analysis of the C/EBP alpha, PPAR gamma 2, resistin, adiponectin, and leptin promoters to examine the kinetics of C/EBP protein binding to adipogenic genes in differentiating cells. In contrast to prior studies, we determined that C/EBP beta and delta were bound to endogenous regulatory sequences controlling the expression of these genes within 1-4 h of adipogenic induction. These results indicated that C/EBP beta and delta bind not only to genes that are induced early in the adipogenic process but also to genes that are induced much later during differentiation, without a delay between induction of C/EBP protein levels and DNA binding by these proteins. We also showed that each of the genes examined undergoes a transition in vivo from early occupancy by C/EBP beta and delta to occupancy by C/EBP alpha at times that correlate with the induction of C/EBP alpha. protein levels, demonstrating the generality of the transition during adipogenesis and indicating that the binding of specific C/EBP isoforms does not correlate with timing of expression from each gene. We have concluded that C/EBP family members bind to adipogenic genes in vivo in a manner that follows the induction of C/EBP protein synthesis.