ABSTRACT During adipogenesis, CCAAT/enhancer binding protein α(C/EBPα) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPα gene and a putative transacting factor (C/EBPα undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass '50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2α. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2α. Both AP-2α protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2α/CUP, an AP-2α1 expression vector, cotransfected with a C/EBPα promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPα gene is repressed by AP-2α/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT/enhancer-binding protein (C/EBP)β is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C/EBP activates expression of C/EBPα and peroxisome proliferator-activated receptorγ, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C/EBPβ is expressed immediately after induction but exhibits delayed acquisition of DNAbinding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C/EBPβ (-/-) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C EBP (LAP) but not dominant-negative C/EBPβ (LIP) in C/EBPβ (-/-) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C/EBPβ is a prerequisite for MCE in the adipocyte-differentiation program.

Hormonal induction of 3T3-L1 preadipocytes triggers a cascade of events that initiate differentiation into adipocytes. CCAAT/enhancer-binding proteinsβ andδ (C/EBPβ/δ) are expressed early in the differentiation program, but are not immediately active. After a long lag, C/EBPβ/δ become competent to bind to the C/EBP regulatory element in the C/EBPa gene promoter, C/EBPa being a transcriptional activator of numerous adipocyte genes. As C/EBPβ/δ acquire binding activity, they become localized to centromeres as preadipocytes synchronously enter S phase at the onset of mitotic clonal expansion. Localization to centromeres occurs through C/EBP consensus-binding sites in centromeric satellite DNA. C/EBPa, which is antimitotic, becomes centromere-associated much later in the differentiation program as mitotic clonal expansion ceases and the cells become terminally differentiated.