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.
Highlights
CCAAT/enhancer binding protein a (C/EBPa) is a basic region-leucine zipper transcription factor expressed within granulocytic and monocytic myeloid cells during hematopoiesis; C/ EBPa is the predominant C/EBP protein in immature myeloid cells [1,2]
Murine lineage-negative marrow cells were transduced with pLKO.1 lentiviral vectors expressing one of several Cebpa shRNAs (B9, B10, or B11) or the empty pLKO.1 vector for 2 days followed by puromycin selection for 2 additional days
Providing new insight into the role of C/EBPa in myeloid lineage specification, we demonstrate that partial Cebpa knockdown impairs granulopoiesis while increasing monopoiesis with further reduction blocking commitment to either myeloid lineage
Summary
CCAAT/enhancer binding protein a (C/EBPa) is a basic region-leucine zipper transcription factor expressed within granulocytic and monocytic myeloid cells during hematopoiesis; C/ EBPa is the predominant C/EBP protein in immature myeloid cells [1,2]. Exogenous C/ EBPa directs granulocytic maturation of the U937, HL-60, or 32Dcl myeloid cell lines but induces monocytic maturation of murine marrow myeloid progenitors or lymphoid cells [2,5,6,7,8]. Cebpa knockdown led to accumulation of an immature population apparently unable to commit to either lineage, with increased in vitro growth and replating capacity, a preleukemic phenotype. Supporting the idea that Cebpa knockdown blocks granulocyte lineage commitment and not granulocytic maturation, we show that 5-fold Cebpa reduction in the 32Dcl cells line allows maturation to the metamyelocyte/band stage in response to G-CSF. Cebpa knockdown unexpectedly increased marrow cell erythropoiesis, potentially reflecting reduced Sfpi1/PU. expression and thereby GATA-1 derepression [9,10]
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