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

RESEARCH ARTICLE In Vivo Deletion of the Cebpa +37 kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in NonHematopoietic Tissues to Impair Granulopoiesis Hong Guo, Stacy Cooper, Alan D. Friedman* Division of Pediatric Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America * OPEN ACCESS Citation: Guo H, Cooper S, Friedman AD (2016) 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. PLoS ONE 11(3): e0150809. doi:10.1371/journal.pone.0150809 Editor: Tadayuki Akagi, Kanazawa University, JAPAN Received: December 18, 2015 Accepted: February 19, 2016 Published: March 3, 2016 Copyright: © 2016 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Institutes of Health grants U01 HL099775, P30 CA006973, and T32 CA60441, www.nih.gov. The U01 grant includes funds designated for Dr. Friedman's laboratory and personnel support. The P30 grant provides Oncology enter core services. The T32 grant is a training grant which helps fund Dr. Cooper's salary. the Alex's Lemonade Stand Foundation and Damon Runyon Cancer Research Foundation support Dr. Cooper's salary and provide 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. PLOS ONE | DOI:10.1371/journal.pone.0150809 March 3, 2016 1 / 23 The +37 kb Cebpa Enhancer Is Critical for Myelopoiesis supply support. The Giant Food Children's Cancer Research Fund, no grant number, www.giantfood. com. Giant foods makes an annual charitable donation to our Pediatric Oncology division and the monies are used in part for maintaining laboratory equipment. National Institutes of Health grant R01 HL130034 to Alan D. Friedman. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Giant foods is a commercial funder. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. 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 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]. As GMP mature, high-level C/EBPα expression is required for granulopoiesis while reduced levels allow monopoiesis [4]. 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 also impairing trans-activation [5]. The Cebpa promoter is directly activated by C/EBPα and RUNX1 [6, 7]. In addition, 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 H3K4me1 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]. Runx1, C/EBPα, Pu.1, Erg, Fli-1, GATA2, Scl, Meis1, and Gfi-1b bind chromatin in the region of this enhancer in hematopoietic cells as determined by ChIP-Seq [9, 10], Runx1, C/EBPα, Pu.1, Fli-1, Erg, Ets1, c-Myb, GATA2, and Scl bind conserved enhancer cis elements in gel shift assays, and mutation of the Runx1, C/EBP, Ets, Myb, GATA, or E-box sites each reduce enhancer activity in 32Dcl3 myeloid cells in reporter assays [7, 11]. 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]. To determine whether the +37 kb Cebpa enhancer is also critical for regulating Cebpa expression in vivo, we have now generated and characterized mice in which loxP sites flank the enhancer, designated as Enh(f/f) mice. Germline deletion using CMV-Cre revealed marked reduction of Cebpa expression in marrow but not in other tissues, including liver, adipose, and lung, that normally express (...truncated)