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.

ABSTRACT During adipocyte differentiation, the expression of CyEBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the CyEBPagene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoterluciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPa gene prior to induction of the adipocyte differentiation program.