A short conserved motif is required for repressor domain function in the myeloid-specific transcription factor CCAAT enhancer-binding protein epsilon

CCAAT/enhancer-binding protein epsilon (C/EBP epsilon) is expressed almost exclusively in the myeloid lineage of the hematopoietic system and function s during terminal differentiation of neutrophils and macrophages, and in th e regulation of cytokine gene expression in macrophages and T cells. We hav e undertaken a series of structure/function studies on the murine C/EBP eps ilon polypeptide to investigate the mechanism by which C/EBP epsilon activa tes transcription. Studies with deletion mutants and fusion proteins consis ting of C/EBP epsilon sequences joined to the Gal4 DNA-binding protein iden tified two transcriptional activation domains in C/EBP epsilon, Removal of sequences between the two activation domains or sequences between the secon d activation domain and the C-terminal DNA binding domain significantly inc reased the activity of C/EBP epsilon, suggesting the presence of two separa te regulatory domains (designated RD-1 epsilon and RD-2 epsilon), RD-1 epsi lon behaved as a classic active repressor domain being capable of inhibitin g adjacent activation domains irrespective of their origin and when linked to a heterologous DNA binding domain. Mutagenesis studies revealed a short motif in RD-1 epsilon that appears to be a target site for protein-protein interactions and is conserved in repressor domains from C/EBP beta, Sp3, c- Fos, and FosB, The juxtaposition of activation and repressor domains may pe rmit C/EBP epsilon to function as a transcriptional activator or repressor at different stages of myeloid differentiation or as an inducible transcrip tional activator of cytokine genes.