Identification of a novel enhancer of CEBPE essential for granulocytic differentiation.

Abstract

CCAAT/enhancer binding protein epsilon (CEBPE) is an essential transcription factor for granulocytic differentiation. Mutations of CEBPE occur in individuals with neutrophil-specific granule deficiency (SGD), which is characterized by defects in neutrophil maturation. Cebpe knockout mice also exhibit defects in terminal differentiation of granulocytes, a phenotype reminiscent of SGD. Analysis of DNase-Seq data revealed an open chromatin region 6kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified interaction between this +6 kb region and the core promoter of Cebpe using 4C-seq. To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using dCas9-KRAB and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6 kb region. To investigate further the role of this novel enhancer in myelopoiesis, we generated mice with deletion of this region using CRISPR/Cas9 technology. Germline deletion of the +6 kb enhancer resulted in reduced levels of CEBPE and its target genes and caused a severe block in granulocytic differentiation. We also identified binding of CEBPA and CEBPE to the +6 kb enhancer which suggests their role in regulating the expression of Cebpe. In summary, we have identified a novel enhancer crucial for regulating expression of Cebpe, and required for normal granulocytic differentiation.