CCAAT/Enhancer-Binding Protein ε Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding.

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε) as an antagonist of GATA-1 transactivation of the eosinophil's major basic protein-1 (MBP1) P2-promoter, showing C/EBPε and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε mapping its repressor domains, comparing it to C/EBPε and C/EBPε, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε but independent of sumoylation of the RDI core "VKEEP" sumoylation site. These findings identify the N-terminus of C/EBPε as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε repression of GATA-1 occurs via a combination of both C/EBPε-GATA-1 protein-protein interaction and C/EBPε binding to a C/EBP site in the MBP1 promoter. The C/EBPε isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.