C/EBPa-Mediated Activation of MicroRNAs 34a and 223 Inhibits Lef1 Expression To Achieve Efficient Reprogramming into Macrophages

Abstract

MicroRNAs (miRNAs) exert negative effects on gene expression and influence cell lineage choice during hematopoiesis. C/EBPa-induced pre-B cell-to-macrophage transdifferentiation provides an excellent model to investigate the contribution of miRNAs to hematopoietic cell identity, especially because the two cell types involved fall into separate lymphoid and myeloid branches. In this process, efficient repression of the B cell-specific program is essential to ensure transdifferentation and macrophage function. miRNA profiling revealed that upregulation of miRNAs is highly predominant compared with downregulation and that C/EBPa directly regulates several upregulated miRNAs. We also determined that miRNA 34a (miR-34a) and miR-223 sharply accelerate C/EBPa-mediated transdifferentiation, whereas their depletion delays this process. These two miRNAs affect the transdifferentiation efficiency and activity of macrophages, including their lipopolysaccharide (LPS)-dependent inflammatory response. miR-34a and miR-223 directly target and downregulate the lymphoid transcription factor Lef1, whose ectopic expression delays transdifferentiation to an extent similar to that seen with miR-34a and miR-223 depletion. In addition, ectopic introduction of Lef1 in macrophages causes upregulation of B cell markers, including CD19, Pax5, and Ikzf3. Our report demonstrates the importance of these miRNAs in ensuring the erasure of key B cell transcription factors, such as Lef1, and reinforces the notion of their essential role in fine-tuning the control required for establishing cell identity.