A Short Conserved Motif Is Required for Repressor Domain Function in the Myeloid-specific Transcription Factor CCAAT/Enhancer-binding Protein ε

Nicholas D Angerer,Yang Du,Demet Nalbant,Simon C Williams

doi:10.1074/jbc.274.7.4147

Abstract

CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) 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/EBPepsilon polypeptide to investigate the mechanism by which C/EBPepsilon activates transcription. Studies with deletion mutants and fusion proteins consisting of C/EBPepsilon sequences joined to the Gal4 DNA-binding protein identified two transcriptional activation domains in C/EBPepsilon. 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/EBPepsilon, suggesting the presence of two separate regulatory domains (designated RD-1epsilon and RD-2epsilon). RD-1epsilon 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-1epsilon that appears to be a target site for protein-protein interactions and is conserved in repressor domains from C/EBPbeta, Sp3, c-Fos, and FosB. The juxtaposition of activation and repressor domains may permit C/EBPepsilon to function as a transcriptional activator or repressor at different stages of myeloid differentiation or as an inducible transcriptional activator of cytokine genes.

Highlights

Transcription factors play important roles in the commitment of precursor cells to particular hematopoietic lineages and the terminal differentiation of specific cell types [1]
The CCAAT/enhancer-binding protein (C/EBP)⑀ Protein Contains Two Regions That Function as Transcriptional Activation Domains—To identify functional domains that mediate transcriptional activation by C/EBP⑀, we compared the ability of wild type C/EBP⑀ and two proteins lacking N-terminal segments to activate the (DEI)4-35AlbLUC reporter construct in HepG2 human hepatocarcinoma cells (Fig. 1A)
We previously demonstrated that amino acids 33– 64 of C/EBP⑀ functions as a relatively weak activation domain when fused to the DNA binding domain of the yeast transcriptional activator protein Gal4 [18]

Summary

The abbreviations used are

C/EBP, CCAAT/enhancer-binding protein; DBD, DNA binding domain; ADM, activation domain module; AD, activation domain; RD, regulatory domain; M-CSFR, macrophage-colony-stimulating factor receptor; CM, conserved motif; PCR, polymerase chain reaction; MAP, mitogen-activated protein. The primary defects observed in C/EBP⑀-deficient mice were in the development of the granulocyte lineage, there is evidence that C/EBP⑀ functions in other hematopoietic cell types. The lack of defects in monocytic development and function in C/EBP⑀-deficient mice may be due to functional redundancy among C/EBP family members expressed in this cell lineage [7]. Two regions within the C/EBP⑀ polypeptide act as repressor domains and are likely to be sites of protein-protein interactions that modulate the activity of C/EBP⑀.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION