LEF-1 Regulates C/EBP α Expression and Neutrophil Differentiation in Normal Myelopoiesis and Congenital Neutropenia by a β-Catenin Independent Mechanism.

Abstract

Lymphoid enhancer-binding factor 1 (LEF-1) belongs to the canonical Wnt signaling pathway acting in transcriptional complexes with β-catenin. LEF-1 can also act independent of β-catenin (i.e. in the TGF-β or Notch pathway). Additionally, recent studies described LEF-1 dominant negative isoform (dnLEF-1), which lack the β-catenin binding domain and functions as either a transcriptional repressor or activator. To date, analysis of the role of LEF-1 in hematopoiesis has been restricted to the lymphoid compartment.Previously we described the crucial role of lymphoid enhancer-binding factor 1 (LEF-1) in granulopoiesis. We found that LEF-1 mediates proliferation, survival, and differentiation of granulocyte progenitor cells. Moreover, abrogated LEF-1 expression is one of the pathomechanism of severe congenital neutropenia CN (Skokowa et al., Nature Medicine, in press). Based on these findings, we aimed to characterize the molecular mechanisms of LEF-1 in the regulation of granulocytic differentiation. C/EBPα is well known as a key transcription factor in granulopoiesis and we found it to be a target gene directly regulated by LEF-1. A screen of the known 566 bp upstream promoter of C/EBPα gene revealed a putative LEF-1 binding site (− 559 bp to − 538 bp). We confirmed LEF-1 binding to C/EBPα promoter in nuclear extracts from CD34+ and CD33+ cells in the transcription factor binding NoShift and ChIP assays. Interestingly, LEF-1 binds to the C/EBPα promoter more efficiently in CD33+ myeloid progenitors than in CD34+ cells. The direct regulation of C/EBPα by LEF-1 was further confirmed in functional studies. We found that in line with down-regulation of LEF-1, expression of C/EBPα was also significantly reduced in CD33+ myeloid progenitors of CN patients. Moreover, LEF-1 rescue of these cells resulted in a marked up-regulation of C/EBPα mRNA expression and in vitro restoration of defective granulocytic differentiation. Remarkably, transduction of CN CD33+ cells with dnLEF-1 isoform, which lacks the ß-catenin-binding domain, resulted in up-regulation of C/EBPα to a similar degree as it was observed with full-length LEF-1. A direct regulatory link between LEF-1 and C/EBPα was additionally confirmed in LEF-1 inhibition experiments. C/EBPα expression was significantly down-regulated in CD34+ cells of healthy individuals, transduced with LEF-1 shRNA. Therefore, we propose that LEF-1 is a key regulator of myeloid differentiation acting in a β-catenin-independent manner, similar as it is known for LEF-1 regulation of T-lymphocyte development.