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

Abstract

CCAAT/enhancer-binding protein ε (C/EBPε) is expressed almost exclusively in the myeloid lineage of the hematopoietic system and functions during terminal differentiation of neutrophils and macrophages, and in the regulation of cytokine gene expression in macrophages and T cells. We have undertaken a series of structure/function studies on the murine C/EBPε polypeptide to investigate the mechanism by which C/EBPε activates transcription. Studies with deletion mutants and fusion proteins consisting of C/EBPε sequences joined to the Gal4 DNA-binding protein identified two transcriptional activation domains in C/EBPε. Removal of sequences between the two activation domains or sequences between the second activation domain and the C-terminal DNA binding domain significantly increased the activity of C/EBPε, suggesting the presence of two separate regulatory domains (designated RD-1ε and RD2E). RD-lε behaved as a classic active repressor domain being capable of inhibiting adjacent activation domains irrespective of their origin and when linked to a heterologous DNA binding domain. Mutagenesis studies revealed a short motif in RD-1ε that appears to be a target site for protein-protein interactions and is conserved in repressor domains from C/EBPβ, Sp3, c-Fos, and FosB. The juxtaposition of activation and repressor domains may permit C/EBPε to function as a transcriptional activator or repressor at different stages of myeloid differentiation or as an inducible transcriptional activator of cytokine genes.