The Adipocyte Differentiation Program: RepressiodDerepression of the C/EBPα Gene

Abstract

The 3T3-L1 adipocyte differentiation system is a reliable in vitro model for adipogenesis in vivo. Under the controlled conditions of cell culture, 3T3-L1 preadipocytes can be induced (with appropriate hormonal agents) to differentiate into cells that have the biochemical and morphological characteristics of tissue adipocytes. We have identified, cloned, and characterized a number of adipocyte genes that are coordinately activated during differentiation and contribute to acquisition of the adipocyte phenotype. C/EBPα serves as a pleiotropic transcriptional activator of these and many other adipocyte genes. Expression of C/EBPα is not only required for, but is sufficient (in combination with PPARγ) to activate the differentiation program of 3T3-L1 preadipocytes without the use of hormonal agents. In view of the importance of C/EBPα in adipocyte differentiation, we have begun to characterize the promoter of the C/EBPα gene. In addition to a C/EBP regulatory element in the proximal promoter of the gene, we have identified three repressor binding sites. A differentially expressed nuclear factor that binds to two of these sites has been isolated and characterized. This factor, referred to as CUP (C/EBPα Undifferentiated Protein), is expressed by preadipocytes but not adipocytes. Both the mouse and human C/EBPα genes possess two CUP binding sites, one in the 5′-flanking and another in the 5′-untranslated region of the genes. Mutation of either CUP site individually has little effect on reporter gene transcription mediated by the promoter; however, when both CUP sites are mutated marked synergistic “derepression” occurs. We have purified CUP from 3T3-L1 preadipocytes and shown it to be an isoform of the transcription factor AP-2α. Forced expression of the AP-2α1 isoform in adipocytes, which normally do not express this factor, inhibits reporter gene transcription mediated by the C/EBPα promoter. Our findings indicate that CUP/AP-2α is a repressor of the C/EBPα gene promoter and suggest that this factor maintains the gene in a repressed state prior to differentiation. A binding site (a GC box) for another repressor has been identified at the 5′-end of the C/EBP regulatory element in the C/EBPα promoter. The transcription factor Sp1, which is expressed by preadipocytes, was found to bind at this site. By binding at this site, Sp1 sterically can block access to the C/EBP binding site by members of the C/EBP family. Thus, transcriptional activation of the C/EBPα a gene by C/EBPβ (and possibly C/EBPδ), which is thought to occur early in the adipocyte differentiation program, would be prevented. Consistent with this view, transactivation of C/EBPα promoter by C/EBPβ is inhibited by Sp1. Preliminary evidence indicates that the differentiation inducers increase the rate of turnover of Sp1. Lowering the Sp1 level early in the differentiation program would facilitate binding of C/EBPβ to the C/EBP regulatory element and, thereby, transcriptional activation of the C/EBPα gene.