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

Abstract

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 Cebpa 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 CEBPA knockdown in human marrow CD34+ cells. These results apparently reflect altered myeloid lineage specification, as similar knockdown allowed nearly complete 32Dcl3 granulocytic maturation. Cebpa 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 Cebpa 3-fold in differentiating cells but 6-fold in accumulating blasts. Indicating that Cebpa is the relevant shRNA target, shRNA-resistant C/EBPα-ER rescued marrow myelopoiesis. Cebpa knockdown in murine marrow cells also increased in vitro erythropoiesis, perhaps reflecting 1.6-fold reduction in PU.1 leading to GATA-1 derepression. Global gene expression analysis of lineage-negative blasts that accumulate after Cebpa knockdown demonstrated reduction in Cebpe and Gfi1, known transcriptional regulators of granulopoiesis, and also reduced Ets1 and Klf5. 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. Cebpa, Cebpe, Gfi1, Ets1, and Klf5 RNAs were increased in the c-Kit+GCSFR+ and Klf4 and Irf8 in the c-Kit+MCSFR+ populations, with PU.1 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 Cebpa gene expression in granulocyte compared with monocyte progenitors.

IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production

“…In contrast, c/EBPa is expressed by all GPs and MPs but only a subset of GMPs (supplemental Figure 8). These data are consistent with a requirement for both of these transcription factors in neutrophil and monocyte differentiation (c/EBPa homodimers direct neutrophil fate, whereas heterodimers with AP-1 proteins promote monocyte fate 20,21 ), with higher levels of PU.1 being required for monocyte differentiation. [22][23][24] IRF8 regulates the survival and differentiation, but not production, of GPs and MPs…”

Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity

“…The C/EBP family of transcription factors regulates differentiation of various cell types, including myeloid cells 31 . IPA upstream regulator analysis also predicted CEBPB activation (Table S2).…”

C/EBPα in normal and malignant myelopoiesis

“…Cebpa (−/−) fetal liver or marrow from Cebpa (f/f);Mx1-Cre adult mice exposed to pIpC to induce Cre expression and biallelic deletion of the Cebpa ORF have markedly reduced GMP and myeloid colony-forming units (CFU), with increased CMP, LSK, and megakaryocyte/erythroid progenitors (MEP), and their peripheral blood has markedly reduced neutrophils and monocytes and absence of eosinophils, with twofold elevated platelets and mild lymphocytosis [60, 63]. Beyond the GMP, threefold Cebpa knockdown prevents granulopoiesis but not monopoiesis, while sixfold knockdown prevents commitment to either lineage and increases erythropoiesis [64]. …”

“…C/EBPα induces transcription of several regulatory proteins required for subsequent lineage maturation, including the transcription factors C/EBPε, Gfi-1, and KLF5 [64, 69–72]. Absence of C/EBPε leads to secondary granule deficiency [73], Gfi-1(−/−) mice develop severe neutropenia [74, 75], and KLF5 contributes to 32Dcl3 granulopoiesis [76].…”