During adipocyte differentiation, CCAAT/enhancer-binding protein alpha (C/EBP alpha) functions as a pleiotropic transcriptional activator of numerous adipocyte genes. The promoter of the C/EBP alpha gene has an E-box upstream of C/EBP binding site. Deletion or mutation of the E-box decreases promoter activity, suggesting that the E-box participates in the regulation of C/EBP alpha expression. Protein binding to the E-box during the adipocyte differentiation is increased as indicated by EMSA and UV cross-linking. Purification of the E-box binding proteins from differentiated 3T3-L1 adipocytes, showed that USF and AP-4 are associated with the E-box. Supershift analysis showed that USF1 and USF2 bind to this element as heterodimers, whereas the addition of anti-AP-4 antibody enhanced the binding complex, suggesting that AP-4 negatively regulates the promoter activity. The expression of AP-4 is reciprocally regulated with USF-1 during adipocyte differentiation. These findings suggest that USF-1 and 2 play roles in C/EBP alpha expression, whereas the AP-4 represses it. (c) 2007 Elsevier Inc. All rights reserved.