Derepression of the C/EBPα Gene during Adipogenesis: Identification of AP-2α as a Repressor

• Published in: Proceedings of the National Academy of Sciences - PNAS 1998-03, Vol.95 (7), p.3467-3471
• Main Authors: Jiang, Man-Shiow, Tang, Qi-Qun, McLenithan, John, Geiman, Deb, Shillinglaw, Wendy, Henzel, William J., Lane, M. Daniel
• Format: Article
• Language: English
• Subjects: Adipocytes; Amino acids; Antibodies; Binding sites; Biological Sciences; Genes; Genetic vectors; Oligonucleotide probes; Oligonucleotides; Protein isoforms; Reporter genes
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.7.3467

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-2α 1 expression vector, cotransfected with a C/EBP α 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.