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Originally published In Press as doi:10.1074/jbc.M011546200 on February 6, 2001

J. Biol. Chem., Vol. 276, Issue 21, 17739-17746, May 25, 2001
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Structural and Functional Studies of CCAAT/Enhancer-binding Protein epsilon *

Jian-Guo Tang and H. Phillip KoefflerDagger

From the Division of Hematology/Oncology, Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California 90048

CCAAT/enhancer-binding protein (C/EBP) epsilon  is a critical transcription factor for differentiation of myeloid cells. Structural and functional relationships of C/EBPepsilon were explored by recombinant protein studies, gene mutation, and transactivation assays. Evidence strongly suggested that C/EBPepsilon does not have disulfide bonds. Transactivation analysis of C/EBPepsilon 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/EBPepsilon was not detected using both reducing and non-reducing SDS-polyacrylamide gel electrophoresis with Western blot analysis from either bacterial or mammalian expressed C/EBPepsilon . Furthermore, C/EBPepsilon 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/EBPbeta 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/EBPepsilon . 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/EBPepsilon 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/EBPepsilon showed that the C/EBPalpha and -delta but not -beta ADM1 markedly enhanced the chimeric C/EBPepsilon 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/EBPepsilon 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.

* This work was supported in part by the National Institutes of Health as well as by the C. and H. Koeffler Fund, Horn Foundation, Parker Hughes Fund, KO-SO Foundation, and Lymphoma Foundation of America.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Holder of the endowed Mark Goodson Chair of Oncology Research. To whom correspondence should be addressed: Cedars-Sinai Medical Center/UCLA School of Medicine, 8700 Beverly Blvd., BM 109, Los Angeles, CA 90048. Tel.: 310-423-4609; Fax: 310-423-0443; E-mail: koeffler@cshs.org.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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