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

CCAAT/enhancer-binding protein (C/EBP) epsilon is a critical transcription factor for differentiation of myeloid cells. Structural and functional rela tionships of C/EBP epsilon were explored by recombinant protein studies, ge ne mutation, and transactivation assays. Evidence strongly suggested that C /EBP epsilon does not have disulfide bonds. Transactivation analysis of C/E BP epsilon having mutations of each of three conserved cysteines (C345, C14 8S, 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 detect ed using both reducing and non-reducing SDS-polyacrylamide gel electrophore sis with Western blot analysis from either bacterial or mammalian expressed C/EBP epsilon, Furthermore, C/EBP epsilon mutant C280S gave a gel band sim ilar to that for wild type, suggesting that this C-terminal, conserved cyst eine is not involved in disulfide bond formation in vivo, even though previ ous data for C/EBP beta suggested that dimers may form in vitro utilizing t his conserved cysteine residue. Mutational studies of conserved residues in the activating domain 1 (ADM1) and ADM2 of the amino region of the gene in dicated that negative charge is critical for transactivational activity of C/EBP epsilon, Mutational analyses of hydrophobic amino acids in ADM1 sugge sted that these residues do not play a key role in transactivational activi ty, Further mutational studies indicated that, although the N-terminal 32-a mino acid peptide of C/EBP epsilon isoform p32 did not greatly influence th e transactivation activity compared with p30 isoform, this peptide does mod ulate transactivation activity, Domain swapping experiments substituting th e ADM1 domain of various C/EBPs for C/EBP epsilon showed that the C/EBP alp ha and -delta but not -beta ADM1 markedly enhanced the chimeric C/EBP epsil on transcriptional activity. Based on mutational data and possible mRNA str ucture, we hypothesized about the effect of mRNA structure on translation o f the two major C/EBP epsilon isoforms: p32 and p30, The data suggested a v ery stable 8-base pair double helical structure with one strand sequence in cluding the initial codon for p32 and complementary strand with the initial codon for p30.