Structural and functional studies of CCAAT/enhancer-binding protein ε

CCAAT/enhancer-binding protein (C/EBP) epsilon is a critical transcription factor for differentiation of myeloid cells. Structural and functional relationships of C/EBP epsilon were explored by recombinant protein studies, gene mutation, and transactivation assays. Evidence strongly suggested that C/EBP epsilon does not have disulfide bonds. Transactivation analysis of C/EBP epsilon having mutations of each of three conserved cysteines (C345, C148S, and C280S) indicated that the three mutant proteins had almost the same activity as the wild type. Dimer formation of C/EBP epsilon was not detected using both reducing and non-reducing SDS-polyacrylamide gel electrophoresis with Western blot analysis from either bacterial or mammalian expressed C/EBP epsilon, Furthermore, C/EBP epsilon mutant C280S gave a gel band similar to that for wild type, suggesting that this C-terminal, conserved cysteine is not involved in disulfide bond formation in vivo, even though previous data for C/EBP beta suggested that dimers may form in vitro utilizing this conserved cysteine residue. Mutational studies of conserved residues in the activating domain 1 (ADM1) and ADM2 of the amino region of the gene indicated that negative charge is critical for transactivational activity of C/EBP epsilon, Mutational analyses of hydrophobic amino acids in ADM1 suggested that these residues do not play a key role in transactivational activity, Further mutational studies indicated that, although the N-terminal 32-amino acid peptide of C/EBP epsilon isoform p32 did not greatly influence the transactivation activity compared with p30 isoform, this peptide does modulate transactivation activity, Domain swapping experiments substituting the ADM1 domain of various C/EBPs for C/EBP epsilon showed that the C/EBP alpha and -delta but not -beta ADM1 markedly enhanced the chimeric C/EBP epsilon transcriptional activity. Based on mutational data and possible mRNA structure, we hypothesized about the effect of mRNA structure on translation of the two major C/EBP epsilon isoforms: p32 and p30, The data suggested a very stable 8-base pair double helical structure with one strand sequence including the initial codon for p32 and complementary strand with the initial codon for p30.