p300 Coactivates the adipogenic transcription factor CCAAT/enhancer-binding protein alpha

Despite the knowledge that CCAAT/enhancer-binding protein alpha (C/EBP alph a) plays an important role in preadipocyte differentiation, our understandi ng of how C/EBP alpha interacts with nuclear proteins to regulate transcrip tion is limited. Based on the hypothesis that evolutionarily conserved regi ons are functionally important and likely to interact with coactivators, we compared the amino acid sequence of C/EBP alpha from different species (fr og to human) and identified four highly conserved regions (CR1-CR4) within the transactivation domain. A series of amino-terminal truncations and inte rnal deletion constructs were made creating forms of C/EBP alpha which lack single or multiple conserved regions. To determine which regions of the C/ EBP alpha transactivation domain are important in its ability to induce spo ntaneous differentiation of 3T3-L1 preadipocytes, we infected preadipocytes with expression vectors encoding the C/EBP alpha conserved region mutants and observed their ability to induce differentiation. We found that CR2 fus ed 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/EBP alpha because a combination of CR1 and C R3 can also induce adipogenesis. Because the transcriptional coactivator p3 00 participates in the signaling of many transcription factors to the basal transcriptional apparatus, we examined whether functional interaction exis ts between C/EBP alpha and p300. Cotransfection of p300 with p42C/EBP alpha results in a synergistic increase in leptin promoter activity, indicating that p300 acts as a transcriptional coactivator of C/EBP alpha. Analyses us ing C/EBP alpha conserved region mutants suggest that multiple regions (CR2 and CR3) of the C/EBP alpha transactivation domain functionally interact w ith p300.