REPRESSION OF TRANSCRIPTION MEDIATED BY DUAL ELEMENTS IN THE CCAAT ENHANCER BINDING-PROTEIN ALPHA-GENE/

During adipocyte differentiation, the expression of C/EBP alpha is act ivated, which in turn serves to transcriptionally activate numerous ad ipocyte genes. A previous search for cis elements that regulate transc ription of the C/EBP alpha gene led to the identification of a potenti al repressive element within the proximal 5' flanking region of the ge ne. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, we re found to contain a factor, CUP (C/EBP alpha undifferentiated protei n), that binds to this site (the CUP-1 site). In the present investiga tion, we show that C/EBP alpha promoter-luciferase constructs containi ng both the proximal 5' flanking and the entire 5' untranslated region s of the gene exhibit an expression pattern during adipocyte different iation comparable to that of the endogenous C/EBP alpha gene. Mutation of the CUP-1 site in these constructs had little effect on reporter g ene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocy tes was dramatically up-regulated. Consistent with this finding, a sec ond CUP binding site (the CUP-2 site) was identified in the 5' untrans lated region. Although mutation of either CUP element in constructs co ntaining both the 5' flanking and 5' untranslated region had little ef fect on reporter gene transcription, mutation of both CUP elements mar kedly activated transcription. Thus, it appears that dual CUP regulato ry elements repress transcription of the C/EBP alpha gene prior to ind uction of the adipocyte differentiation program.