Identification of a novel enhancer of CEBPE essential for granulocytic differentiation

• Published in: Blood Vol. 133; no. 23; pp. 2507 - 2517
• Main Authors: Shyamsunder, Pavithra, Shanmugasundaram, Mahalakshmi, Mayakonda, Anand, Dakle, Pushkar, Teoh, Weoi Woon, Han, Lin, Kanojia, Deepika, Lim, Mei Chee, Fullwood, Melissa, An, Omer, Yang, Henry, Shi, Jizhong, Hossain, Mohammad Zakir, Madan, Vikas, Koeffler, H. Phillip
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.06.2019
• Subjects: Animals; CCAAT-Enhancer-Binding Proteins - biosynthesis; CCAAT-Enhancer-Binding Proteins - genetics; Cell Differentiation - genetics; Gene Expression Regulation - genetics; Granulocytes - metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelopoiesis - genetics
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.2018886077

CCAAT/enhancer binding protein ε (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, we have identified a novel enhancer crucial for regulating expression of Cebpe and required for normal granulocytic differentiation. •Expression of murine Cebpe is regulated by an enhancer located 6 kb downstream from its transcriptional start site.•Deletion of the +6-kb enhancer in mice leads to a complete block in terminal differentiation of granulocytes. [Display omitted]