Granulopoiesis Requires Increased C/EBPα Compared to Monopoiesis, Correlated with Elevated <i>Cebpa</i> in Immature G-CSF Receptor versus M-CSF Receptor Expressing Cells

<div><p>C/EBPα is required for the formation of granulocyte-monocyte progenitors; however, its role in subsequent myeloid lineage specification remains uncertain. Transduction of murine marrow with either of two <i>Cebpa</i> shRNAs markedly increases monocyte and reduces granulocyte colonies in methylcellulose or the monocyte to neutrophil ratio in liquid culture. Similar findings were found after marrow shRNA transduction and transplantation and with <i>CEBPA</i> knockdown in human marrow CD34⁺ cells. These results apparently reflect altered myeloid lineage specification, as similar knockdown allowed nearly complete 32Dcl3 granulocytic maturation. <i>Cebpa</i> knockdown also generated lineage-negative blasts with increased colony replating capacity but unchanged cell cycle parameters, likely reflecting complete differentiation block. The shRNA having the greatest effect on lineage skewing reduced <i>Cebpa</i> 3-fold in differentiating cells but 6-fold in accumulating blasts. Indicating that <i>Cebpa</i> is the relevant shRNA target, shRNA-resistant C/EBPα-ER rescued marrow myelopoiesis. <i>Cebpa</i> knockdown in murine marrow cells also increased <i>in vitro</i> erythropoiesis, perhaps reflecting 1.6-fold reduction in <i>PU.1</i> leading to GATA-1 derepression. Global gene expression analysis of lineage-negative blasts that accumulate after <i>Cebpa</i> knockdown demonstrated reduction in <i>Cebpe</i> and <i>Gfi1</i>, known transcriptional regulators of granulopoiesis, and also reduced <i>Ets1</i> and <i>Klf5</i>. Populations enriched for immature granulocyte or monocyte progenitor/precursors were isolated by sorting Lin⁻Sca-1⁻c-Kit⁺ cells into GCSFR⁺MCSFR⁻ or GCSFR⁻MCSFR⁺ subsets. <i>Cebpa</i>, <i>Cebpe</i>, <i>Gfi1</i>, <i>Ets1</i>, and <i>Klf5</i> RNAs were increased in the c-Kit⁺GCSFR⁺ and <i>Klf4</i> and <i>Irf8</i> in the c-Kit⁺MCSFR⁺ populations, with <i>PU.1</i> levels similar in both. In summary, higher levels of C/EBPα are required for granulocyte and lower levels for monocyte lineage specification, and this myeloid bifurcation may be facilitated by increased <i>Cebpa</i> gene expression in granulocyte compared with monocyte progenitors.</p>