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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, Shi Jizhong, Md 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 TSS.

  • Deletion of the +6 kb enhancer in mice leads to a complete block in terminal differentiation of granulocytes

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.

  • Submitted November 15, 2018.
  • Revision received March 26, 2019.
  • Accepted April 2, 2019.