Identification of a novel enhancer of CEBPE essential for granulocytic differentiation

CCAAT/enhancer binding protein (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 I hypersensitive sites sequencing data revealed an open chromatin region 6 kb downstream of the transcriptional start site of Cebpe in murine myeloid cells. We identified an interaction between this +6-kb region and the core promoter of Cebpe using circular chromosome conformation capture sequencing (4C-seq). To understand the role of this putative enhancer in transcriptional regulation of Cebpe, we targeted it using catalytically inactive Cas9 fused to Krüppel-associated box (KRAB) domain and observed a significant downregulation of transcript and protein levels of CEBPE in cells expressing guide RNA targeting the +6-kb region. To further investigate the role of this novel enhancer further 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,...

Source: Blood - June 5, 2019 Category: Hematology Authors: Shyamsunder, P., Shanmugasundaram, M., Mayakonda, A., Dakle, P., Teoh, W. W., Han, L., Kanojia, D., Lim, M. C., Fullwood, M., An, O., Yang, H., Shi, J., Hossain, M. Z., Madan, V., Koeffler, H. P. Tags: Hematopoiesis and Stem Cells, Phagocytes, Granulocytes, and Myelopoiesis Source Type: research