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3834 Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

Program: Oral and Poster Abstracts
Session: 502. Hematopoiesis: Regulation of Gene Transcription, Cytokines, Signal Transduction, Apoptosis, and Cell Cycle Regulation: Poster III
Hematology Disease Topics & Pathways:
Biological Processes, epigenetics, hematopoiesis
Monday, December 3, 2018, 6:00 PM-8:00 PM
Hall GH (San Diego Convention Center)

Pavithra Shyamsunder, PhD1*, Mahalakshmi Shanmugasundaram1*, Anand Mayakonda1*, Weoi Woon Teoh1*, Lin Han, MSc1,2*, Mei Chee Lim1*, Melissa Fullwood1*, Omer An1*, Henry Yang, PhD1*, Shi Jizhong1*, Md.Zakhir Hossain1*, Vikas Madan, PhD1* and H. Phillip Koeffler, MD1,3,4

1Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore
2Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
3Cedars-Sinai Medical Center, Division of Hematology/Oncology, UCLA School of Medicine, Los Angeles, CA
4Department of Hematology-Oncology, National University Cancer Institute of Singapore (NCIS), National University Hospital, Singapore, Singapore

CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins.

Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE.

Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp).

To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice.

In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH