Abstract
Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.
Highlights
§ These authors contributed to this work. ¶ To whom correspondence should be addressed: Cedars-Sinai Research Institute, UCLA School of Medicine, 8700 Beverly Blvd., B121, Los Angeles, CA 90048
The CCAAT/enhancer-binding protein (C/EBP)⑀ Activation Domain Is Localized in the N Terminus—We previously demonstrated that C/EBP⑀ can activate transcription from the myeloid-specific mim-1 and human myeloperoxidase promoters [12]
Sequence analysis revealed that the basic region and the leucine zipper domains are located between amino acid residues 162 and 235 [11, 12]
Summary
GAL4-C/EBP⑀ Hybrid Constructs—Cloning vector pGALM (a gift from Drs Gregory Kato and Chi Dang) used for all recombinant plasmids in this study is similar to pSG424 [40] and contains the DNA binding domain from GAL4 (amino acids 1–147) driven by the SV40 early promoter and followed by a polylinker containing restriction sites for subcloning. After proper treatment with restriction enzymes, the VP16 AD and the C/EBP⑀ fragments were inserted into the cloning vector pGALM by a three way ligation and confirmed by sequencing. (i) For CMV-⑀⌬116 –234, PCR-derived DNA fragments encoding amino acids 1–70 and 235–249 of C/EBP⑀ were digested with restriction enzymes SalI and EcoRI and ligated together after the two fragments were blunted by a fill-in reaction using Klenow. This produced an in frame fusion of the two C/EBP⑀ fragments that removed residues 116 –234. The neutrophil elastase promoter containing a consensus C/EBP binding site 1 base pair upstream of a Myb binding site [44] was fused to the luciferase gene (NE-Luc) for use as a reporter gene
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