p300 Coactivates the Adipogenic Transcription Factor CCAAT/Enhancer-binding Protein α

• Published in: The Journal of biological chemistry 2001-05, Vol.276 (19), p.16348-16355
• Main Authors: Erickson, Robin L., Hemati, Nahid, Ross, Sarah E., MacDougald, Ormond A.
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Adipocytes - cytology; Adipocytes - metabolism; Animals; Base Sequence; CCAAT-Enhancer-Binding Protein-alpha - chemistry; CCAAT-Enhancer-Binding Protein-alpha - genetics; CCAAT-Enhancer-Binding Protein-alpha - metabolism; Cell Line; Cloning, Molecular; DNA Primers; E1A-Associated p300 Protein; Genes, Reporter; Genetic Vectors; Humans; Luciferases - genetics; Mice; Molecular Sequence Data; Nuclear Proteins - metabolism; Polymerase Chain Reaction; Recombinant Fusion Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Retroviridae; Trans-Activators - metabolism; Transcriptional Activation; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M100128200

Despite the knowledge that CCAAT/enhancer-binding protein α (C/EBPα) plays an important role in preadipocyte differentiation, our understanding of how C/EBPα interacts with nuclear proteins to regulate transcription is limited. Based on the hypothesis that evolutionarily conserved regions are functionally important and likely to interact with coactivators, we compared the amino acid sequence of C/EBPα from different species (frog to human) and identified four highly conserved regions (CR1–CR4) within the transactivation domain. A series of amino-terminal truncations and internal deletion constructs were made creating forms of C/EBPα which lack single or multiple conserved regions. To determine which regions of the C/EBPα transactivation domain are important in its ability to induce spontaneous differentiation of 3T3-L1 preadipocytes, we infected preadipocytes with expression vectors encoding the C/EBPα conserved region mutants and observed their ability to induce differentiation. We found that CR2 fused to the DNA binding domain is able to induce spontaneous differentiation independent of the other conserved regions. However, CR2 was not necessary for the adipogenic action of C/EBPα because a combination of CR1 and CR3 can also induce adipogenesis. Because the transcriptional coactivator p300 participates in the signaling of many transcription factors to the basal transcriptional apparatus, we examined whether functional interaction exists between C/EBPα and p300. Cotransfection of p300 with p42C/EBPα results in a synergistic increase in leptin promoter activity, indicating that p300 acts as a transcriptional coactivator of C/EBPα. Analyses using C/EBPα conserved region mutants suggest that multiple regions (CR2 and CR3) of the C/EBPα transactivation domain functionally interact with p300.