DEREPRESSION OF THE C EBP-ALPHA GENE DURING ADIPOGENESIS - IDENTIFICATION OF AP-2-ALPHA AS A REPRESSOR/

During adipogenesis, CCAAT/enhancer binding protein alpha (C/EBP alpha ) serves as a pleiotropic transcriptional activator of adipocyte genes , Previously, we identified dual repressive elements in the C/EBP alph a gene and a putative transacting factor (C/EBP alpha undifferentiated protein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements, In the present investigation, CUP was purifie d 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes, Amino aci d sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass approximate to 50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP- 2 alpha. Electrophoretic mobility shift and Western blot analysis on p urified CUP and preadipocyte nuclear extracts confirmed the identity o f CUP as AP-2 alpha. Both AP-2 alpha protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes, Consistent wi th a repressive role of AP-2 alpha CUP, an AP-2 alpha 1 expression vec tor, cotransfected with a C/EBP alpha promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription, Taken toget her with previous results, these findings suggest that In preadipocyte s the C/EBP alpha gene is repressed by AP-2 alpha/CUP, which, upon ind uction of differentiation, is down-regulated, allowing expression of t he gene.