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Identification of a novel enhancer of CEBPE essential for granulocytic differentiation

Pavithra Shyamsunder, Mahalakshmi Shanmugasundaram, Anand Mayakonda, Pushkar Dakle, Weoi Woon Teoh, Lin Han, Deepika Kanojia, Mei Chee Lim, Melissa Fullwood, Omer An, Henry Yang, Jizhong Shi, Mohammad Zakir Hossain, Vikas Madan and H. Phillip Koeffler

Key Points

  • 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.

Abstract

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.

  • Submitted November 15, 2018.
  • Accepted March 25, 2019.
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