Abstract
CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil’s major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 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/EBPε27 but independent of sumoylation of the RDI core “VKEEP” sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein–protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 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/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.
Highlights
IntroductionC/EBPε responsible for neutrophil specific granule deficiency [7,8], which includes a deficiency in eosinophil development [9], demonstrate an essential role for the C/EBPε32 isoform in the terminal differentiation and maturation of granulocytes in both mice and humans, and the ability of this transcription factor to regulate various granulocyte promoters in a lineage-specific fashion [5,7,10,11]
We previously reported that the ability of the C/EBPε27 isoform to inhibit eosinophil differentiation and gene expression occurs via its unique ability to repress GATA-1-mediated transactivation of eosinophil gene promoters, while the other activator and repressor isoforms, C/EBPε32 and C/EBPε14, lack this activity [11]
Our results show that C/EBPε27 functions in vivo as a repressor of endogenous eosinophil gene transcription, that two repression domains contribute to its attenuation of GATA-1 activity— its unique N-terminal domain, that repression of GATA-1 is independent of sumoylation of C/EBPε27, and that it does not require DNA
Summary
C/EBPε responsible for neutrophil specific granule deficiency [7,8], which includes a deficiency in eosinophil development [9], demonstrate an essential role for the C/EBPε32 isoform in the terminal differentiation and maturation of granulocytes in both mice and humans, and the ability of this transcription factor to regulate various granulocyte promoters in a lineage-specific fashion [5,7,10,11]. Transcriptional repression mediated by the RDI domain of the murine equivalent of full-length C/EBPε32 was reported to be dependent upon sumoylation of a conserved SUMO consensus site (VKEEP) [19]. Our results show that C/EBPε27 functions in vivo as a repressor of endogenous eosinophil gene transcription, that two repression domains contribute to its attenuation of GATA-1 activity— its unique N-terminal domain, that repression of GATA-1 is independent of sumoylation of C/EBPε27 , and that it does not require DNA binding to a proximal C/EBP site
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