In Vivo Deletion of the Cebpa +37 kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis.

Hong Guo,Stacy Cooper,Alan D Friedman,Tadayuki Akagi

doi:10.1371/journal.pone.0150809

Hong Guo, Stacy Cooper + Show 2 more

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https://doi.org/10.1371/journal.pone.0150809

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Journal: PloS one	Publication Date: Mar 3, 2016
Citations: 23	License type: CC BY 4.0

Affiliation: Johns Hopkins Medicine, Johns Hopkins University

Abstract

The murine Cebpa gene contains a +37 kb, evolutionarily conserved 440 bp enhancer that directs high-level expression to myeloid progenitors in transgenic mice. The enhancer is bound and activated by Runx1, Scl, GATA2, C/EBPα, c-Myb, Pu.1, and additional Ets factors in myeloid cells. CRISPR/Cas9-mediated replacement of the wild-type enhancer with a variant mutant in its seven Ets sites leads to 20-fold reduction of Cebpa mRNA in the 32Dcl3 myeloid cell line. To determine the effect of deleting the enhancer in vivo, we now characterize C57BL/6 mice in which loxP sites flank a 688 bp DNA segment containing the enhancer. CMV-Cre mediated germline deletion resulted in diminution of the expected number of viable Enh(f/f);CMV-Cre offspring, with 28-fold reduction in marrow Cebpa mRNA but normal levels in liver, lung, adipose, intestine, muscle, and kidney. Cre-transduction of lineage-negative marrow cells in vitro reduced Cebpa mRNA 12-fold, with impairment of granulocytic maturation, morphologic blast accumulation, and IL-3 dependent myeloid colony replating for >12 generations. Exposure of Enh(f/f);Mx1-Cre mice to pIpC led to 14-fold reduction of Cebpa mRNA in GMP or CMP, 30-fold reduction in LSK, and <2-fold reduction in the LSK/SLAM subset. FACS analysis of marrow from these mice revealed 10-fold reduced neutrophils, 3-fold decreased GMP, and 3-fold increased LSK cells. Progenitor cell cycle progression was mildly impaired. Granulocyte and B lymphoid colony forming units were reduced while monocytic and erythroid colonies were increased, with reduced Pu.1 and Gfi1 and increased Egr1 and Klf4 in GMP. Finally, competitive transplantation indicated preservation of functional long-term hematopoietic stem cells upon enhancer deletion and confirmed marrow-intrinsic impairment of granulopoiesis and B cell generation with LSK and monocyte lineage expansion. These findings demonstrate a critical role for the +37 kb Cebpa enhancer for hematopoietic-specific Cebpa expression, with enhancer deletion leading to impaired myelopoiesis and potentially preleukemic progenitor expansion.

Highlights

CCAAT/enhancer binding protein α (C/EBPα) is a basic region-leucine zipper transcription factor expressed preferentially within granulocytic and monocytic myeloid cells during hematopoiesis [1]
C/EBPα levels increase as long-term hematopoietic stem cells (LT-HSC) progress to the common myeloid progenitor (CMP) and subsequently to the granulocyte-monocyte progenitor (GMP), with Cebpa open reading frame (ORF) deletion preventing GMP formation associated with accumulation of upstream CMP and the Lin-Sca-1+c-kit+ (LSK) stem/progenitor subsets [2, 3]
Mutation of its seven Ets sites led to the greatest reduction in enhancer activity, and CRISPR/Cas9-mediated replacement of the endogenous enhancer alleles with a variant harboring point mutations in these Ets sites led to 20-fold reduced Cebpa mRNA expression in 32Dcl3 myeloid cells [11]

Summary

Introduction

CCAAT/enhancer binding protein α (C/EBPα) is a basic region-leucine zipper transcription factor expressed preferentially within granulocytic and monocytic myeloid cells during hematopoiesis [1]. C/EBPα expression or activity is commonly diminished in acute myeloid leukemia (AML) cases, including CEBPA point mutations impacting trans-activation or DNA-binding, RUNX1-ETO expression reducing CEBPA transcription, and C/EBPα(S21) phosphorylation impairing trans-activation [5]. We identified a 440 bp DNA segment centered at +37.5 kb in the murine Cebpa gene, with 85% homology to the +42 kb region of the human CEBPA locus, harboring enhancer specific H3K4me histone marks and together with the promoter capable of directing high-level hCD4 transgene expression to GMP, CMP, and LSK cells but not to multiple non-hematopoietic tissues [7, 8]. Mutation of its seven Ets sites led to the greatest reduction in enhancer activity, and CRISPR/Cas9-mediated replacement of the endogenous enhancer alleles with a variant harboring point mutations in these Ets sites led to 20-fold reduced Cebpa mRNA expression in 32Dcl myeloid cells [11]

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